Brain Mechanisms in Mental Retardation

U C L A F O R U M I N M E D I C A L S C I E N C E S

EDITORIAL BOARD Mary A. B. Brazier, Editor-in-Chief

Nathaniel A. Buchwald Carmine D. Clemente Louise M. Darling John L. Fahey Victor E. Hall James V. Maloney

Carl M. Pearson Charles H. Sawyer Emil L. Smith Reidar F. Sognnaes W. Eugene Stern Bradley R. Straatsma

Emery Zimmerman

U C L A F O R U M IN M E D I C A L S C I E N C E S

N U M B E R 18

Brain Mechanisms in Mental Retardation

N A T H A N I E L A . B U C H W A L D Mental Retardation Research Center, Brain Research Institute, and

Departments of Anatomy and Psychiatry

University of California Los Angeles

Los Angeles, California

M A R Y A . B . B R A Z I E R

Brain Research Institute

University of California Los Angeles


Proceedings of a Conference in the Series on Mental Retardation Sponsored by The National Institute of Child Health and Human Development

Mental Retardation Research Centers Series

A C A D E M I C P R E S S N e w Y o r k S a n F ranc i sco L o n d o n 1975

A Subsidiary of Harcourt Brace Jovanovich, Publishers

EDITED BY

COPYRIGHT © 1 9 7 5 , B Y ACADEMIC P R E S S , INC. A L L RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY B E REPRODUCED OR TRANSMITTED IN ANY F O R M OR B Y ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING F R O M THE PUBLISHER.

A C A D E M I C P R E S S , I N C . I l l Fifth Avenue, New York, New York 10003

United Kingdom Edition published by A C A D E M I C P R E S S , I N C . ( L O N D O N ) L T D . 24/28 Oval Road, London NW1

Library of Congress Cataloging in Publication Data

Main entry under title:

Brain mechanisms in mental retardation.

(UCLA forum in medical sciences ; v. 18) Includes bibliographies and indexes. 1. Mental deficiency-Congresses. 2. Bra in -

Congresses. I. Buchwald, Nathaniel A. II. Brazier, Mary Agnes Burniston, (date) III. United States. National Institute o f Child Health and Human Develop-ment. IV. Series. [DNLM: 1. Mental retardation-Physiopathology-Congresses. 2. Neurophysiology-Congresses. W3 U17 no. 18 1974 / WM300 B8145 1974] RC570.B72 616 .8 '588 75*3581 ISBN 0 - 1 2 - 1 3 9 0 5 0 - X

PRINTED IN THE UNITED STATES OF AMERICA

List of Contributors and Participants

Names of contributors to this volume are marked with an asterisk.

*Anthony M. Adinolfi, Mental Retardation Research Center and Neuropsychiatric Insti-tute, University of California Los Angeles, Los Angeles, California 90024

*Joseph Altaian, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47906

*June E. Ayling, Mental Retardation Research Center and Department of Biological Chem-istry, University of California Los Angeles, Los Angeles, California 90024

*Robert E. Barrett, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

*John Blass, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

Mary A. B. Brazier, Editor for the UCLA Forum in Medical Sciences, Brain Research Institute, University of California Los Angeles, Los Angeles, California 90024

*G. A. M. Breen, 1 Mental Retardation Research Center and Department of Anatomy, University of California Los Angeles, Los Angeles, California 90024

^Jennifer S. Buchwald, Mental Retardation Research Center and Department of Phys-iology, School of Medicine, University of California Los Angeles, Los Angeles, California 90024

*N. A. Buchwald, Mental Retardation Research Center, Brain Research Institute, and Departments of Anatomy and Psychiatry, University of California Los Angeles, Los Angeles, California 90024

*Gerald Cohen, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

*Lucien Cote, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

1 Present address; Molecular Biology Department, Roswell Park Memorial Institute, Buffalo, New York 14203.

xi

xii List of Contributors and Participants

*J. de Vellis, Mental Retardation Research Center and Department of Anatomy, University of California Los Angeles, Los Angeles, California 90024

*Marc A. Dichter, 2 Department of Medicine, Beth Israel Hospital, Boston, Massachu-setts 02215

*John Dobbing, Department of Child Health, University of Manchester, Manchester, England

*Albert Dorfman, Joseph P. Kennedy, Jr . , Mental Retardation Research Center,

Department of Pediatrics, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637

*Philip E. Duffy, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

*D. R. G. Fuller, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

*John Garcia, Department of Psychology, University of California Los Angeles, Los Angeles, California 90024

*Patricia S. Goldman, Laboratory of Psychology and Psychopathology, National Institute of Mental Health, Bethesda, Maryland 20014

*Richard Heikkila, Department of Neurology, College of Physicians and Surgeons,

Columbia University, New York, New York 10032 *Alfred Heller, Department of Pharmacological and Physiological Sciences, The

University of Chicago, Chicago, Illinois 60637 *Philip C. Hoffmann, Department of Pharmacological and Physiological Sciences, The

University of Chicago, Chicago, Illinois 60637 ^Chester D. Hull, Mental Retardation Research Center, Brain Research Institute, and

Departments of Anatomy and Psychiatry, University of California Los Angeles, Los Angeles, California 90024

*Peter R. Huttenlocher, Department of Pediatrics, The University of Chicago, Chicago, Illinois 60637

David L. Joftes, National Institute of Child Health and Human Development, Bethesda, Maryland 20014

*Seymour Kaufman, Laboratory of Neurochemistry, National Institute of Mental Health,

Bethesda, Maryland 20014

*Hayato Kihara, The Neuropsychiatric Institute, Pacific State Hospital, Research Group, Pomona, California

*Anne Kitsikis, Department of Physiology, School of Medicine, Laval University,

University City, Quebec, Canada

James L. Lessard, Children's Hospital Medical Center, Cincinnati, Ohio

*M. S. Levine, Department of Psychiatry, University of California Los Angeles, Los

Angeles, California 90024

*Gary Lynch, Department of Psychobiology, University of California Irvine, Irvine, California 92664

*J. F. McGinnis, Mental Retardation Research Center and Department of Anatomy, University of California Los Angeles, Los Angeles, California 90024

*Luis Marco, Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

*Robert J . Marcus, Mental Retardation Research Center and Department of Psychiatry, University of California Los Angeles, Los Angeles, California 90024

2 Present address: Department of Neurology, Beth Israel Hospital, Boston, Massachusetts

02215.

List of Contributors and Participants xiii

^Catherine Mytilineou, Department of Neurology, College of Physicians and Surgeons,

Columbia University, New York, New York 10032

"James Olds, Division of Biology, California Institute of Technology, Pasadena, California 91109

*George Popjak, Mental Retardation Research Center and Department of Biochemistry, University of California Los Angeles, Los Angeles, California 90024

*Dominiek P. Purpura, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, New York 10461

*Pasko Rakic, Department of Neuropathology, Harvard Medical School, and Department of Neuroscience, Children's Hospital Medical Center, Boston, Massachusetts 02115

*Guenter Rose, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

Fred E. Samson, Ralph L. Smith Mental Retardation Research Center, University of Kansas, Kansas City, Kansas

Richard J . Schain, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

*S. Stefan Soltysik, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

*John F. Tallman, Developmental and Metabolic Neurology Branch, National Institutes of Health, Bethesda, Maryland 20014

George Tarjan, Director of Mental Retardation and Child Psychiatry Program, Neuro-psychiatry Institute, University of California Los Angeles, Los Angeles, California 90024

T h o m a s A. Tedesco, 3 Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

""Virginia M. Tennyson, Department of Pathology, Division of Neuropathology, and Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

*Jaime R. Villablanca, Mental Retardation Research Center and Department of Psychi-atry, University of California Los Angeles, Los Angeles, California 90024

Lesnick E. Westrum, Child Development and Mental Retardation Center, University of Washington, Seattle, Washington

*Charles D. Woody, Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024

Stephen Zamenhof, Department of Microbiology and Immunology, University of Cali-fornia Los Angeles, Los Angeles, California 90024

3 Present address: Department of Pediatrics, University of South Florida, College of Medicine, Tampa, Florida 33620.

Foreword

T h i s v o l u m e has b e e n sponsored jo in t ly b y the Men ta l Re ta rda t ion

Program of the Nat iona l Ins t i tu te of Ch i ld Heal th and H u m a n Deve l -

o p m e n t and b y the Un ive r s i t y of Cal i fornia Los Ange l e s F o r u m in M e d -

ical Sc i ences . For the N . I . C . H . D . Men ta l Re ta rda t ion P rog ram, it is the

fourth b o o k b a s e d on conferences o rgan ized b y one of the twelve

Menta l Re ta rda t ion Resea rch Cen te r s . T h e staff of the Men ta l Reta rda-

t ion P rog ram has en thus ias t ica l ly suppor ted the Cen te r s ' Resea rch

Ser ies vo lumes . In part icular , Dr. Theodo re T j o s s e m , Di rec tor of the

Menta l Re ta rda t ion P rog ram, Dr . M i c h a e l J . B e g a b , H e a d , Men ta l R e -

tardat ion Resea rch Cen te r s , and Dr . D a v i d Jof tes , B i o m e d i c a l Sc i ences

Admin is t ra to r , p rov ided e n c o u r a g e m e n t and suppor t for th is vo lume .

Dr . Jof tes , in addi t ion , devo ted m u c h hard work pr ior to and dur ing

the conference w h i c h p rov ided the mater ia l for our b o o k .

T h e Menta l Re ta rda t ion Resea rch Cente rs have b e e n set up at major

un ivers i t i es th roughou t the U n i t e d Sta tes . T h e y w e r e des igned to con-

duct research di rec ted toward an unde r s t and ing of menta l re tardat ion

and related aspects of h u m a n deve lopment . T h e Menta l Re ta rda t ion

Research Cen te r at U C L A is a mul t id i sc ip l inary organiza t ion w h o s e

interes ts ex tend from an thropolog ica l and socio logica l inves t iga t ions

of cross-cul tural aspects of men ta l re tardat ion to molecular chemica l

s tudies of m e m b r a n e p h e n o m e n a related to me tabo l i c def ic iencies

w h i c h under l ie the m o s t severe cogn i t ive abnormal i t i e s . T h e U C L A

Cente r is h o u s e d in a four-s tory add i t ion to the preex is t ing U C L A

Neuropsych ia t r i c Ins t i tu te . It has an impor tan t adjunct research group

at Pacific State Hospi ta l in P o m o n a , Cal i fornia , w h e r e ep idemio log ica l

xiv

Foreword xv

s tudies of men ta l re tardat ion and b i o c h e m i c a l and neurophys io log ica l

research , r equ i r ing the p r e sence of a popu la t ion of re tarded pa t ien t s ,

are carr ied out. T h e Resea rch Cen te r is c losely a l igned wi th a s is ter

un i t devo ted to c l inical aspec ts of men ta l re tardat ion and to in te rd is -

c ip l inary t ra in ing of profess ionals in the field. T h e Cl in ica l U n i t and

the Resea rch Cen te r compr i s e a b road ly b a s e d Men ta l Re ta rda t ion Pro-

gram at U C L A . T h i s p rog ram, founded and o rgan ized unde r the lead-

ersh ip of Dr . G e o r g e Tar jan , r epresen t s U C L A ' s approach to the un-

ders tand ing , p reven t ion , and t rea tment of the cogn i t ive def ic iencies

w h i c h afflict several mi l l ions of A m e r i c a n c i t i zens .

T h i s v o l u m e is the ou tg rowth of a confe rence he ld at O x n a r d , Cal i -

fornia , in January , 1974 . T h e pu rpose of th is confe rence was to p resen t

and to d iscuss neurob io log ica l f indings w h i c h m i g h t lead to an under -

s tand ing of b a s i c p rocesses under ly ing the p h e n o m e n a of menta l

def ic iency. M o s t of the data repor ted at O x n a r d we re der ived from

labora tory researches . H o w e v e r , several of the speakers , all w i th long

h i s to r ies of a c c o m p l i s h m e n t in the labora tory , concen t ra ted the i r pre-

sen ta t ions on research on h u m a n b e i n g s or examina t ion of h u m a n

pa thologica l mater ia l .

T h e cosponso r of th is v o l u m e , the U C L A F o r u m in Med ica l Sc i -

e n c e s , w a s set up b y Dr . S h e r m a n Mel l inkoff , D e a n of the S c h o o l of

M e d i c i n e , to enab le pub l i ca t ion of research mater ia l reflective of the

var ie ty of inves t iga t ive efforts at the U C L A Cen te r for the Hea l th S c i -

ences . T h i s is the e igh teen th v o l u m e in the F o r u m Ser i e s . W e are

grateful to D e a n Mel l inkof f and to the Edi tor ia l Boa rd of the F o r u m for

the i r part in the p roduc t ion of th is b o o k . M o s t especia l ly , thanks go to

Doc to r M a r y A . B . Braz ie r , the efficient and unde r s t and ing Edi tor - in-

C h i e f of the U C L A forum, for he r e n c o u r a g e m e n t and exper t i se dur ing

the p l ann ing and carrying out of the ent i re project .

NATHANIEL A. BUCHWALD

G E O R G E T A R J A N

Professor of Psychiatry and Director of Mental Retardation and Child Psychiatry Program, Neuropsychiatric Institute

University of California Los Angeles Los Angeles, California

Welcoming Remarks

Thi s confe rence seeks to e x a m i n e wha t w e k n o w , at p resen t , abou t

the b ra in m e c h a n i s m s under ly ing menta l re tardat ion. Progress in the

s tudy of men ta l re tardat ion and h u m a n deve lopmen t has , of course ,

accelera ted marked ly s ince Congres s legis la ted the e s t ab l i shmen t of

the Menta l Re ta rda t ion Resea rch Cen te r s . T h e s e Cen te r s and the avail-

abi l i ty of research suppor t in deve lopmenta l b i o l o g y and chemis t ry for

sc ient i s t s not affiliated wi th the Cente rs have b rough t a w h o l e n e w

popula t ion of researchers and a w h o l e n e w set of approaches to

s tudies i m p i n g i n g on p r o b l e m s of men ta l re tardat ion. T h e excel lent

qual i ty of the workers a t t racted to th is field is a t tes ted to b y the peop le

represen ted at th i s conference and b y the difficulty i n c h o o s i n g t h e m

from the large pool of ded ica ted researchers in the field.

T h e r e is an old idea abou t sc ien t i s t s b e i n g un in te re s t ed in the real

p r o b l e m s of the wor ld , the idea that s c i ence is good for its o w n sake ,

and that i f it i s useful , so m u c h the be t te r , bu t that ut i l i ty is not a

r equ i remen t . I don ' t t h ink m o s t good sc ien t i s t s accept th is v i e w any-

more . T h e fact that so m a n y of you are devot ing your efforts to

p rob l ems of s igni f icance to h u m a n b e i n g s nega tes th is idea . E v e n

m o r e impor tan t , I t h ink , is the fact that you h a v e b e e n ab le to do so

wi thou t sacrif icing the level of your scientif ic efforts. L o w grade sc ien-

tific research , even i f specif ical ly a i m e d at a target such as men ta l re-

tardat ion, has little va lue . It is hea r t en ing , i n d e e d , that fine sc ient i s t s

have b e c o m e in te res ted in p r o b l e m s of men ta l re ta rda t ion and re la ted

aspects of deve lopmen t . I look forward to your confe rence and to the

t ime w h e n the resul ts of you r efforts wil l impac t u p o n the p r o b l e m s of

the menta l ly re tarded.

GEORGE TARJAN

1 Timing of Major Ontogenetic Events

in the Visual Cortex of the Rhesus Monkey

P A S K O R A K I C

Department of Neuropathology, Harvard Medical School,

and Department of Neuroscience, Children's Hospital Medical Center,

Boston, Massachusetts

A. INTRODUCTION

T h e neocor tex i n m a n has r eached an e n o r m o u s s ize and complex i ty w h i c h

are approached only in s o m e of the s u b h u m a n p r ima tes . T h e de t e rmina t ion of

the s e q u e n c e and t i m i n g of the cellular even t s that occur dur ing the deve lop-

m e n t of th i s h u g e s tructure is essen t ia l for the unde r s t and ing of the cort ical

abnormal i t i e s that m i g h t lead to men ta l re tardat ion. A l though m a n y facts and

bas i c concep t s abou t neocor t ica l deve lopmen t we re in i t ia l ly o b t a i n e d from the

s tudies of the h u m a n e m b r y o s ( 2 3 , 3 1 , 7 6 ) , the p r imary source of our k n o w l e d g e

in recent years has b e e n expe r imen ta l w o r k in roden ts . Labora to ry an ima l s

were used in these s tudies b e c a u s e au torad iography and e lect ron mic roscopy ,

two informat ive expe r imen ta l p rocedures , canno t b e appl ied to h u m a n e m -

bryos . T h e s e m e t h o d s have p rov ided impor tan t n e w in format ion and con-

firmed s o m e old h y p o t h e s e s w i th m o r e re l iable data. T h u s , a few bas i c con-

cepts and deve lopmen ta l even t s i l lustrated schemat ica l ly in F igure 1 are n o w

rather wel l e s t ab l i shed : (a) cort ical neu rons are genera ted in prol i ferat ive

zones close to the vent r icu lar surface ra ther than in the cortex itself, (b) after

the i r last d iv i s ion y o u n g n e u r o n s a s s u m e a b ipo la r shape and migra te radial ly

to the cort ical pla te , (c) n e u r o n s genera ted first are u l t imate ly s i tua ted in the

deepes t cort ical layers as neu rons genera ted later b y p a s s earl ier genera ted

ones and a s s u m e m o r e superficial pos i t i ons , (d) the deepe r n e u r o n s differen-

tiate earl ier than those s i tuated m o r e superficial ly, and (e) in roden t s all cor-

3

4 Pasko Rakic

A B C D E FIGURE 1. Schematic drawing of the major cellular events during the development of the mam-malian cortical plate (CP). The basic principles of cell behavior during proliferation migration and differentiation stages apply, with some modifications, also to the development of other regions of the vertebrate central nervous system; this figure was used by the Boulder Committee (8) to illus-trate recommended neuroembryonic terminology for ventricular (V), sub ventricular (S), interme-diate (I), and marginal (M) zones. Further explanation in text.

t ical neu rons are genera ted dur ing the last several days of ges ta t ion and in

s o m e spec ies a shor t pe r iod after b i r th .

M o s t of these pr inc ip les have b e e n der ived from the s tudy of small l i s sen-

cephal ic b ra ins . It i s impor t an t to es tab l i sh w h e t h e r the neocor tex in a large

gyrencepha l ic b ra in l ike that o f the m a n develops accord ing to the s ame pr in-

ciples . At wha t t ime do cort ical neu rons or ig ina te dur ing the protracted devel-

o p m e n t of p r imate b r a in? W h e r e is such an e n o r m o u s n u m b e r of neu rons

genera ted and wha t is the rate of the i r mig ra t ion in such a large b ra in? W h a t is

the re la t ionsh ip b e t w e e n the t ime of or ig in and the pos i t ion of neu rons in the

cortical l aminae in the sharply layered p r imate neocor tex? H o w is genes i s of

neu rons related to the format ion of fissures and gyr i? T h e present s tudy,

w h i c h deals w i t h these bas i c i s sues in the rhesus m o n k e y , represen ts the first

s tep in an o n g o i n g , more deta i led analys is o f neocor t ica l genes i s in pri-

ma tes .

Severa l p roper t ies m a k e the p r imary v isua l cor tex of rhesus m o n k e y a very

sui table mode l for the s tudy of co r t i cogenes i s . T h e hor izonta l stratif ication of

neu rons in to separa te layers in th is spec ies is very sharp (Figure 2B) and area

17 of B r o d m a n n (9) can b e d i s t i ngu i shed from adjacent cort ical areas at rela- '

t ively early e m b r y o n i c s tages . R e c e n t morpho log ica l (15 ,26 ,27 ,33 ,75 ) and

phys io log ica l s tud ies (e .g . , 2 5 , 8 1 ) of the v isual cor tex in m o n k e y have focused

a t tent ion to th is reg ion . T h e prot rac ted span of deve lopmen t increases the res-

olut ion of tempora l s e q u e n c e s in n e u r o g e n e s i s (51 ,52) and the large size of the

m o n k e y fetus al lows adequa te fixation for e lect ron mic ro scopy (49 ,50) .

1. Timing of Major Ontogenetic Events 5

FIGURE 2. A. Coronal section through the occipital lobe and cerebellum of a 3-month-old monkey. The two arrows indicate a strip of visual cortex about 10 mm long in the depth of the cal-carine fissure, where the time of neuron origin was analyzed in the present study. The area in the rectangle is enlarged in Figure 2B and on the left side of Figure 8. Thirty-micrometer section stained with cresyl violet. B. Cytoarchitectonics of the monkey visual cortex (area 17) in the depth of calcarine fissure indicated by rectangle in Figure 2A. Roman numerals indicate cortical layers according to the Brodmann's (9) classification adopted in this study. The photograph demon-strates the sharp delineation of the cortical layers in monkey with three clearly indicated horizon-tal fiber-rich strata (layers I, IVB, and V) dividing the cortex into three cell-dense zones. In this region white matter (WM) situated between cortex and an almost obliterated lateral ventricle (LV) is very thin.

A

6 Pasko Rakic

B. TIME OF NEURON ORIGIN

T h e t ime of o r ig in of a neu ron canno t b e de t e rmined b y direct examina t i on

of h is to logica l p repara t ions of the deve lop ing b ra in . T h e on ly avai lable

m e t h o d w h i c h can p rov ide re l iable data on the t ime of neu ron or ig in is 3 H -

t h y m i d i n e au torad iography (66) . T h e procedure involves expos ing b ra in cells

to 3 H - t h y m i d i n e at different deve lopmenta l s tages . Fo l lowing an in t r avenous

in jec t ion th is nuc leo t ide ci rculates in the b loods t r eam of the m o n k e y for on ly a

short t ime , that i s , as a pulse , and b e c o m e s incorpora ted in to the D N A of all

d iv id ing cells (41) . A n i m a l s are ki l led at matur i ty and all " h e a v i l y l a b e l e d "

neu rons in the au torad iograms are those w h i c h were in the last cell d iv i s ion at

the t ime of the in jec t ion . T h e m i n i m u m n u m b e r of gra ins for c lassif icat ion of

neu rons as " h e a v i l y l a b e l e d " for p r imate s p e c i m e n s w a s arbi t rar i ly deter-

m i n e d for each s p e c i m e n as half o f the m a x i m u m grain count found in

neu rons of that s p e c i m e n (51 ,52) . Cel ls w h i c h have d iv ided only once or twice

after the in jec t ion are l ight ly l abe led , w h e r e a s cells w h i c h had thei r last

d iv i s ion before in jec t ion as wel l as all cells w h i c h have di lu ted their radioac-

t ivi ty th rough m a n y s u b s e q u e n t d iv i s ions are un labe led . B y in jec t ing a ser ies

of p regnan t an ima l s , the t ime o f o r ig in of cort ical n e u r o n s has b e e n deter-

m i n e d in several rodent spec ies ( 2 , 6 , 1 8 , 6 5 ) . T h e p resen t au toradiographic

s tudy in the m o n k e y b ra in represen ts the first ana lys is o f the t ime of neu ron

or ig in of the neocor t ex in any non- roden t spec ies .

P regnan t m o n k e y s w e r e in jec ted once each wi th 3 H - t h y m i d i n e at the forti-

eth e m b r y o n i c day (E40) and at E 4 5 , E 5 0 , E 5 4 , E 6 2 , E 7 0 , E 8 0 , E 9 0 , E 1 0 2 , E 1 2 0 ,

and E 1 4 0 . Ges ta t ion age w a s b a s e d on the a s sumpt ion that ovula t ion and con-

cept ion occurred on the twelfth day of the mens t rua l cycle . P r e g n a n c y in the

rhesus m o n k e y lasts 165 days . All fetuses we re del ivered normal ly and ki l led

at 2 to 5 m o n t h s of age . M o s t cort ical cells have already a t ta ined thei r final

pos i t ion and can b e classif ied as n e u r o n s or gl ia in these " j u v e n i l e " m o n k e y s

(52) . T w o addi t ional m o n k e y s were in jec ted at the second and e igh teen th

postnata l days , respec t ive ly , and sacrif iced in the th i rd mon th . [For m o r e de-

tails abou t p r imate au torad iography , see Rak ic (51) and N o w a k o w s k i and

Rakic (41) . ]

N o heav i ly labeled n e u r o n s are p resen t in the au torad iograms of the v isua l

cor tex in an imals in jec ted at £ 4 0 * or after E 1 0 2 . H o w e v e r , all an imals in jec ted

at i n t e rven ing ages con ta ined heav i ly l abe led neu rons in s o m e layer of the

* No heavily labeled neurons were seen in the visual cortex of the "juvenile" monkey that had

been exposed to 3 H-thymidine on E40 but a few lightly labeled neurons, located exclusively in the

deepest part of the cortex, were found in this specimen (52). Since heavily labeled cells were

found elsewhere in the same brain, these lightly labeled cortical neurons are probably the prod-

ucts of several cell divisions subsequent to the time of injection. Thus it is unlikely that any visual

cortical neurons were born at E40. The first neuron of the visual cortex, therefore, may actually be

generated somewhere between E40 and E45.


visual cortex. O n l y a few neu rons are l abe led b y in jec t ion at E45 or E 1 0 2 ;

the rate of p roduc t ion of neu rons for the v i sua l cor tex is at m a x i m u m in te rms

of n u m b e r of l abe led n e u r o n s per un i t area of the cor tex at E 7 0 . O n the

bas i s of th is ana lys i s it w a s conc luded that the en t i re popu la t ion of cort ical

n e u r o n s in the m o n k e y ' s v isua l cor tex is genera ted dur ing the 2 - m o n t h per iod

from a round E45 to E102 (52) . It is of no te that p re l imina ry e x a m i n a t i o n has

s h o w n that the neu rons of o ther neocor t ica l areas in the neocor t ex of rhesus

m o n k e y are genera ted dur ing approx imate ly the s ame per iod w i t h on ly a

sl ight shif t ing in the t ime from area to area (P. R a k i c , u n p u b l i s h e d ) . Al-

t hough no neu rons are l abe led in an imals in jec ted dur ing the last 2 m o n t h s

of ges ta t ion or after b i r th , n u m e r o u s as t rocytes w e r e l abe led th roughou t the

cortex (see Sec t ion F ) .

In the course of the s tudy it b e c a m e apparent that h i s t ogenes i s o f p r imate

neocor tex differs in s o m e quant i t a t ive and qual i ta t ive aspec ts from that of the

previous ly s tud ied rodent spec ies . S o m e differences are pure ly numer i ca l and

relate to the larger s ize of the m o n k e y cor tex and to the longer t ime necessa ry

for i ts deve lopment . T h i s po in t is v iv id ly i l lustrated in F igure 3 w h i c h d e m o n -

strates the dramat ic g rowth of the m o n k e y bra in . W h e n 3 H - t h y m i d i n e is in-

j ec ted in to the p regnan t m o n k e y at E 4 8 , the fetal b r a in is less than 1 % of the

v o l u m e to b e a t t a ined b y P 9 0 . Fur the rmore the fetal v isua l cor tex is repre-

sen ted b y a relat ively smal ler fraction of the total h e m i s p h e r i c surface than at

P90 : it actually enlarges several h u n d r e d t imes dur ing the in t e rven ing m o n t h s

(P. R a k i c , u n p u b l i s h e d ) . S i n c e n e u r o n s in the last cell d iv i s ion at E48 are later

to b e sought as heav i ly l abe led cells at P90 in e n o r m o u s l y e x p a n d e d cor tex,

l ight mic roscop ic ana lys is is l i terally " a search for a need le in the h a y s t a c k . " In

m i c e , w h e r e the b ra in g rows only 3-4 t imes in v o l u m e dur ing the compa-

rable deve lopmenta l pe r iod (e .g . , b e t w e e n E14 and P 3 0 ) , m a n y m o r e l abe led

neu rons are encoun te red per sec t ion . T h e total t ime pe r iod of cort ical neu ron

produc t ion in the m o n k e y is 10 t imes longer than in rodents , 60 days in

m o n k e y (52) ve r sus on ly 6 days in m i c e (2) . S i n c e S phase and the cell genera-

t ion cycle last approx imate ly the s a m e t ime in all m a m m a l s e x a m i n e d so far

( 5 , 2 9 , 6 6 ) , the pe rcen tage of cort ical neu rons expec ted to b e labe led in adults

after a s ingle in jec t ion of 3 H - t h y m i d i n e at e m b r y o n i c ages shou ld b e 10 t imes

smaller in m o n k e y s than in m i c e . In prac t ice , h o w e v e r , the pe rcen tage of

labe led n e u r o n s is e v e n smal ler b e c a u s e of the br iefer pe r iod dur ing w h i c h 3 H - t h y m i d i n e is ava i lab le in the c i rcula t ing b lood of the p regnan t m o n k e y s

compared to that in roden ts (41) and , in addi t ion , the effect of D N A turnover

m a y b e e n h a n c e d b y the long survival t ime . T h e pract ical o u t c o m e is that a

larger n u m b e r of au to rad iograms m u s t b e p repared and ana lyzed in the case

of the m o n k e y .

O the r differences in the h i s t ogenes i s o f p r imate and rodent neocor tex ap-

pear to b e b io logica l ly m o r e s ignif icant . For example , the fact that n o n e w

neurons are genera ted in the last two fetal m o n t h s or after b i r th is surpr i s ing

8 Pasko Rakic

E48 P90 FIGURE 3. External features of the lateral (upper row) and medial (lower row) surfaces of the monkey brain at forty-eighth (E48) embryonic day and at third postnatal month (P90). Aldehyde-fixed brains were bisected in the midsagittal plane and photographed and reproduced at the same magnification.


s ince , in roden t s , neocor t ica l neu rons are p roduced dur ing the late fetal ages

or even for shor t pe r iods after the b i r th of the an imal . T h u s , in the m o u s e ,

neu rons des t ined for the neocor tex are genera ted b e t w e e n E l l and E17 (2) , in

the rat b e t w e e n E14 and E21 (18) , and in the go lden h a m s t e r b e t w e e n approxi -

mate ly E l l and P2 (65) . In the m o n k e y vir tual ly all neocor t ica l n e u r o n s are

genera ted in the midd le third of the ges ta t ional pe r iod . It is impor tan t to

e m p h a s i z e that b i r th is an arbi t rary po in t in the life of an an ima l and it is not

direct ly re la ted to the per iod of neu ron produc t ion (e .g . , 1 ,20 ,49 ,50) . Therefore

the deve lopmen t o f synapses and o ther cr i ter ia of neurona l matura t ion shou ld

b e used in eva lua t ing inf luences of sensory e n v i r o n m e n t or o ther factors on

the deve lop ing neocor t ex in neona te s of different spec ies .

S i n c e our u l t imate goal is to learn m o r e abou t deve lopmen t of neocor t ex in

m a n , an impor tan t q u e s t i o n is w h e t h e r the genes i s of cort ical n e u r o n s in m a n

is comple ted as early, re lat ive to b i r th , as in the rhesus m o n k e y . A c o m p a r i s o n

of some of the morpho log ica l features of the v i sua l cor tex in h u m a n fe tuses of

different ages w i t h that of the m o n k e y at E 1 0 0 , w h e n neu ron p roduc t ion is al-

mos t comple te , m a y he lp to answer th is ques t ion . O u r p re l imina ry results

compar ing Niss l - and Go lg i - s t a ined mater ia l in b o t h spec ies ind ica te that the

h u m a n v isua l cor tex also different iates early in ges ta t ion and that p r o b a b l y all

neu rons are genera ted wel l before b i r th , pe rhaps even b y the midges ta t iona l

per iod . T h e s e f indings are in ag reemen t w i t h p rev ious Golg i s tudies of cor-

tical deve lopmen t in m a n (35 ,44 ,45 ,46 ) . [For r ev iew on th is sub jec t , see

S i d m a n and Rak ic (69) . ] S tudy of total D N A syn thes i s in the deve lop ing

h u m a n b ra in suppor ts the no t ion that the major i ty of n e u r o n s in m a n migh t

b e already bo rn b y the midd le of the ges ta t ion per iod ( D o b b i n g , Chap te r 14) .

O t h e r cr i ter ia of the b ra in matura t ion also s h o w the s ame t e n d e n c y toward

early appearance in m a n . For example , synapses in the h u m a n fetal neocor tex

are wel l r ep resen ted al ready dur ing the s econd fetal m o n t h (37) , wh i l e rat

neocor tex still s h o w s very few synapses at t e rm (A. Pe te rs , pe rsona l c o m -

mun ica t i on ) . S imi la r ly , the format ion of mye l i n in the b ra in b e g i n s before

b i r th in m a n (24 ,30 ,80 ) and rhesus m o n k e y (3) , w h e r e a s it occurs pos tnata l ly

in the rodent b ra in (28 ,74) .

C. CORRELATION BETWEEN TIME OF ORIGIN AND EVENTUAL POSITION OF NEURONS

IN THE CORTICAL LAMINAE

As ini t ia l ly sugges ted from Golg i s tud ies (31 ,56 ,76 ) and more recent ly con-

firmed b y 3 H - t h y m i d i n e au to rad iography in rodents (2) n e u r o n s des t ined to

be s i tuated in the deepe r cort ical layers are genera ted earl ier than those of the

more superficial o n e s . T h e re la t ionsh ip b e t w e e n the t ime of cell o r ig in and its

final pos i t ion in the cort ical l aminae of the m o n k e y v i sua l cor tex w a s s tud ied

in the same ser ies of s p e c i m e n s desc r ibed in the p rev ious sec t ion . T h e pos i -

10 Pasko Rakic

t ions of heav i ly labe led neu rons (Figures 4 - 7 ) we re recorded wi th the aid of a

Z e i s s mic roscope e q u i p p e d wi th a ca l ibra ted d rawing tube . To ob ta in s e m i -

quant i ta t ive data all heav i ly l abe led neu rons encoun te red wi th a str ip of

v isua l cor tex 2 .5 m m long se lec ted r andomly in the area of the calcar ine fissure

(Figure 2A) we re recorded (52) . T h e locat ion of the cort ical layers and the d i s -

FIGURE 4. Photomicrograph of an autoradiogram of the visual cortex in a 58-day-old "juvenile" monkey that had been injected with 3 H-thymidine at forty-fifth embryonic day (E45-P58). Field shows deep portion of future cortical layer (VI) and underlying white matter (WM). Arrows point to the three heavily labeled neurons.


FIGURE 5. Autoradiograms of the monkey visual cortex in a 50-day-old "juvenile" animal that

had been injected with 3 H-thymidine at sixty-second embryonic day (E62-P50). Roman numerals V

and VI indicate cortical layers according to Brodmann's (9) classification as illustrated in Figure 1.

Toluidine blue-stained autoradiograms are not suitable in general for further classification of

neurons but so-called giant solitary pyramids of Meynert (36) are easily distinguishable (M).

Some of these cells were labeled by injection at E62, as illustrated in B .

tance from the pial surface were precise ly d e t e r m i n e d for each l abe led cell and

its pos i t ion plot ted a long a radial vec tor perpendicular ly t ravers ing the cort ical

plate (Figure 8 ) . T h e heav i ly l abe led neu rons in the b ra in of a " j u v e n i l e "

m o n k e y that had b e e n in jec ted at E45 (the earl iest in jec t ion w h i c h labels heav -

ily some n e u r o n s in the v isua l cortex) w e r e local ized in a na r row z o n e in the

deeper por t ion of layer V I (Figure 4 ) . Sca t te red neu rons s i tuated in the whi t e

mat ter , b e l o w layer V I , are also labe led in th is s p e c i m e n . In mos t fields a

12 Pasko Rakic

n u m b e r of l ight ly l abe led cells we re de tec ted superficially to the heav i ly l abe led

o n e s , an ind ica t ion that the later genera ted cells take up m o r e external pos i -

t ions . T h i s w a s conf i rmed b y the f inding that heav i ly labe led neu rons in the

an imal in jec ted at E54 are loca ted s o m e w h a t m o r e superficially, a l though still

w i t h i n layer V I (Figure 8 ) . T h e major i ty o f neu rons genera ted at E62 c o m e to

b e s i tuated in the uppe r two- th i rds of layer V I , wh i l e s o m e are local ized in

layer V (Figures 5 and 8 ) . Cel ls w i th long efferent axons pass ing from area 17

to the m i d b r a i n (11) , the so-cal led g ian t sol i tary pyramida l neu rons of M e y n e r t

(36) , s i tuated in the rhesus m o n k e y in layers V and VI were also labe led b y an

in jec t ion at E62 (Figure 5 B ) .

A n in jec t ion at E70 p redominan t ly labels neu rons that later take up res i -

E70-P98 - J E8CPP48 jgjp » *~

4 .

FIGURE 6. Photographs of autoradiograms of the visual cortex in two "juvenile" monkeys ex-posed to 3 H-thymidine at slightly different gestational ages. Animal illustrated in A had been in-jected at E70 and killed at P98; animal in B received 3 H-thymidine at E80 and was killed at P48. The overall distribution of labeled neurons within layers V , IVB, and IVC is different as graphi-cally represented in Figure 8. Arrows point to heavily labeled neurons.

V A


i «

FIGURE 7. Autoradiograms of visual cortex in a 65-day-old "juvenile" monkey whose mother had

been injected with 3 H-thymidine at E102. Photographs A to C depict the border zone between

layers I and II. Only three heavily labeled cells (arrows) in more than 50 slides examined in this

case were classified as neurons.*

dence in layer V bu t also m a n y cells in layer I V C (Figures 5 A and 8 D ) .

N e u r o n s genera ted at E80 b e c o m e d i s t r ibu ted over the ent i re w i d t h of layer

IV , w i th the h ighes t concen t ra t ion in layer I V B (Figures 5 B and 8 E ) ; a few

radioact ive cells are s i tua ted in layer III . In jec t ion at E90 labels n e u r o n s in b o t h

layers III and II (Figure 8F ) .

B y E102 a lmost all n e u r o n s in the v isua l cortex have b e e n b o r n , s ince only a

ve ry few neurons* located at the ve ry borde r b e t w e e n layers II and the cell-

sparse layer I are l abe led in the 3 -mon th -o ld m o n k e y that h a d b e e n in jec ted on

th is day (Figures 7 and 8 G ) . H o w e v e r , in th is s p e c i m e n s o m e smal l nuc le i si t-

* Only three labeled neurons were found in 80 2.5-mm long strips of the visual cortex situated

in the depth of calcarine fissure examined in 40 sections of a specimen injected with 3 H-thymidine

at E102 and killed at P65. It was calculated that less than 1 in 10 6 neurons was labeled in this spec-

imen (52).

14 Pasko Rakic

FIGURE 8. Diagrammatic representation of the positions of heavily labeled neurons in the visual cortex of juvenile animals which had been injected with 3H-thymidine at various embryonic days (E) indicated at the top of each vertical line. [From Rakic (52) with permission of Science. Copyright 1974 by the American Association for the Advancement of Science.] On the left, for orientation, is a photomicrograph of a 30-//,m cresyl violet-stained section photographed at the same magnifica-tion used for plotting of the labeled neurons with the drawing tube. Division into cortical layers, indicated by Roman numerals, are according to Brodmann (9). Horizontal markers on each vertical vector except G indicate positions of all heavily labeled neurons encountered in a randomly selected 2.5-mm long strip of the calcarine cortex. The three labeled neurons whose positions are represented in vector G were found only after examination of 80 areas of calcarine cortex each 2.5-mm wide in 40 autoradiograms from a single monkey. LV, Obliterated posterior horn of the lateral ventricle.

I

II

&

III

'iVA

IVB

IVC

V

VI

A B C D E F G

E45 E54 E62 E70 E80 E90 EI02

I

II

&

III

IVA

IVB

IVC

V

VI


uated p redominan t ly in the deeper half of the cor tex are rad ioac t ive ; these

were classif ied as glial (see S e c t i o n F and F igure 18) .

T h e neu rons in the p lexi form layer I we re no t l abe led in any of the spec i -

m e n s in th is se r ies (52) . T h e s e neu rons e i the r are genera ted before E 4 0 , or

they ar ise in s o m e relat ively short t ime in terval b e t w e e n the ages s ampled in

the presen t ser ies of an imals .

As rep resen ted graphica l ly in F igure 8, the pos i t i on of heav i ly labe led

neu rons in the j uven i l e m o n k e y ' s v i sua l cor tex correla tes w i t h the t ime of cell

or ig in in the fe tus ; cells des t ined for deep cort ical pos i t i ons are gene ra t ed first,

and m o r e superf icial o n e s progress ive ly later. T h u s , m o s t o f the n e u r o n s of

layer V I are b o r n b e t w e e n E45 and E 6 0 , layer V b e t w e e n E60 and E 7 0 , layer I V

b e t w e e n E70 and E 8 0 , and layers III and II b e t w e e n E80 and E 1 0 0 . As dis-

cussed in S e c t i o n E , these data per ta in on ly to the t i m e o f cell o r ig in and do

not reveal w h e n the cells actually a t ta ined the i r p e r m a n e n t pos i t i ons .

Auto rad iograph ic resul ts in th is p r imate b ra in cor robora te the " i n s i d e - o u t "

pat tern of cell d i spos i t ion desc r ibed prev ious ly in roden t s ( 2 , 6 , 1 8 , 6 5 ) . C o m -

par ison of the data in F igure 8 w i t h those of a s tudy in m i c e (10) s h o w s that

mos t of the s imul t aneous ly genera ted n e u r o n s in the m o n k e y , par t icular ly

those genera ted early b e c o m e eventua l ly conf ined to relat ively n a r r o w strata of

the cor tex , i . e . , the " i n s i d e - o u t " p r inc ip le is m o r e r ig id ly fo l lowed in the

m o n k e y . T h i s m a y b e the deve lopmen ta l ba s i s for the sharper b o u n d a r i e s of

cort ical layers in the v isua l cor tex of adult p r imates (52) .

D. PLACE OF NEURON ORIGIN

T h e place w h e r e cort ical n e u r o n s or ig ina te w a s s tud ied in ano the r se r ies of

exper imen ta l an imals p repared for au torad iographic s tudy. P regnan t m o n k e y s

we re in jec ted w i t h 3 H - t h y m i d i n e once each at E 4 1 , E 4 5 , E 5 0 , E 5 8 , E 6 9 , E87 ,

E 9 0 , E 1 2 0 , and E140 and 1 hou r later the i r fe tuses we re taken b y hys t e ro tomy

and ki l led b y vascular perfus ion. In add i t ion o n e neona ta l m o n k e y w a s ki l led

at P 3 , 1 h o u r after 3 H - t h y m i d i n e in jec t ion . To lu id ine b lue - s t a ined , 1-jum plas-

tic sec t ions across e i ther the en t i re t h i ckness or on ly the i n n e r hal f of the

cerebral wall w e r e p repared as desc r ibed prev ious ly (49) .

At E41 the cerebra l wal l in the occipi ta l r eg ion is on ly 150 /xm th ick and con-

sists of ventr icular ,* i n t e rmed ia t e , and marg ina l zones (Figure 9 A ) . O n e h o u r

after exposure to 3 H - t h y m i d i n e , l abe led nuc le i are concen t ra ted in the ou ter

por t ion of the vent r icu lar z o n e , w h e r e a s mi to t ic figures are a lmost exc lus ive ly

located at the vent r icu lar surface (Figure 9 A ) . S i n c e an ima l s in jec ted at th i s age

and ki l led at pos tnata l ages con t a ined no l abe led n e u r o n s (see above) p r e sum-

ably all the labe led nuc le i at E41 b e l o n g to a popu la t ion of prol i ferat ing cells

* Nomenclature recommendations of the Boulder Committee (8) have been adopted. See also

Figure 1.

16 Pasko Rakic

FIGURE 9. A. Autoradiograms of the cerebral wall at the posterior pole of the occipital lobe. 3 H -Thymidine was injected at E41, and the embryo was killed 1 hour later. Labeled cells are located in the outer third of the ventricular zone (V), very few are in the intermediate (I), marginal (M) zones or at the ventricular surface where numerous mitotic figures are present. B. Section across cerebral wall in the occipital lobe at E45. The fundamental embryonic layers are clearly delineated: ventricular (V), subventricular (S), intermediate (I), and marginal (M) zones, with incipient cor-


w h i c h are e n g a g e d in in te rk ine t ic nuc lear mig ra t i on , as obse rved in the ven-

tricular zone of o ther spec ies ( 6 1 , 6 2 , 6 7 , 7 7 ) .

At E45 an i nc ip i en t cort ical p la te cons i s t i ng o f 2 - 3 rows o f n e u r o n s has

formed in the pos te r io r part of a cerebra l ves ic le (Figure 9 B ) . T h i s cort ical plate

of on ly a few squa re mi l l imete rs in surface area in the m o s t pos te r io r por t ion

of the occipi ta l lobe a l ready con ta ins some pos tmi to t i c neu rons of the p rospec-

t ive v isua l cor tex. Au to rad iog rams of th is E45 s p e c i m e n , ki l led 1 hou r after 3 H -

t h y m i d i n e in jec t ion , reveal no labe led cells in the cort ical plate i tse l f a l though

n u m e r o u s rad ioac t ive nuc le i are p resen t in the ventr icular zone . Smal l

n u m b e r s of l abe led nuc le i are s i tuated in a n e w cell b a n d w h i c h deve lops

b e t w e e n vent r icu lar and in te rmed ia te zones . T h e p re sence of occas iona l m i -

totic figures in th i s loca t ion ind ica tes that the prol i ferat ing cells in th is b a n d

do not m o v e to the vent r icular surface for the i r d iv i s ion . T h i s relat ively i ncon-

sp icuous layer of cells represent the b e g i n n i n g of the subvent r icu la r zone

w h i c h later wi l l b e c o m e m u c h more mass ive .

T h e on ly c o n s p i c u o u s morpho log ica l c h a n g e in the cerebra l wall du r ing the

next 5 to 10 days is the deve lopmen t of a d i s t inc t ive and m u c h th icker s u b v e n -

tricular zone . At E53 it has approx imate ly the s ame th ickness as the vent r icular

zone and can b e r ecogn ized as a layer of loosely o rgan ized cells located exter-

nally to a zone of m o r e closely packed , vert ical ly a r ranged, cells c o m p o s i n g the

ventr icular z o n e (Figure 9 C ) . Bo th vent r icular and subvent r icu la r zones in the

fetus in jec ted at E53 and ki l led 1 hour later con ta in n u m e r o u s labe led cel ls ,

whe reas the cort ical plate i tself is no t labe led (Figure 9 D ) . O n e - m i c r o m e t e r

plastic sec t ions s ta ined wi th to lu id ine b lue demons t ra te that mi to t ic figures

of the cerebra l wall (apart from those of endothe l ia l cells) are conf ined exclu-

s ively to two wel l -def ined loca t ions , in the vent r icular zone at the ve ry surface

of the lateral vent r ic le and th roughout the subvent r icu la r zone (Figure 9 E ) .

T h e s e f indings are in accord wi th e s t ab l i shed v i e w s of cell b e h a v i o r in pro-

l iferative zones of the m a m m a l i a n central ne rvous sys t em. Ven t r i cu la r cells

dur ing prol i ferat ive phase s r ema in pe rmanen t ly a t tached to the vent r icular

surface to w h i c h the nuc leus m o v e s for d iv i s ion ( 2 1 , 2 2 , 6 1 , 6 7 ) , w h e r e a s sub -

tical plate (CP) already developed. Tissue was fixed by intracardiac perfusion with a glu-teraldehyde (1.25%)-paraformaldehyde (1%) mixture in phosphate buffer (pH 7.3), and by post-fixation in 2 % O s 0 4 , embedded in Epon-Araldite, sectioned at 1 jiim and stained with alkaline toluidine blue. C. Cerebral wall in the posterior region of occipital lobe at E53. Note the 2-fold increase in the thickness of the subventricular and intermediate zones and 4-fold increase in the width of the cortical plate as compared to specimen at E45 illustrated in B. Abbreviations and method of tissue preparation the same as in B . D. Autoradiogram made from a section at a compa-rable location in the opposite hemisphere of the specimen illustrated in C. This animal was in-jected with 3 H-thymidine at E53 1 hour before sacrifice. Labeled cells are concentrated in the outer third of the ventricular zone or scattered throughout subventricular zone—but none is present in the cortical plate. Abbreviations as in B. E. Higher magnification of the ventricular (V) and part of subventricular (S) zones. Mitotic figures in the cerebral wall are present exclusively at the ven-tricular surface (arrow) or throughout subventricular zone (double arrow).

18 Pasko Rakic

ventr icular cells do not contact e i ther cerebra l surface [Figure 16 and Rak ic et al.

(54)] and d iv ide in situ.

Both vent r icular and subven t r i cu la r zones pers i s t in parallel and p roduce

n u m e r o u s cells dur ing the nex t 5 w e e k s as demons t r a t ed in au to rad iograms of

the fe tuses in jec ted at E 5 8 , E 6 9 , E 7 5 , and E87 and ki l led 1 h o u r later. A p -

parent ly these two zones represen t the on ly sources of cort ical neu rons s ince

in these s p e c i m e n s the cort ical plate i tself con ta ins ve ry few labe led cells (F ig-

FIGURE 1 0 . A. Autoradiogram of the cerebral wall in the region of visual cortex of a monkey fetus

injected with 3 H-thymidine at E 5 8 and killed 1 hour later. Labeled cells are located in the ven-

tricular (V) and subventricular (S) zones but not in the intermediate zone or in the cortical plate

(CP). Occasional radioactive nuclei which can be encountered within the cortex itself belong to

endothelial cells as illustrated in B and C at higher magnification. M, Marginal zone.

1. Timing of Major Ontogenetic Events 1 9

ure 10A) . Closer e x a m i n a t i o n s h o w s that the occas iona l ly l abe led nuc le i scat-

tered th roughou t the cor tex in these s p e c i m e n s b e l o n g e i the r to endothe l ia l

capil lary cells (Figure 10B and C) or , par t icular ly toward the e n d of th i s pe r iod ,

to cells of glial l ineage (Figure 18) . D u r i n g th is t ime per iod the subven t r i cu la r

zone gradually b e c o m e s re la t ively w i d e r b e c a u s e the vent r icular zone is nar-

rowing . B o t h the n u m b e r of mi to t ic figures (Figure 11A) and the relat ive

n u m b e r of l abe led cells (F igure 11B) at later s tages ind ica te that the subven -

tricular z o n e p roduces a progress ive ly larger n u m b e r of cells than does the

vent r icular zone .

B y E 9 0 , the vent r icu lar zone b e c o m e s vir tual ly exhaus ted so that thereafter

the subvent r icu la r zone is the on ly source of addi t ional n e u r o n s des t ined for

the v isua l cor tex. I ndeed , in an imals in jec ted at E90 and ki l led 1 hou r later the

subvent r icu lar z o n e still con ta ins m a n y labe led cel ls . A n apparen t compl ica -

t ion is that w h e n in jec t ion is g iven at later s tages , inc reased n u m b e r s of

labeled cells are p resen t also in the cor tex itself, b u t these are classif ied as pro-

l iferat ing glial cells (see Sec t ion F and F igure 18) . T h u s , in an ima l s in jec ted at

E120 , E 1 4 0 , and P3 and ki l led 1 hou r later, a f ew labe led cells are still p resen t

FIGURE 11. A. Photomicrograph of the inner part of the cerebral wall in the occipital lobe at E81. Ventricular zone (V) is attenuated and contains only a few mitotic figures (crossed arrow) com-pared to earlier stages. The subventricular zone (S) remains relatively wide and full of dividing cells (arrows). B. Autoradiogram of animal injected at E87 and killed 1 hour later. Most radioactive cells are in the subventricular zone (S).

20 Pasko Rakic

in the ves t ige of the subvent r icu la r z o n e , and m a n y m o r e are found through-

out the cortex. H o w e v e r , n o n e of these are neurona l precursors , s i nce in spec i -

m e n s in jec ted at these ages and ki l led postnata l ly n o neu rons are labe led in

the visual cortex (see S e c t i o n B ) .

O n the b a s i s o f these obse rva t ions , it can b e conc luded that in the rhesus

m o n k e y , neu rons of the v isua l cor tex are genera ted in b o t h the vent r icular and

the subvent r icu lar zones . H o w e v e r , the relat ive propor t ion of neu rons gen -

erated i n these two zones changes w i t h t ime . At the earl iest s tages , n e u r o n s

or ig inate a lmost exclus ively in the vent r icular zone ; later they are p roduced in

bo th ventr icular and subven t r i cu la r z o n e s , and b y the end of the pe r iod of

neu rogenes i s the subvent r icu la r zone b e c o m e s the p r e d o m i n a n t source of n e w

cells. Fur ther , it is no t k n o w n w h a t popula t ion o f the prol i ferat ing ventr icular

cells is des t ined at any g iven stage to b e c o m e neu rons versus glia.

E. RATE AND MODE OF NEURONAL MIGRATION

Y o u n g neu rons genera ted in the ventr icular and subvent r icu la r zones

eventual ly b e c o m e d isp laced to the cort ical plate. T h i s p rocess conven t iona l ly

t e rmed cell migra t ion ,* involves m o v i n g an e n o r m o u s n u m b e r of young

neurons across inc reas ing ly longer d is tances from the proliferat ive zones to

the external marg in of the deve lop ing cortical plate. Disorders of neurona l

migra t ion in m a n m a y b e r e spons ib l e in part for cort ical mal fo rmat ions such

as microgyr ia , pachygyr ia , l i s sencepha ly , and nodular ec top ias (12) , bu t these

disorders m a y also cause m o r e sa l ient abnormal i t i e s unde tec tab le b y the usual

pa thologica l m e t h o d s . S ing le gene muta t ion ( 1 0 , 5 3 , 6 8 ) , X - r a y i r rad ia t ions

(13 ,19 ,39 ) , v i ruses (40) , or drugs (32,78) m a y interfere w i th the process of

p roduc t ion and migra t ion of neu rons in the m a m m a l i a n te lencephalon .

A l though such defects in neurona l migra t ion to the neocor tex p robab ly con-

t r ibute impor tan t ly to the e t io logy of men ta l re tardat ion, the unders t and ing of

migra t ion is f ragmentary . A pre l iminary examina t ion of the still i ncomple te

ser ies of au torad iograms from m o n k e y fetuses p rov ides some ins igh t s into

this complex p rob l em.

T h e migra t ion rate o f y o u n g n e u r o n s in the m o n k e y ce r eb rum w a s s tud ied

* In this report and in previous publications (47,48,49,53,68) the view has been adopted that until there is evidence to the contrary, "migration" is the most suitable term, whether the cell as a whole changes position [as in late stages of neocortical genesis (48,49)] or extends a cytoplasmic process outward toward and then parallel to the pia from an immediately postmitotic rounded form at the embryonic ventricular surface (21), or extends a process radially inward while holding its earlier generated axonal process in a fixed position [as in the case of the cerebellar granule cell (55)]. It would seem inappropriate, until more facts are available, to use different terms for the migration of an outward-directed process of a postmitotic ventricular cell along the surface of adja-cent columnar ventricular cells (8), for the migration of a young cerebellar granule cell process and soma along a Bergmann fiber (47), or for the migration of a growing axon tip along a solid substratum in vitro (79).

1. Timing of Major Ontogenetic Events 2 1

in au to rad iograms prepared from fetuses of va r ious ages ki l led e i the r 1 h o u r or

3 , 7, or 14 days fo l lowing 3 H - t h y m i d i n e in jec t ion . In a fetus in jec ted at E45 and

kil led at E 4 8 , a lmost all o f the heav i ly l abe led cells are found in the ou te rmos t

s t ra tum o f the cort ical plate. O n l y very few heav i ly l abe led cells are seen at

in te rmedia te levels b e t w e e n vent r icu lar zone and cort ical plate . In a fetus in-

jec ted at E46 and ki l led at E53 (Figure 12A) labe led cells form a hor izon ta l b a n d

located in the midd le of the cort ical plate . Externa l to the labe led cells l ie sev-

eral rows of weak ly labe led or un labe led cells w h i c h w e r e genera ted fol lowing

the t h y m i d i n e in jec t ion . There fore , at early s tages y o u n g n e u r o n s m o v e to the

cort ical plate re lat ively synchronous ly ; m o s t of t h e m reach the i r des t ina t ion in

less than 3 days , and a lmos t all w i t h i n 7 days .

FIGURE 12. Autoradiograms of the cerebral wall in fetuses injected with 3 H-thymidine and killed at various times afterward. Photographs of the outer portion of the cerebral wall include molecular layer (M), cortical plate, intermediate zone, and portion of subventricular zone taken in all three fetuses at the comparable regions. Further explanation in text. A. E46-E53. B. E52-E55. C. E65-E72.

2 2 Pasko Rakic

Examina t ion of s imi lar ly p repared sec t ions from sl ight ly older m o n k e y fe-

tuses shows a cons ide rab le difference in the t imes w h e n young neu rons from

a s imul taneous ly genera ted set finally reach the i r p e r m a n e n t pos i t ions . For ex-

ample , in a fetus in jec ted at E52 and ki l led 3 days later at E 5 5 , on ly a few of the

heavi ly labe led cells are found at the i r final des t ina t ion in the superficial por-

t ion of the cort ical plate.* M o s t heav i ly labe led cells are still located in the

in te rmedia te and subvent r icu la r zones . Apparen t d i s soc ia t ion of cells into

fast, synchronous ly migra t ing , and s low, a synchronous ly migra t ing c lasses is

well demons t r a t ed in a fetus in jec ted at E65 and ki l led 7 days later at E 7 2 . In

this s p e c i m e n s o m e labe led cells have a t ta ined the i r final pos i t ion in the

superficial s t ra tum of the cort ical plate bu t m a n y are still in the in t e rmed ia te

zone (Figure 12C) . S o m e neu rons genera ted on E65 have reached the i r des t ina-

t ion b y E72 bu t the major i ty had no t m o v e d very far from thei r s i te of o r ig in

dur ing the 7-day per iod . T h i s difference in migra to ry b e h a v i o r b e c o m e s even

more consp icuous toward the end of cor t i cogenes i s . T h u s , in the fetus in jec ted

at E92 and ki l led at E95 only an occas iona l cell has arr ived at the superficial

strata of the cortex (Figure 13A) . S o m e labeled cells are pass ing th rough deep

cortical layers (Figure 13B) and the major i ty still res ide in the subvent r icu lar

and in te rmedia te zones (Figure 13C) . S i n c e in a fetus in jec ted at E90 and ki l led

at E97 m a n y labe led cells are still s i tuated in the subvent r icu la r and in t e rmed i -

ate zones (Figure 1 3 C ) , apparent ly at th is age they take m o r e than 7 days to ar-

r ive at the i r des t ina t ion . T h e dynamics of cell m o v e m e n t and the exact t ime of

arrival at the cor tex wil l b e d e t e r m i n e d w h e n addi t ional an imals are p repared

wi th longer intervals b e t w e e n in jec t ion and sacrifice.

Essent ia l ly s imi lar changes in migra t ion ra tes , bu t t e lescoped into a shor ter

t ime interval , have b e e n obse rved dur ing genes i s o f the rat neocor t ex (18) . In

this spec ies at early s tages ( E 1 4 - E 1 8 ) y o u n g n e u r o n s arrive at the cort ical plate

in abou t 2 days . H o w e v e r , m a n y y o u n g neu rons w h i c h or ig ina te toward the

end of ges ta t ion ( E 1 9 - E 2 1 ) con t inue to migra te dur ing the first w e e k after

b i r th . T h e first group of cells gene ra ted at E20 reach the cor tex in abou t 3 days ,

the last in abou t 10 days . T h u s the rate of arrival at the cortex a m o n g the

s imul taneous ly genera ted cells is var iab le .

If the t ime necessa ry for y o u n g neu rons to reach the superficial por t ion of

the cort ical plate depends on ly on the length of the migra t ion pa thway , then

all s imul taneous ly genera ted cells should arrive synchronous ly at the i r des t i -

na t ion . Ye t at any g iven late s tage of cor t i cogenes i s o f the rhesus m o n k e y ,

some cells reach thei r des t ina t ion several t imes more rapidly than o thers . T h e

* The relative thickness of the lateral and medial cerebral wall is different at various fetal ages. For example at E70 the lateral cerebral wall of the occipital lobe is about two times thicker than the medial, and this difference increases to more than 4 times at E120. Since the time of the arrival of simultaneously generated neurons to the cortical plate is affected by the length of their pathway, the comparison of the autoradiograms in this study was made on roughly corresponding areas of the medioventral portion of the cerebral wall, which has an intermediate thickness.


FIGURE 1 3 . Autoradiograms of an animal injected at E 9 2 and killed 3 days later at E 9 5 to indicate

position of heavily labeled cells (arrows). A. Molecular layer and superficial cortex. B . Deep cortex.

C. Deep cortex and intermediate zone.

t ime of arrival m i g h t inf luence the final cell pos i t ion in the cort ical plate .

T h u s , n e u r o n s genera ted o n a g iven day ear ly in deve lopmen t c o m e to occupy

a re la t ively na r row zone of the ma tu re cor tex, wh i l e those genera ted at a g iven

later age c o m e to b e d i s t r ibu ted over a s o m e w h a t w i d e r zone ; also, neu rons

bo rn 8 to 10 days apart m a y over lap s o m e w h a t in pos i t ion a long the radial axis

[Rakic (52) and F igure 8 ] .

O n e explana t ion for the w i d e d i s t r ibu t ion of cells genera ted s imul t aneous ly

is that m o r e s lowly m o v i n g neu rons reach the superficial por t ion of the cor tex

w h e n it has a l ready inc reased several h u n d r e d mic rome te r s in t h i ckness b y

the add i t ion of m a n y n e u r o n s , s o m e of w h i c h w e r e actual ly genera ted later

bu t had migra ted m o r e rapid ly (52) . A s a c o n s e q u e n c e the m o r e s lowly

mov ing y o u n g neu rons at tain in the mature b ra in m o r e superficial pos i t ions

than the rapidly m o v i n g n e u r o n s gene ra t ed s imul taneous ly . At later deve lop-

menta l s tages the difference in the rate of cell mig ra t ion a m o n g s imul taneous ly

2 4 Pasko Rakic

genera ted cell inc reases , as does the span of the i r d i s t r ibu t ion a long the radial

vectors across the cor tex (Figure 8 ) . A poss ib le l eng then ing of the cell genera -

t ion cycle at later ages (29) m i g h t also inf luence the t ime interval b e t w e e n in-

j ec t i on and arrival of y o u n g n e u r o n s i n the cor tex. H o w e v e r , th i s effect is

p robab ly small b e c a u s e heav i ly labe led cells we re located at var ious levels o f

the in te rmedia te zones in fe tuses ki l led 3 and 7 days after in jec t ion , thus in -

dica t ing that cells a l ready had d iv ided . T h e t imes sampled in the presen t

mater ia l are not su i tab le for d i s t i ngu i sh ing w h e t h e r the difference in rate of

cell arrival in the cor tex i s due to a differential mig ra t ion rate or to the dif-

ferential lags in in i t i a t ion of migra t ion . W h y the difference in t ime of arrival to

the cortex a m o n g s imul taneous ly genera ted cells b e c o m e s apparent on ly at the

midd le and late s tages of cor t i cogenes i s and is it in some w a y rela ted to the

m e c h a n i s m of cell m o v e m e n t ? T h e s e ques t ions are cons ide red b e l o w in the

l ight of s o m e s tud ies o n the m o d e o f n e u r o n migra t ion .

O n e of the several m e c h a n i s m s p roposed to expla in cell d i sp lacements

w i th in the central ne rvous sys tem s tems from the idea that the t issue is

o rgan ized as a pseudost ra t i f ied ep i the l ium (61) . S i n c e the early neu roep i the -

l ium is c o m p o s e d of co lumnar epi thel ia l cells w h i c h , at least in s o m e phases of

thei r in te rmi to t ic cycle , s tretch from the ventr icular to the external surface, it

was sugges ted that the same a r rangement m i g h t con t inue to exis t at later

deve lopmenta l s tages and that only the cell nuc leus actually m o v e s w i t h i n the

e longa ted b u t fixed cy top lasmic cy l inder (7 ,38) . S i n c e dur ing m i t o s i s v e n -

tricular cells lose thei r external p rocess ( 2 1 , 2 2 , 7 2 ) , they wou ld have to r eg row

and w i thd raw it a l ternat ively dur ing the cell genera t ion cycle unt i l finally the

external process is u sed for nuc lear t rans locat ion . T h i s m e c h a n i s m m i g h t

apply at early s tages of co r t i cogenes i s w h e n the migra t ion pa ths b e t w e e n ven -

tricular and pia l surfaces are re la t ively short .

In rhesus m o n k e y fetuses at early s tages , b e t w e e n E40 and E 5 5 , m a n y

b ipolar cells w h o s e nuc le i are s i tuated in the ventr icular zone have a smoo th

radial p rocess w h i c h spans the ent i re cerebra l wall and te rmina tes at the ex-

ternal surface in one or several endfeet (64) . At these early s tages of develop-

ment , ex is t ing morpho log ica l m e t h o d s do not p rov ide features pe rmi t t ing clas-

sif ication of these cells as glial or neurona l precursors . S o m e of these radial ly

o r ien ted cells m i g h t i n d e e d b e pos tmi to t ic y o u n g neu rons in w h i c h the nu-

cleus eventual ly m o v e s to a m o r e superficial pos i t ion as p roposed b y M o r e s t

(38) . Howeve r , at s l ight ly later s tages , cells w h i c h span the ent i re cerebral wall

a s sume the typical morpho logy of radial glial cells (Figures 14 and 19) . T h e i r

somata are s i tuated close to the vent r icu lar surface, and have an e longated ,

radially o r ien ted glial p rocess w h i c h spans the full t h i ckness of the deve lop ing

cerebra l wall [ S c h m e c h e l and Rak ic (64) and Sec t ion F o f th i s chap te r ] .

A different m e c h a n i s m of cell migra t ion has b e e n postula ted in rhesus

m o n k e y fetuses at late s tages of co r t i cogenes i s (48 ,49) . T h e migra t ing n e u r o n s

appear to b e relat ively s imple and s m o o t h b ipo la r cells w i th a l ead ing process

FIGURE 14. A. Camera lucida drawing of a Golgi-impregnated coronal section across the cerebral vesicles of a 97-day monkey fetus. From Rakic (49). Indentation on the left represents incipient calcarine fissure. The caliber of the radial fibers is slightly exaggerated in order to illustrate their arrangement at such a low magnification (scale equals 1 mm). Arrows indicate cerebral wall. B. Photomicrograph of the Golgi preparation used for the drawing in A taken from the area outlined by the rectangle. C and D. Photomicrographs of two migrating young neurons (MN) oriented par-allel to radial glial fibers (RF) in 95-day monkey fetus. E. Photographically enlarged segment of the radial glial fiber passing through the intermediate zone. Note numerous lamellate expansions present at E95.

25

26 Pasko Rakic

A B IOOM


w h o s e leng th is on ly a fract ion of the total migra to ry p a t h w a y of the expanded

cerebra l wall at these ages (Figure 14B) . Elec t ron mic roscop ic e x a m i n a t i o n of

migra t ing cells sugges t s they find the i r w a y to the cor tex b y us ing radial ly

o r ien ted glial f ibers as gu ides (48 ,49) . T h r e e - d i m e n s i o n a l recons t ruc t ion of

e lect ron mic roscop ic ser ial sec t ions o f 58 -day fetal m o n k e y c e r e b r u m s h o w s

that b o t h the t ra i l ing and the l ead ing p rocesses of s o m e migra t ing cells are

conf ined to the in te rmedia te zone and do no t approach e i ther cerebra l surface

(54). Rad ia l e l emen t s of the fetal cerebra l wal l are c o m m o n l y o rgan ized in fas-

cicles (Figure 15) cons i s t ing of o n e or two radial glial p rocesses , the leading

and trai l ing p rocesses of several mig ra t ing n e u r o n s , and poss ib ly afferent or

efferent axons (49) . T h e lead ing process of the mig ra t ing cell f requent ly

b r a n c h e s and ex tends a long m o r e than o n e radial fascicle (Figure 16E) . S u c h a

process is p r o b a b l y capab le of b e i n g ret racted b e c a u s e , i f i ts form and n u m b e r

were fixed, the cell w o u l d b e unab le to advance b e y o n d fiber bund le s ly ing

t ransverse ly b e t w e e n its b r a n c h e s (Figure 1 6 G ) .

Mul t ip le neu rona l p rocesses are s een at a g iven level o f the in te rmed ia te

zone or the cort ical plate s ince several neu rons m o v e in succes s ion a long a

s ingle radial pa th (Figure 15) . W h e r e they over lap , the later ar r iv ing n e u r o n s

m a y b e m o v i n g a long the surfaces o f the i r p redecessor s ra ther t han a long the

glial f iber specif ical ly (Figure 15B) . A s they en te r the cort ical plate the neu rons

that fol low a g iven radial pa th b e c o m e a l igned in a s ingle ver t ical co lumn , an

a r rangement w h i c h is a p r o m i n e n t morpho log ica l feature o f the fetal neocor t ex

(Figure 17) . T h e a l ignmen t of the migra t ing neu ron a long the radial glial fiber

FIGURE 15. A. Camera lucida drawing of the part of cerebral wall indicated by arrows in Figure 14A. Modified from Rakic (49). Composite illustration is combined from Golgi section (black profiles of cell images) and on adjacent section counterstained with toluidine blue (outlined profiles of cell nuclei). The middle 2000 ptm of the intermediate zone, similar in structure to the sectors drawn, are omitted. The rectangle marked with an arrowhead shows the approximate position of the three-dimensional reconstruction in Figure 14B. Abbreviations: C, cortical plate; I, intermediate zone; M, molecular layer; MN, migrating neuron; OR, optic radiation; RF, radial fiber; SV, subventricular zone; V, ventricular zone. B . Three-dimensional reconstruction of mi-grating neurons, based on electron micrographs of semiserial sections. From Rakic (49). The reconstruction was made at the level of the intermediate zone indicated by the rectangle and arrowhead in Figure 15A. The subventricular zone lies some distance below the reconstructed area, whereas the cortex is more than 1000 fxm above it. The lower portion of the diagram con-tains uniform, parallel fibers of the optic radiation (OR) and the remainder is occupied by more variable and irregularly disposed fiber systems; the border between the two systems is easily recognized. Except at the lower portion of the figure, most of these fibers are deleted from the diagram to expose the radial fibers (striped vertical shafts ( R F 1 - 6 ) and their relationships to the migrating cells (A, B , and C) and to other vertical processes. The soma of migrating cell A, with its nucleus (N) and voluminous leading process (LP), are within the reconstructed space, except for the terminal part of the attenuated trailing process (TP) and the tip of the vertical ascending pseudopodium. Cross sections of cell A in relation to the several vertical fibers in the fascicle are drawn at levels " a " to "d" at the right side of the figure. The perikaryon of cell B is cut off at the top of the reconstructed space, whereas the leading process of cell C is shown just penetrating between fibers of the optic radiation (OR) on its way across the intermediate zone.

FIGURE 1 6 . Computer-aided three-dimensional reconstruction of the cells in the occipital lobe of fetal monkey brain. Modified from Rakic et al. (54). A . Outline of a coronal section across the oc-cipital lobe of a 58-day-old monkey embryo to indicate the area in which cells were reconstructed. The cerebral ventricle (CV) is relatively large at this stage and the lateral wall (L) is much thicker than the medial wall (M) on which the incipient calcarine fissure is slightly indented. The shaded area between the arrowheads indicates the position of the block of tissue that was processed for elec-tron microscopic study. B . Drawing of a 1-fjum thick, toluidine blue-stained section across the en-tire area shaded in Figure 1 6 A . The cerebral wall from the ventricular surface at the bottom to the external surface at the top of the drawing consists of ventricular zone (VZ), subventricular zone (SV), intermediate zone (IZ), and cortical plate (CP). The area outlined by the rhomboid was cut serially for electron microscopic examination. The overlapping squares represent individual fields

2 8

A B C D

E F G H

FIGURE 17. A. A portion of the cortical plate of a 97-day monkey fetus which illustrates vertical columnar organization of the cortex at this developmental stage. The top of the figure is closer to the pial surface. Photomicrograph of a l-fim toluidine blue-stained section. B . Electron micro-graph of the deep portion of the prospective visual cortex of 97-day monkey fetus. Migrating neuron (MN), its leading process (LP) aligned with a radial fiber ( R F ) , bypasses more mature deep neurons (DN) on its way to superficial layers. The shaft of the radial fiber ( R F ) is only partially present within this section. It also does not have the usual amount of glycogen particles (42) because of en bloc staining with uranyl acetate used in preparing tissue (49).

photographed for the reconstruction on photomontages. Arrows A to F point to the positions of six reconstructed cells illustrated in C-H, respectively. Cells were drawn with the aid of superim-posed outlines of cell profiles in serial sections at different levels traced onto transparent Mylar sheets. Some of the fibers which lie in contiguity with the migrating cells were also traced and reconstructed but most of the neighboring cells and processes were omitted. For details, see Rakic et al. (54).

2 9

30 Pasko Rakic

in the cort ical plate has b e e n conv inc ing ly demons t ra t ed also in the rat te len-

cepha lon at late s tages of cor t i cogenes i s (42) . In addi t ion , the radial a l ignment

o f dendr i tes o f pyramida l neu rons desc r ibed in adult an imals (7 ,14 ,43) m a y

reflect the m e c h a n i s m of cell mig ra t ion to the cor tex. T h i s sub jec t also mer i t s

inves t iga t ion in t e rms of the funct ional co lumns desc r ibed in the v i sua l cor tex

(26 ,27) . W h e t h e r the t ime of neu ron arrival at i ts final pos i t ion in the cort ical

plate cor responds to the t ime of i ng rowth of specific afferents from the lateral

genicula te b o d y also r ema ins to b e de te rmined .

T h e m e c h a n i s m of cell mig ra t ion mer i t s further inves t iga t ion b y specia l

me thods . H o w e v e r , on the bas i s of the scanty data n o w avai lable it is poss ib le

to speculate that two different m e c h a n i s m s of cell d i sp lacement m igh t exist :

one in opera t ion at the early s tages of cort ical format ion and the o ther ut i l ized

at later s tages . At early s tages w h e n the migra tory p a t h w a y usual ly does no t

exceed 2 0 0 - 3 0 0 /xm, the external p rocess of the ventr icular cell m igh t s tretch

across the ent i re mig ra t ion d is tance . T h e nuc leus m i g h t then t ranslocate

w i th in its o w n cyl inder of cy top lasm (38) , a poss ib i l i ty w h i c h w o u l d correlate

wi th the rapid , synch ronous m o v e m e n t obse rved in our autoradiographic

mater ia l . T h i s migra to ry m e c h a n i s m m a y b e reserved for ventr icular cells only .

Dur ing the later s tages of cor t i cogenes i s w h e n m a n y more y o u n g neurons ,

mos t ly of subvent r icu la r o r ig in , m o v e over a d i s tance w h i c h m a y b e m o r e

than 10 t imes the length of the i r l ead ing p rocess , mig ra t ing cells follow radial

glial gu ides across the expanded in te rmedia te zone as they pass from the si te

of genes i s to the superficial layers o f the cort ical plate. A s desc r ibed a b o v e ,

au toradiographic ana lys is ind ica tes that cell d i sp lacement at late s tages , w h e n

the major i ty of neu rons are genera ted in the subvent r icu la r z o n e , p roceeds

less synchronous ly and requ i res more total t ime . It is poss ib le that glial gu ides

are used mos t ly b y subvent r icu la r cells. It is impor tan t to no te that cerebel lar

granule cells genera ted in the external granular layer , w h i c h represen ts a

der ivat ive of the subvent r icu la r zone , migra te a long s imi lar gu ides formed b y

B e r g m a n n glial fibers (47) .

F. GENESIS OF RADIAL GLIAL CELLS AND ASTROCYTES

A general ly accepted v i e w w h i c h s tems from the classic w o r k of H i s (23) is

that a g iven reg ion of the central ne rvous sys tem b e c o m e s popula ted b y glial

cells on ly after the major i ty of n e u r o n s are genera ted and have a s s u m e d the i r

final pos i t ions . Th i s concep t r e m a i n e d u n s h a k e n b y the au torad iographic

s tudies of the past decade , b e c a u s e relat ively few labe led glial cells we re

recorded in adult an imals in jec ted w i t h 3 H - t h y m i d i n e at e m b r y o n i c s tages

w h e n the major i ty of neu rons in a par t icular r eg ion are b e i n g genera ted . If one

does no t take in to account the l imi ta t ions of the au toradiographic m e t h o d , it

wou ld appear that results in the rhesus m o n k e y also suppor t the classic con-


cept (52) . I n d e e d few glial cells are labe led in " j u v e n i l e " an imals w h i c h had

b e e n in jec ted at E 1 2 0 , E 1 4 0 , P 2 , or P 1 8 , that i s , after neu ron p roduc t ion has

f in ished. T h e s e heav i ly l abe led cells we re scat tered th roughou t the ent i re

t h i ckness of the cor tex and were classif ied as glia on the bas i s of small nuc lear

s ize , a b s e n c e of Niss l subs t ance , and satel l i te pos i t i on to n e u r o n s (Figure 18) .

H o w e v e r , it should b e e m p h a s i z e d that au torad iographic data o f th i s sort m a y

b e mi s l ead ing i f u sed to de t e rmine the onse t o f g l i agenes i s b e c a u s e the usual

schedule o f in jec t ion and ki l l ing canno t resolve th i s q u e s t i o n for a con t inu -

ously d iv id ing cell popula t ion . In s u b s e q u e n t d iv i s ions the radioac t iv i ty of the

first genera ted glial cells is d i lu ted b e y o n d the th resho ld of r ecogn i t ion in au-

to rad iograms (66) . T h u s , our resul ts s h o w on ly those cells w h i c h were gen-

erated last ra ther than the first in a g iven glial l ine . To de t e rmine the first

FIGURE 18. Autoradiograms of the visual cortex in "juvenile" animals injected with 3 H -thymidine at various embryonic ages. A, E102; B , E120; C, E140. Neurons, characterized by a large pale nucleus and Nissl substance in their cytoplasm, are not radioactive, whereas numerous glial cells characterized by small dark nucleus and unstained cytoplasm are heavily labeled (arrows).

32 Pasko Rakic

appearance of glial cel ls , au torad iographic mater ia l has to b e supp lemen ted

wi th fetal b ra in t i ssue s ta ined wi th a Golg i m e t h o d w h i c h adequa te ly impreg-

na tes immatu re glial e l emen t s .

To analyze g l i agenes i s in m o n k e y visual cor tex, occipi ta l l obes of an imals

aged from E48 to P1003 we re p rocessed according to R io Hor tega ' s rap id Golg i

m e t h o d (60) modif ied b y S tensaas (70) . W h e n used on t i s sue fixed p rev ious ly

b y perfus ion wi th a ldehyde mix tu re , th is m e t h o d g ives good impregna t ions of

imma tu re glial e l emen t s in the m o n k e y ce rebe l lum (47) and c e r e b r u m

(49 ,63 ,64) .

At early s tages ( E 4 8 - E 7 0 ) radial ly o r ien ted , b ipo la r cells w h i c h span the en-

tire cerebra l wall o f the te lencephal ic ves ic le have s m o o t h somata and radial

p rocesses w h i c h t e rmina te at the pial surface in o n e or several endfeet (Figure

19A) . W e are no t ab le to define the l ineage of any of these cells. Howeve r ,

already at th i s early age , s o m e radial p rocesses w h i c h end w i t h i n the cerebral

wall at tach to b lood vesse ls w i t h character is t ic endfeet (Figure 19A) , an ind ica-

t ion of the i r glial nature . Th i s p rov ides the first ev idence for the p resence of

glial cells dur ing early s tages o f cort ical deve lopmen t in the rhesus m o n k e y

(64) . After E 7 0 , m o s t b ipo la r cells spann ing the m o n k e y cerebral wall possess

lamellate expans ions a long the s egmen t o f radial fiber ly ing in the inter-

media te zone . The i r endfee t at the pial surface s ta in w i th the per iod ic ac id-

Schi f f m e t h o d and the i r appearance in Golg i prepara t ions (Figures 14 and 19)

correlates w i t h a class o f 0 . 8 - 1 . 2 - j a m th i ck fibers p rev ious ly s h o w n b y elec-

tron mic roscopy to possess m a n y glial character is t ics (49) . The i r morpho logy

r e sembles radial glial cells desc r ibed in o the r spec ies ( 4 , 1 6 , 1 7 , 3 4 , 3 5 , 4 2 , 5 6 ,

5 7 , 5 8 , 7 1 , 7 2 , 7 6 ) . In the rhesus m o n k e y they b e c o m e mos t p r o m i n e n t after E80

and the i r radial f ibers are eas i ly d i s t ingu i shed from the p rocesses of migra t ing

neurons (Figure 14B and C ) .

M o s t radial glial cells in m a m m a l i a n neocor tex we re cons ide red b y R a m o n y

Cajal (56) to t ransform th rough in te rmedia ry forms in to pro toplasmic and

fibrillary as t rocytes . Trans i t iona l forms b e t w e e n radial glial cells and as-

trocytes are first obse r ve d in the m o n k e y at E 6 5 , w h e n the somata of s o m e

radial glial cells de tach from the ventr icular surface and b e c o m e displaced to

the in te rmedia te zone (64) . B e t w e e n E75 and E 1 2 0 , the major i ty of radial glial

cells acqui re n u m e r o u s and b u s h y lamellate expans ions (Figure 19C) . T rans i -

t ional forms from radial glia to as t rocytes are first s een in the deep cort ical

layers (at E65) then in the m o r e superficial layers and in subpia l pos i t ion in

layer I (E85) . T h u s , imma tu re as t rocytes appear in the deve lop ing cort ical plate

at the t ime w h e n n e u r o n produc t ion is at i ts peak (52) . Vir tual ly all the radial

glial cells contac t ing b l o o d vesse ls apparent ly t ransform into pro top lasmic as-

t rocytes b y E120 . H o w e v e r , m a n y of those radial glial cells w h i c h span the

cerebral wall pers is t longer and s o m e con t inue to t ransform into as t rocytes .

After b i r th the n u m b e r of radial glial cells con t inues to decrease s teadi ly . Un t i l

P20 it is poss ib le to verify the i r ent i re course across the cerebra l wall , in the


E

FIGURE 19. Semischematic illustration of gliagenesis in monkey telencephalon. Composite drawing is combined from impregnated cells in Golgi sections (black profiles of cell images) and cell nuclei as they appear in adjacent sections stained with toluidine blue (open profiles). In the diagrams, cell silhouettes and radial processes are represented larger than their actual size in order to illustrate their morphology at such a low magnification. Cell images in each column are typical of approximately the following age ranges A, E50-E60; B , E60-E70; C, E70-E80; D , E80-E120; E, E120-P20. Detailed description and documentation is published elsewhere (64). Abbreviations: CP, cortical plate; E, ependymal layer; M, molecular (marginal) layer; I, intermediate zone; S, sub-ventricular zone; V, ventricular zone; W, white matter.

34 Pasko Rakic

depth of the calcar ine fissure w h e r e the t i ssue r ema ins less than 2 m m th ick

(64) . E v e n he re , no radial glial cells s p a n n i n g the cerebra l wall we re seen in 11

an imals e x a m i n e d b e t w e e n P50 and P200 despi te the p re sence of we l l - impreg-

na ted t rans i t ional forms near the ventr icular surface and myr iad as t rocytes

th roughout the h e m i s p h e r e .

T h i s s tudy ind ica tes that cer ta in glial e lements—radia l glial cel ls—are

present m u c h earl ier in deve lopmen t than prev ious ly recogn ized . Dur ing fetal

cortical deve lopmen t radial glial cells m a y serve specia l funct ions such as scaf-

folding for the fragile e m b r y o n i c ne rvous t i ssue (56) or a gu ide for late m i -

grat ing cells (48 ,49) . T o w a r d the e n d of ges ta t ion and shor t ly after b i r th m o s t

of the radial glial cells t ransform into immatu re as t rocytes and pe rhaps d iv ide

again several t imes , thus p rov id ing the s tem for b o t h pro top lasmic and fibril-

lary as t rocytes of adult an imals .

G. RELATIONSHIP BETWEEN TIME OF NEURON ORIGIN AND THE APPEARANCE OF CONVOLUTIONS

IN THE OCCIPITAL LOBE

T h e m e c h a n i s m under ly ing the deve lopmen t of f issures and gyri , w h i c h at-

tain such p r o m i n e n c e in the p r imate ce r eb rum and ce rebe l lum r e m a i n s a m y s -

tery. It is general ly a s s u m e d wi thou t any exper imenta l ev idence that the gen-

era t ion o f large n u m b e r s o f cort ical neu rons in p r imates con t r ibu tes m o r e to

an increase in cort ical area than to an inc rease in i ts th ickness . T h e expanded

growth of the h e m i s p h e r i c surface w i t h i n a l imi ted cranial space w a s though t

to lead to the format ion of fissures and gyri . H o w e v e r , as r ev i ewed e l sewhere

(69) , it i s difficult to expla in the deve lopmen t of fissures and gyri in such

s imple mechan ica l t e rms . S i n c e the occipi ta l lobe in rhesus m o n k e y has rela-

t ively deep sulci and p r o m i n e n t gyr i , it is useful to correlate the t ime of thei r

format ion wi th the t ime of neurona l p roduc t ion .

Toward the end of the per iod of neu ron genes i s on ly a relat ively sha l low

and wide ly gap ing calcar ine fissure exis ts on the med ia l cerebral wall (Figure

2 0 A ) . The par ie to-occip i ta l no tch is jus t r ecogn izab le on the dorsal cerebra l

surface (Figure 20 A ' ) . T h e lateral surface of the occipi ta l lobe is still comple te ly

smooth . B y E l 12 the b ra in is on ly s l ight ly larger. T h e calcar ine and par ie to-oc-

cipi tal f issures n o w sl ight ly deeper and be t t e r ou t l ined , r ema in the on ly in-

denta t ions in the occipi ta l lobe (Figure 20B and B ' ) . T h i s f inding is remarkab le

if one takes in to account that vir tual ly all cort ical neu rons are present b y E100 .

It appears , therefore , that the e n o r m o u s increase in surface area, w h i c h s u b -

sequen t ly results in the d e e p e n i n g of p r imary fissures and the deve lopmen t o f

secondary f issures occurs after neu rons have b e e n genera ted . Therefore , the

process of f issurat ion o f the t e l encepha lon in m o n k e y co inc ides no t w i t h the

genes i s of neu rons , bu t w i th the deve lopmen t of the i r p rocesses and connec -


FIGURE 20. External configuration of the lateral (A-C) and medial ( A ' - C ) surfaces of the monkey

cerebrum at E97 (A,A') , E112 (B,B ' ) , and P150 ( C , C ) . All brains were fixed similarly by vascular

perfusion with glutaraldehyde-formaldehyde mixture in phosphate buffer, taken from the skull 1

hour later and photographed at the same magnification.

t ions as wel l as w i t h the inc rease in glial cell n u m b e r s . H o w e v e r , s o m e addi-

t ional factors such as the regional differences in the mig ra t ion rate of late

genera ted neu rons and the t ime w h e n they at ta in the i r p e r m a n e n t pos i t i on are

still u n k n o w n and have to b e worked out in re la t ion to the format ion of fis-

c1

IQmm

c

PI50

B

B'

EII2

A A*

E97

36 Pasko Rakic

sures . T h e s e po in t s have re levance for the unde r s t and ing of m e c h a n i s m s un-

der lying s o m e h u m a n cort ical dysgeneses such as mic rogyr i a and l i s sen-

cepha ly (12 ,59) .

H. SUMMARY

T h e place and t ime of o r ig in , the migra t ion and eventual d i spos i t ion of

neurons of the m o n k e y v isua l cor tex we re s tud ied b y au torad iography in

animals ki l led at var ious intervals after 3 H - t h y m i d i n e pulse labe l ing at e m b r y -

onic (E) and early pos tna ta l (P) s tages . All n e u r o n s des t ined for the v isua l

cortex are genera ted dur ing abou t a 2 - m o n t h pe r iod b e t w e e n E45 and E102 .

Neu ron pos i t i on in the cort ical l aminae correlates sys temat ica l ly wi th t ime of

cell o r ig in ; neu rons des t ined for deeper cort ical pos i t ions are genera ted ear-

l ier , and m o r e superficial ones progress ive ly later. T h u s , mos t neu rons in layer

V I are b o r n b e t w e e n E 4 5 and E 6 0 , in layer V b e t w e e n E 6 0 and E 7 0 , in layer IV

b e t w e e n E70 and E 8 0 , and in layers III and II b e t w e e n E80 and E102 . N o

neu rons , bu t n u m e r o u s glia, are genera ted w i th in the cort ical plate itself. In i -

tially y o u n g n e u r o n s are p roduced a lmost exclus ive ly in the ventr icular zone .

Later t hey are p robab ly genera ted in bo th ventr icular and subvent r icu lar

zones , and b y the end of the prol iferat ive pe r iod the subvent r icu lar zone

b e c o m e s the p r e d o m i n a n t source of n e w cells . At the t ime w h e n all neu rons

des t ined for the m o n k e y v i sua l cor tex have a l ready b e e n p roduced , p r imary

fissures are bare ly ind ica t ed on the cerebra l surface and n o secondary fissure

appears as yet .

Au to rad iograph ic ana lyses ind ica te that at early s tages y o u n g neu rons m o v e

to the cort ical plate relat ively synchronous ly and at a fast rate, w h e r e a s at later

s tages there are cons ide rab le dif ferences in the rates o f cell migra t ion . A t early

s tages w h e n the mig ra t ion p a t h w a y is relat ively short , the external process of

the ventr icular cell m a y stretch across a lmost the ent i re migra tory d is tance . It

is pos s ib l e that nuc le i m o v e w i thou t in te r rupt ion wi th in the i r o w n cyl inders of

cy top lasm, a m e c h a n i s m w h i c h m i g h t account for the rapid , synchronous

m o v e m e n t of cell b o d i e s as s een in the au toradiographic mater ia l . Du r ing

later s tages w h e n y o u n g n e u r o n s , mos t ly of subvent r icu la r o r ig in , m o v e across

a d is tance w h i c h is m o r e than 10 t imes the length of thei r l ead ing process ,

migra t ing cells fol low radial glial gu ides across the w i d e n e d in te rmedia te zone

and th rough dense ly packed cort ical plate. T h i s type of cell d i sp lacement

s e e m s to p roceed less synch ronous ly and requ i re m o r e t ime . It i s p roposed

that two different m e c h a n i s m s of cell d i sp lacemen t m i g h t exis t : one in opera-

t ion at early s tages of cort ical format ion w h e n mos t ly ventr icular cells migra te ,

and ano ther ut i l ized b y subvent r icu la r cells at later s tages .

T h e first glial e l emen t s are ident i f iable at E48 as b ipo la r radial cells w h i c h

te rminate wi th endfee t on b l o o d vesse ls . Typica l radial glial cells w h i c h span


the ent i re cerebra l wall are ident i f ied b y E 7 0 . Trans i t iona l forms from radial

glial cells to i m m a t u r e as t rocytes in the deve lop ing v isua l cor tex are first

de tec ted at E 6 5 , b e c o m e m o s t p r o m i n e n t from E80 to b i r th , sharply decrease

postnata l ly , and vir tual ly cea se at 7 m o n t h s of age .

Corre la t ion of h u m a n and m o n k e y visual cor t ices s t a ined wi th b o t h Niss l

and Golg i m e t h o d s ind ica tes that in m a n the full c o m p l e m e n t of neocor t ica l

neu rons is also p r o b a b l y genera ted wel l before b i r th .

ACKNOWLEDGMENT

This work was supported by Grant No. NS 11233 from the National Institutes of

Health.

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1. Timing of Major Ontogenetic Events •39

Radiation Biology of the Fetal and Juvenile Mammal. Proc. 9th Annu. Hanford Biol. Symp. US At. Energy Comm., Washington, D.C. , 1969: 755-767 .

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2 Effects of Interference with Cerebellar

Maturation on the Development of Locomotion. An Experimental Model

of Neurobehavioral Retardation

J O S E P H A L T M A N

Department of Biological Sciences, Purdue University,

West Lafayette, Indiana

INTRODUCTION

T h e exper imenta l ana lys i s of the causa t ive factors of men ta l re tardat ion in

m a n is h inde red b y n u m e r o u s pract ical and mora l cons ide ra t ions . T h i s j u s -

tifies the use of an ima l mode l s , even t hough these mode l s rarely, i f ever ,

mi r ror adequa te ly the complex i t i e s of h u m a n men ta l re tardat ion , w h e r e b i o -

logical va r iab les are in t e rming led wi th mul t i far ious cultural factors.

W e have b e e n engaged for several years in a mul t id i sc ip l ina ry s tudy of the

postnata l deve lopmen t of the ce rebe l lum and locomot ion in rats unde r normal

cond i t ions and fol lowing var ious exper imen ta l t rea tments . S o m e of the treat-

men t s that w e have used , such as undernu t r i t ion or the admin i s t r a t ion of hor-

m o n e s and drugs , have long b e e n suspec ted a m o n g the factors that can lead to

menta l re tardat ion. W e recogn ize that exper imen ta l in ter ference w i t h cere -

bel lar and m o t o r d e v e l o p m e n t does not , str ict ly speak ing , represen t " m e n t a l "

re tardat ion. H o w e v e r , it does cons t i tu te an ins t ance of neura l and behav io ra l

re tardat ion, one that offers m a n y exper imen ta l advantages over the re tardat ion

of o ther neura l and behav io ra l sys t ems . T h e ce rebe l lum is an eas i ly access ib le

and wide ly s tud ied s tructure w h i c h deve lops in the rat to a large extent after

b i r th w h e n exper imen ta l man ipu l a t i ons can b e eas i ly appl ied . M o r e o v e r , the

objec t ive s tudy of overt m o t o r deve lopmen t , as o p p o s e d to m a n y covert

menta l p h e n o m e n a , offers m a n y exper imen ta l advantages .

4 1

42 Joseph Altman

T h i s r ev iew is res t r ic ted in scope , b e i n g essent ia l ly l imi ted to a s u m m a r y of

recent work from our labora tory in w h i c h focal X- i r rad ia t ion w a s used to in -

terfere w i t h the no rma l course of cerebel lar deve lopment .

NORMATIVE STUDY OF THE POSTNATAL DEVELOPMENT OF THE RAT CEREBELLUM

T h e t i ssue of the ce rebe l lum expands subs tant ia l ly from b i r th to 2 1 days , the

t ime w h e n rat pups are usual ly w e a n e d from the i r mo the r s . A quan t i t a t ive

h is to logica l s tudy s h o w e d (2) that in the sagit tal p lane the area of the cerebe l -

lum inc reases dur ing th is pe r iod over 20-fold (Figure 1) and that th i s increase

is pr imar i ly due to the expans ion of the cerebel la r cor tex. Dur ing the first

w e e k after b i r th the prol i ferat ive zone of the cerebel lar cor tex , the subpia l ex-

ternal ge rmina l layer , g rows rapidly [see A l tman (2 , F igure 3 ) ] , wh i l e the

growth of the o ther layers (which are essent ia l ly un fo rmed at b i r th) is

s luggish. T h i s ind ica tes that few of the cells that c o m e in to ex is tence are dif-

ferent ia t ing at th is t i m e and that the mul t ip ly ing cells are mere ly inc reas ing

the s t em cell popu la t ion o f the external ge rmina l layer . T h e growth of the

granular and molecular layers b e g i n s at an accelera ted rate toward the b e g i n -

n i n g of the s e c o n d w e e k and ends b y the e n d o f the third w e e k (F igure

T h e increase in cerebe l la r m a s s dur ing th is rapid g rowth pe r iod is due to

m a n y deve lopmenta l p rocesses . A recent s tudy e m p l o y i n g quant i t a t ive h is to l -

ogy, h i s tochemis t ry , au torad iography , and electron mic ro scopy (4 -6 ) led us to

the concep tua l iza t ion that pos tnata l cerebel lar neu rogenes i s can b e s u b d i v i d e d

into five, largely sequen t i a l even t s that w e shall refer to as (1) cy togenes i s , (2)

m o r p h o g e n e s i s , (3) synap togenes i s , (4) g l iogenes i s , and (5) m y e l o g e n e s i s . W e

have s tudied in s o m e detai l the first three of these events .

Postnatal Cerebellar Cytogenesis

Cytogenes i s refers to the first s tep in n e u r o g e n e s i s , the prol i ferat ion of

precursor cells w h i c h have the potent ia l to differentiate later in to neu rons . T h e

s tudy of cy togenes i s has b e e n greatly a ided in recent years b y the in t roduc t ion

and ex tens ive u se of 3 H - t h y m i d i n e au torad iography w h i c h al lows the visual-

iza t ion of n e w l y formed cells w h o s e D N A is tagged. Autorad iograph ic s tud ies

from several l abora tor ies (32 ,33 ,46) e s t ab l i shed that w h e r e a s n e u r o n s of the

deep cerebel lar nuc le i and the Purk in je cells o f the cerebel lar cortex are formed

prenatal ly in m i c e and rats , the baske t and stellate cells of the molecular layer

and the granule cells of the granular layer are formed postnatal ly . O u r s tud ies

have indica ted (1 ,2 ,4 ,17 ) that the first con t ingen t of different iat ing cells do no t

migra te (see be low) bu t sett le in the lower ha l f of the molecular layer w h e r e

1 3 5 7 9 11 13 15 1 7 1 9 2 1 2 3 2 5 2 7 3 0 9 0

AGE IN-DAYS ( B L O C K S )

FIGURE 1. A. Tracings of sagittal, matched sections of the cerebellum from rats of different ages, as indicated (d = days). Outer band, ex-

ternal germinal layer; black, granular layer; white above black, molecular layer; white below black, subcortical regions, including medullary

layer. B . Planimetric data of the area growth of the cerebellum in the sagittal plane shown, together with a laminar analysis. Each point rep-

resents means from several animals. Growth of external germinal layer not shown. Slightly modified from Altman ( 2 ) .

2. Interference w

ith C

erebellar Maturation

43

44 Joseph Altman

9

6

• PYRAMIS A GRANULE CELLS o "STELLATE" CELLS

X • "BASKET* CELLS

— a 1 . JL k Jt 6 h 2 d 6 d 13d 2 ld 3 0 d I 2 0 d

AGE AT INJECTION

FIGURE 2. Mean number of intensely labeled cells (cells that ceased to divide soon after the day

that the radiochemical was administered) in the granular and molecular layers of the pyramis in

120-day-old rats that were injected with 3 H-thymidine on the days indicated. Granule cells were

counted in areas 130 fxm2; the much less numerous cells of the molecular layer were counted in

strips 650 /Am long. Intensely labeled cells in the lower half of the molecular layer were designated

as basket cells; those in the upper half as stellate cells. The proportion of glia cells in the samples

is unknown. Slightly modified after Altman (2).

FIGURE 3. Summary of differential counts of labeled granule cells (intensely as well as lightly labeled) in the vermis of an adult rat injected with repeated doses of 3H-thymidine from day 11 to 16. The obtained percentages [for details see Figure 11a, in Altman (2)] indicate regional varia-tions in the proportion of granule cells acquired after 11 days of age. Light diagonal lines, early maturing regions; heavy diagonal lines, intermediate regions; black, late maturing regions. From Altman (2).

12 •

3 -

6

2 . Interference with Cerebellar Maturation 45

Ml)

I HR

ommomm • • o o o o

PM OOOO

o o o o

6 HRS

• o # o # # o o # # # o • o « o # o • • 0 « 0 « * 0 0 O O ^ O o o o «

24 HRS © © © © © © ©©o©o© © © © © © © © © © © © © < ^ o o o o o o ^ • o o o

3 DAYS © © o o © o o © o © © o ©©©©©© ©©©©©©

o o o o o o o o o o o o o o o o

FIGURE 4. Diagrammatic representation of the pattern of cell labeling and movement in the

cerebellar cortex of infant rats as revealed by autoradiography. The data relate to animals injected

with 3 H-thymidine on postnatal day 6 [see Altman (1)] and killed at intervals thereafter, as in-

dicated. In animals killed 1 hour after injection only the cells of the proliferative zone of the ex-

ternal germinal layer (MU) are labeled. In animals surviving for 6 hours, many of the premigratory

cells (PM) of the germinal layer have become labeled. By this time free 3 H-thymidine is no longer

available, indicating that the labeled cells in the premigratory zone are differentiating elements.

By 24 hours after injection there is considerable label dilution in the cell population of multiplying

cells and their derivatives, and some of the labeled cells are seen in the molecular layer as horizon-

tally or vertically oriented bipolar cells. By 3 days after injection, label dilution is extensive

throughout the external germinal layer. Some labeled basket cells are seen in the molecular layer

(ML) and some labeled granule cells have descended through the layer of Purkinje cells (PC) and

settled in the granular layer (GL). Modified after Altman (4).

they b e c o m e different iated as baske t cells toward the e n d of the first w e e k

(Figure 2 ) . T h e cells that are located in the uppe r ha l f of the molecu la r layer,

inc lud ing stellate cel ls , c o m e in to ex i s tence dur ing the s e c o n d w e e k . T h e

granule cells migra te to the granular layer and differentiate dur ing the s econd

and third w e e k , w i t h 25 to 8 0 % of t h e m b e i n g formed b e t w e e n 11 and 2 1 days

d e p e n d i n g on sys temat ic reg iona l var ia t ions (Figure 3 ) . T h u s , g ranule cells in

the dep th of ve rmal f issures different iate earl ier than in the sulci ; the ventra l

lobules ( l ingula and nodulus ) mature before the an ter ior l o b e ; the last m a -

tur ing vermal lobu les are the tuber , dec l ive , and cu lmen . T h e cerebel la r h e m i -

spheres , w i th s o m e excep t iona l reg ional var ia t ions ma tu re later than the

ve rmi s , and the paraf locculus is the last r eg ion to mature (2) . T h e majo r even t s

in the mul t ip l ica t ion and mig ra t ion of cells in the cerebel lar cor tex are s u m -

mar ized in F igure 4 .

Postnatal Cerebellar Morphogenesis

T h e term m o r p h o g e n e s i s in th is context refers to the in i t ia l s tep in neurona l

different ia t ion, n a m e l y , the acqu i s i t i on of specia l morpho log ica l p roper t i es b y

46 Joseph Altman

different c lasses of cells . For i n s t ance , o n e type of cell der ived from the ex-

ternal ge rmina l layer , the granule cell , deve lops a T - shaped axon. T h i s in -

volves the "ex t rus ion ' 7 of parallel f ibers in the coronal p lane and the mig ra t ion

of the soma (the axon b e i n g spun in the process) vert ical ly in to the granular

layer. A n o t h e r type of cell , the ba ske t cell , has i ts ou tg rowing dendr i tes

o r ien ted at a r ight angle to the parallel f ibers , and the s o m a sett les in the

molecu la r layer . T h i s s econd s tep in n e u r o g e n e s i s w a s s tud ied in a ser ies o f

inves t iga t ions w i th h i s tochemis t ry , au torad iography , and , in part icular , e lec-

tron mic roscopy .

T h e external ge rmina l layer is c o m p o s e d of two zones (Figure 4 ) . A n upper

zone c o m p o s e d of round i sh cells w i th f requent mi to t ic f igures that are labeled

direct ly b y 3 H - t h y m i d i n e is the prol i ferat ive zone . T h e under ly ing zone is

c o m p o s e d of sp ind le - shaped cells w h o s e long axis is o r ien ted hor izonta l ly in

the coronal p lane. T h e s e sp ind le - shaped cells rarely, if ever , have mi to t ic fig-

ures and are not l abe led unt i l several hours have e lapsed after in jec t ion (by

w h i c h t ime the in jec ted 3 H - t h y m i d i n e has b e e n excre ted) . O b s e r v a t i o n s w i t h

e lectron m i c r o s c o p y sugges ted that th i s is a p remigra to ry different iat ing z o n e

(4). Apparen t ly the hor izonta l b ipo la r cells " e x t r u d e " cy top lasm that b e c o m e s

the hor izonta l b r anch of the future parallel f ibers (Figure 5 ) . It i s p r e s u m e d

that w h e n the hor izon ta l f ibers reach the i r final l ength , the cell b o d y migra tes

d o w n w a r d th rough the molecu la r layer and eventual ly reaches the granular

layer w h e r e i ts d i f ferent ia t ion con t inues . A s the parallel f ibers are fo rmed the

undif ferent ia ted mat r ix is p u s h e d upward . T h u s the hor izonta l b r a n c h e s of

the parallel fibers are p rogress ive ly formed on the surface of o thers from the

bo t t om of the molecular layer upward b y a s tacking process . T h i s bu i l d ing

des ign m a k e s poss ib le the a s s e m b l y of a mat r ix of ve ry long , th in , and s t raight

threads of hor izonta l ly o r i en ted parallel f ibers w h i c h pi le up ver t ical ly ac-

cording to the i r t ime of or ig in . T h e vert ical b r anch of the parallel f iber is

<JiL>

9$ 9$ FIGURE 5 . Diagrammatic illustration of the transformation of multiplying round cells into spindle-shaped differentiating (premigratory) cells. It is postulated, on the basis of an increase in the proportion of thin processes from the top of the layer downward, that the spindle-shaped cell produces by extrusion the horizontal portion of the parallel fiber then, as described by Ramon y Cajal ( 5 6 ) , the cell dips downward and forms the vertical branch of the parallel fiber. This diagram also illustrates the principle of the progressive stacking of parallel fibers from the bottom of the molecular layer upward and the continued upward displacement thereby of the external germinal layer. From Altman ( 4 ) .


FIGURE 6. Illustration of the principle that the differentiating basket cells, whose dendrites are oriented at a right angle to the parallel fibers, are rendered immobile the moment they begin to differentiate. In contrast, the cell bodies of the differentiating granule cells can move downward between sheaves of parallel fibers. This migration is postulated to be guided by the processes of Bergmann glia cells that reach the surface of the cerebellar cortex (53). From Altman (4).

formed b y t ra i l ing. Re fe rence w a s m a d e ear l ier to the fact that the b a s k e t cells

do not migra te bu t different iate in situ. T h e dendr i t es o f the baske t cells are

o r ien ted sagi t tal ly at a r ight angle to the s tacks of parallel f ibers w h i c h renders

t h e m i m m o b i l e the m o m e n t they b e g i n to differentiate (Figure 6 ) .

Concur ren t ly w i th the m o r p h o g e n e t i c sculp tur ing that is in p rogress in the

external ge rmina l and molecu la r layers , the Purk in je cells also unde rgo

morphogene t i c changes (Figure 7 ) . Severa l phase s we re d i s t i ngu i shed in the

postnata l d i f ferent ia t ion of these prenata l ly fo rmed e l emen t s (5) . After the

Purkin je cells have b e c o m e d i spe r sed and a l igned in a mono laye r , each

develops a t rans ien t apical en l a rgemen t or cone filled w i t h " g r o w t h " cy-

top lasm that is part icular ly r ich in mi tochondr i a . In the lateral aspec t o f the

s o m a t rans ien t pe r i somat i c p rocesses are formed. In the next phase of devel-

o p m e n t the pe r i somat i c p rocesses d i sappear and the apical cy top lasm s t reams

upward in to the g r o w i n g apical dendr i t e that i nvades the format ive molecu la r

layer . T h e p r imary dendr i te forms secondary and then ter t iary b r a n c h e s , and

gradual ly sp ines are fo rmed w h i c h s u b s e q u e n t l y proliferate in great n u m b e r s .

B y th i s t i m e p e r m a n e n t synapses have appea red on the s o m a of t he Purk in je

cells and are also s een in inc reas ing n u m b e r s in the lower , or o lder part , of the

molecu la r layer.

Postnatal Cerebellar Synaptogenesis

The early synap togen ic matura t ion of the cerebe l la r cor tex has b e e n s tud ied

recently b y m a n y inves t iga tors ( a m o n g o thers , see 4 0 , 4 1 , 4 5 , 4 7 , 4 8 , 5 4 ) . In o n e of

our s tudies (3) w e no t i ced round ves ic les 4 0 0 - 1 4 0 0 A in d i ame te r and fur-

n i shed wi th an outer coat . W e s a w these in va r ious par ts of the cerebel lar

cortex in all y o u n g an ima l s , bu t in part icular ly large n u m b e r s in rats aged

2. Interference with Cerebellar Maturation 49

FIGURE 8 . A. High concentration of open coated vesicles (arrows) in the primary dendrite of a

Purkinje cell opposite parallel fibers (PF). B. Portion of the parallel fiber may be "sucked" into the

open coated vesicle (arrows). Both electron micrographs from the pyramis of a 12-day-old rat.

From Altman ( 5 ) .

1 0 - 1 5 days (3) . T h e s e ves ic les we re ident i f ied in c losed (spher ica l ) , o p e n

(f lasklike), and f lat tened forms. C losed coa ted ves ic les we re s een in large

n u m b e r s in the v i c in i ty of the Golg i appara tus and s o m e t i m e s in direct con t i -

nu i ty w i t h i ts c i s te rns . C losed , open , and flat coa ted ves ic les w e r e s een on the

surface o f cel ls , and in n e u r o n s these w e r e f requent ly oppos i t e cell p rocesses

wi th w h i c h synapses are fo rmed , such as i n the dendr i t es o f Purk in je cells op-

posi te parallel f ibers (Figure 8 ) . Of ten , these open coa ted ves ic les we re s i tua ted

FIGURE 7 . Photomicrographs of the cerebellar cortex from rats aged 0 , 5 , 7 , and 10 days. At birth the Purkinje cells are still scattered throughout the cortex, by day 5 they form a distinct mono-layer. In the 5 - and 10-day-old rats the apical cone of Purkinje cells is very pronounced, heralding the imminent outgrowth of the apical dendritic system which is evident by day 10. Semi-thin sec-tions embedded in Epon and cut in the sagittal plane. Stained with azure B ; oil immersion, X1600. Abbreviations: BAd, differentiating basket cells; BAm, mature basket cells; BG, Bergmann glia cell; BGp, Bergmann glia process; BP, bipolar cell in cross section; DGR, descending granule cell; GM, mitotic glia cell; GO, Golgi cell; GR, granule cell; MOL, molecular layer; MU, prolifer-ative zone of the external germinal layer; PM, premigratory zone of the external germinal layer; PU, Purkinje cells; PUL, Purkinje cell layer; d, day. Slightly modified after Altman ( 4 ) .

50 Joseph Altman

oppos i t e cell p rocesses w h i c h had dense m e m b r a n e s , and s o m e t i m e s they we re

con t inuous w i th the dense m e m b r a n e s of early a t t achment s i tes , or p r e sump-

t ive synapses . Occas iona l ly parts o f the parallel f iber actually pro t ruded in to

the cavi ty o f the Purk in je cell coated ves ic les sugges t ing an exchange of ma-

terial b e t w e e n prospec t ive pre- and pos t synapt ic e l emen t s . It w a s postula ted

that the coated ves ic les are invo lved in the format ion of early a t t achment s i tes

or the dense m e m b r a n e s of synapses , and the fol lowing s e q u e n c e of even t s

was sugges ted . T h r o u g h a b u d d i n g process spher ica l , coated ves ic les are

formed from the m e m b r a n e s of the c is terns of the Golg i appara tus . T h e s e spher-

ical ves ic les migra te to the cell surface and after a t t achment to the m e m b r a n e

open up and unfold over i ts surface. W h e n th is unfo ld ing b e c o m e s comple te the

flat dense m e m b r a n e s con t r ibu te mater ia l to or form the synapt ic m e m b r a n e s .

Coa ted ves ic les m a y also con t r ibu te to the expans ion of the dense m e m b r a n e

surface of already formed synapses .

Examina t i on of the e m e r g e n c e wi th age of mature type of synapses around

the somata of Purk in je cells and in the molecular layer (5) o f the pyramis

provided the fol lowing p ic ture o f the sequen t ia l s teps in cerebel lar synap-

togenes i s . Concur ren t ly w i t h the d i sappearance of pe r i somat ic p rocesses , the

per ikarya of Purk in je cells are su r rounded b y ident i f iable baske t cell synapses .

T h i s is p ronounced abou t 8 - 1 0 days after b i r th . A few days later parallel fibers

b e g i n to form synapses w i th ou tg rowing branch le t s of Purk in je cells in the

lower aspect of the molecu la r layer. Dur ing the next phase , w h i c h occurs after

the d i sappearance of the external ge rmina l layer abou t 20 days , parallel f ibers

es tab l i sh synapses wi th dendr i t ic sp ines in the upper molecular layer. T h e

" m a r c h " of synap togenes i s in the molecu la r layer from the b o t t o m upward is

charac ter ized b y three success ive even ts : an ini t ia l g radient in the appearance

and d i sappearance of coated ves ic les , hera ld ing synap togenes i s ; a s imi lar

s u b s e q u e n t t rend in the format ion of synapses ; and finally, the in te rpos i t ion

in the same s e q u e n c e of glial p rocesses b e t w e e n Purk in je cell dendr i tes and

parallel f ibers , ma rk ing the cessa t ion of synap togenes i s . A s u m m a r y of these

events is p re sen ted in F igure 9 .

T h e fo l lowing were the sa l ient even ts in the matura t ion of the granular layer

(6). A large propor t ion of the granule cells is formed dur ing the second week .

But due to the t ime requ i red for their migra t ion and the lag in the format ion

of dendr i tes , few glomerular synapses w i th m o s s y fibers are formed before the

b e g i n n i n g of the th i rd w e e k , and the process is still i n progress at 30 days ,

long after the d isso lu t ion of the external ge rmina l layer. T h e matura t ion of

Golgi cells is a prot racted process . Ev idence was ob t a ined that m o s s y fibers

form synapses w i th the dendr i tes o f Golg i cells and that Golg i cell axons

synapse w i th granule cell dendr i tes as soon as the g lomerul i b e g i n to mature .

T h e s e obse rva t ions sugges ted that synap togenes i s succeeds the m o r p h o g e -

net ic s tage of matura t ion and that different iat ing granule cells do no t form

synapses unt i l the i r axons are formed and thei r cell b o d i e s reach the i r final

des t ina t ion . H o w e v e r , in complex cel ls , as the Purk in je cell , the synapt ic mat -


FIGURE 9. Diagrammatic illustration of some major events in the maturation of a Purkinje cell and of the molecular layer. The width of the molecular layer (left abscissa) as a function of the animal's age (5 discrete columns) is based on measurements [see Figure 4, in Altman (4)]. Con-sidering the principle of the stacking of parallel fibers from the bottom upward, and disregarding the expansion produced by the invading dendritic processes during synaptogenesis, their location within the molecular layer indicates their "age" or time of origin (right abscissa). If the parallel fibers are traced through the columns from left to right it is seen that the fiber formed on day 7 has no synapses on that day but that it has synapses with basket cells on day 12, and increasing number of synapses from the fifteenth day onward with dendritic spines of Purkinje cells. Like-wise, the parallel fiber formed on day 12 has no synapses at that time but it has synapses with a stellate cell on day 15 and with Purkinje cell dendritic spines on day 21. The parallel fiber formed on day 15 has synapses with Purkinje spines on day 21 , but in the upper one-fourth of the molec-ular layer such synapses are still lacking at this age. The displacement and growth of a climbing fiber is indicated on the left side of the Purkinje cell. The upward "march" of glial sheathing of the Purkinje cell is also indicated. The cell width of the two zones of the external germinal layer [see Figure 1, in Altman (4)] is accurate but they are not drawn to scale. Slightly modified, after Altman (5).

u ra t ion of one r eg ion (the soma) m a y b e g i n before the m o r p h o g e n i c matura-

t ion of the ent i re cell ( the dendr i tes ) is comple ted .

T h i s ana lys i s of the course of pos tnata l cerebel la r n e u r o g e n e s i s served us

wi th normat ive data for the proper a s s e s s m e n t of the effects o f va r ious treat-

men t s that interfere w i t h cerebe l la r deve lopmen t , such as undernu t r i t ion , the

adminis t ra t ion of h o r m o n e s and drugs , and focal i r radia t ion w i t h low-dose

SH

3QU

13-n

Vd

vd

d

O

(39

V)N

I9ld

0d

0 3

WI1

WID

TH

OF

TH

E M

OL

EC

UL

AR

L

AY

ER

days

52 Joseph Altman

X-ray . T h e latter wil l b e desc r ibed first as it represents a se lect ive and graded

m e t h o d of des t roying n e w l y forming cells of the cerebel lar cor tex w i th m i n -

imal effects on o ther cell c o m p o n e n t s o f the ce rebe l lum and wi th little distur-

b a n c e of n e i g h b o r i n g s t ructures .

INTERFERENCE WITH POSTNATAL CEREBELLAR DEVELOPMENT BY FOCAL LOW LEVEL X-IRRADIATION

Hicks and h i s col laborator (35 ,36) have s tud ied for s o m e t ime the effects o f

low level X- i r rad ia t ion on the e m b r y o n i c and per ina ta l deve lopmen t of the

central ne rvous sys tem. T h e y found that a s ingle exposure of the w h o l e b o d y

of m i c e and rats to 200 R ki l led a large p ropor t ion of the mul t ip ly ing cells of

the deve lop ing bra in . T h i s se lect ive ac t ion of l ow level rad ia t ion , Hicks

sugges ted , should al low in ter ference w i t h the deve lopmen t o f a part icular

b ra in region b y appropr ia te t iming of the i r radia t ion.

FIGURE 10. Photomicrograph of a section through the cerebellum of a kitten whose skull was ir-radiated on the left side (L) with five repeated doses of 200 R. AN, ansiform lobule; R, right nonir-radiated side. Note absence of external germinal layer over entire left cerebellum. Arrows point to approximate midline. Magnification about X 6 . 6 ; cresyl violet. From Altman, Anderson, and Wright (13).


In a pilot s tudy (13) w e de t e rmined the op t ima l dose neces sa ry for the se lec-

t ive des t ruc t ion of the cells o f the external ge rmina l layer of the ce rebe l lum.

T h e heads of k i t t ens we re i r radia ted dur ing the first 2 w e e k s o f pos tna ta l life

w i t h five repea ted doses of X- ray wi th s ing le doses rang ing from 50 to 4 0 0 R in

different an ima l s . T h e effects on the i r radia ted and control s ides we re eval-

uated in the ans i fo rm lobule (Figure 10) . R e p e a t e d doses of 5 0 - 1 0 0 R reduced

the cell popula t ion of the external ge rmina l layer , 150 R p roduced a subtota l ,

and 200 R a total des t ruc t ion of the ent i re layer . It w a s conc luded , in agree-

m e n t w i t h H i c k s ' ear l ier resul ts , that 200 R is the op t ima l X - r a y dose for the

select ive des t ruc t ion of precursors of the postnata l ly fo rming granule cells in

the cerebel lar cor tex. T h e differential effects of s ingle and mul t ip le exposures

were e x a m i n e d in s u b s e q u e n t s tudies .

Cell Reduction Coupled with Structural Disorganization

T h e morpho log ica l c o n s e q u e n c e s of bi la tera l i r radia t ion of the ce rebe l lum in

n e w b o r n rats w e r e e x a m i n e d w i t h the n u m b e r of dai ly exposu res r ang ing from

1 to 10 days (14 ,15) . W i t h i n 24 hours after i r radia t ion a s ingle dose of 200 R

reduced drast ical ly the cell popula t ion of the external ge rmina l layer , and

max ima l (subtotal) des t ruc t ion could b e o b t a i n e d wi th an addi t iona l dose of

200 R (Figure 13 ) . H o w e v e r , if 4 or m o r e days e lapsed b e t w e e n the end of the

i r radia t ion sess ion and the ki l l ing of the an imal the external ge rmina l layer

b e g a n to regenera te (F igures 1 1 - 1 3 ) .

W i t h up to 5 success ive dai ly doses the speed of recovery of the external ger-

mina l layer w a s inverse ly related to the n u m b e r of doses rece ived (Figure 13) .

In all of these an imals the external ge rmina l layer w a s no rma l or supe rnormal

in w id th b y the ten th day after the last i r radia t ion sess ion . B u t p lan imet r ic

s tudies revea led that the total area occup ied b y this ge rmina l layer over the

decreased cerebel lar surface (or the total ge rmina l cell popu la t ion) w a s in-

verse ly related to the n u m b e r of doses rece ived . T h e regenera ted external ger-

mina l cells re ta ined the i r po tent ia l to differentiate bu t b e c a u s e fewer cells we re

avai lable as a funct ion of inc reased n u m b e r of t r ea tments , the result w a s a

graded re tardat ion in cerebe l la r g rowth (Figure 14) .

W h e n six or m o r e doses of 200 R were de l ivered dur ing in fancy the external

ge rmina l layer fai led to r egenera te , p r e s u m a b l y b e c a u s e the c o m b i n a t i o n of

p ro longed exposure and de layed recovery p reven ted its recovery w i t h i n the

per iod that it i s no rmal ly present (21 days after b i r th ) . T h e result of the ex-

t ended t rea tment w a s the format ion of a ce rebe l lum devo id of granule cells

and of the o ther pos tnata l ly fo rmed neurona l e l emen t s (F igure 15) .

L igh t and e lec t ron mic roscop ic e x a m i n a t i o n of the o rgan iza t ion of the de-

granulated ce rebe l lum (10,11) s h o w e d that the cell b o d i e s of Purk in je cells

were morpholog ica l ly no rma l (Figure 16) bu t that there were c h a n g e s in the i r

dendri t ic conf igura t ion and that the c i rcui t ry of the cerebe l la r cor tex w a s dras-

FIGURE 1 1 . Low power photomicrographs of sagittal sections of the cerebellum (about 9 0 0 /mm

from midline). A. Cerebellar vermis of a normal 4-day-old rat; B , of a 4-day-old rat irradiated with

2 0 0 R on days 0 , 1, 2 , and 3 and killed 2 4 hours later. Note the absence of an external germinal

c

B

A

54


tically al tered. T h e Purk in je cells had sprouted n u m e r o u s m a s s i v e dendr i tes

(Figure 17) that w e r e s tudded wi th thorns . Bu t the dendr i t es we re r andomly

o r i en ted in space , and thei r a rbor iza t ion was different from no rma l and

d e p e n d e d on the pos i t ion of the cell b o d y w i t h i n the cor tex. Pos t synap t i c

m e m b r a n e t h i cken ings were p r o m i n e n t , even in the a b s e n c e of a p p o s e d paral-

lel f ibers ( the axons of granule cel ls) . T h e s e obse rva t ions ind ica ted cons ide r -

able a u t o n o m y in the morpho log ica l deve lopmen t of Purk in je cells in the

cor tex devo id of baske t , stellate, and granule cells . H o w e v e r , the synapt ic dis-

o rgan iza t ion w a s profound. T h e m o s s y f ibers , w h i c h normal ly t e rmina te in

the granular layer and contact granule cel ls , pene t ra ted as far as the surface of

the cor tex and formed synapses w i t h the s o m a and dendr i t e s of Purk in je cells .

In the a b s e n c e of baske t cel ls , baske t l ike synapses we re fo rmed on the cell

b o d i e s of Purk in je cells b y te rmina ls that we re in te rpre ted to b e l o n g to the

recurrent axon collaterals of Purk in je cells (F igure 18) . Pos t synap t i c m e m b r a n e

th i cken ings on the dendr i tes and thorns of Purk in je cel ls , w h i c h we re s een to

form contac ts w i th c l i m b i n g fibers (which is no rma l ) , w e r e also a b u n d a n t op -

posi te o ther Purk in je dendr i t ic thorns , and even glial p rocesses (Figure 19) .

T h i s sugges ted that m a n y (probab ly the major i ty) of the " c o n t a c t s " fo rmed

were abno rma l o r abor t ive . T h e obse rva t ion of these m i s m a t c h e d c o n n e c t i o n s

m a d e us conc lude that the cellular depopu la t ion of the ce rebe l lum dur ing the

g rowth pe r iod resul ts in profound m o r p h o g e n e t i c and synap togene t i c disor-

gan iza t ion . S imi la r obse rva t ions were recent ly m a d e b y o thers in the degranu-

lated ce rebe l lum of mutan t m i c e (37 ,55) and in a ce rebe l lum degranula ted b y

per inata l vi ra l in fec t ion (34 ,43) or a drug (38) .

Structural Disorganization without Substantial Cell Reduction

M o r e a t ten t ion has b e e n pa id recent ly to the p h e n o m e n o n of nerve cell

r educ t ion as a c o n s e q u e n c e of in ter ference wi th b ra in g rowth (e .g . , 59 ,60 ) than

to m o r p h o g e n e t i c and synap togene t i c d i sorgan iza t ion . Bu t inappropr ia te con-

nec t ions fo rmed b y ex is t ing e l emen t s due to in ter ference w i th the no rma l t ime

course of n e u r o g e n e s i s m i g h t i t se l f lead to faulty neura l func t ion ing .

Even though the direct effect o f i r radia t ion of the deve lop ing ce rebe l lum is

layer excepting as a thin sheet over uvula; C, from a 10-day-old rat, irradiated as B. Note presence of a quasi-normal external germinal layer and developing molecular and granular layers caudally in uvula, to lesser extent in pyramis. More rostrally the external germinal layer is reconstituted excepting over centralis ventralis. There is no granular layer in the rostral lobules. Designation of vermian lobules, according to Larsell (41a): II, Centralis ventralis; III, centralis dorsalis; IV, culmen ventralis; V, culmen dorsalis; VI, declive; VII, tuber; VIII, pyramis; IX, uvula; X, nodulus. Large arrows point to fissura prima; egl, external germinal layer; IC, inferior colliculus; gl, granular layer; mo, molecular layer. Cresyl violet, X100. Slightly modified after Altman, An-derson, and Wright (15).

56 Joseph

Altm

an


6.0

— CONTROL - I x 2 0 0 R

— 2 x 2 0 0 R 3 x 2 0 0 R

— 4 x 2 0 0 R - — 5 x 2 0 0 R

4 6 AGE IN DAYS

8 10

FIGURE 13. Cell thickness of the external germinal layer in the lobulus centralis dorsalis as a func-

tion of number of daily exposures, age, and the survival period after the last irradiation session.

A, 2-hour survival (last experiment); • , 24-hour survival (last experiment); + , 4-day survival (last

experiment); O, 10 days of age. Slightly modified from Altman, Anderson, and Wright

(15).

the dec ima t ion of the precursors of its postnata l ly forming n e u r o n s , s o m e con-

s idera t ions sugges ted that th i s t e c h n i q u e m i g h t b e u s e d to p roduce m a x i m a l

structural d i so rgan iza t ion w i t h relat ively little cell loss . For in s t ance , in agree-

m e n t w i th ear l ier obse rva t ions (30 ,36) w e found (7) that in rats i r radia ted w i th

l imi ted doses of 150 or 200 R from days 3 to 6 onward (but at n o o the r t imes ) ,

FIGURE 12. High power photomicrographs of matched regions of the lobulus centralis ventralis from a normal rat 4 days of age (A) and from rats that were focally irradiated with successive doses of 200 R on days 0, 1, 2, 3, and 4 and were killed at different periods thereafter. B . Survival time, 2 hours after last irradiation session. Note absence of external germinal layer (eg); the ependymal wall of the recess of the fourth ventricle (ep) and the pia-arachroid membranes (pa) are not visibly affected. Arrow points to some surviving radioresistant elements. C. Survival time, 24 hours after last irradiation. Arrows indicate the random orientation of the apical cones of Purkinje cells suggesting that, unlike in normal animals, the Purkinje cells are randomly oriented in space. Note also that the distribution of Purkinje cells into a monolayer (PU) has not taken place. D. Survival time, 4 days after last irradiation session. Arrows point to reappearing clusters of cells repre-senting the regenerating external germinal layer. E . Survival time, 6 days after last irradiation (10 days of age). The regenerated external germinal layer forms a continuous sheet of cells. The layer is fragmented in appearance and the orientation of the cells of the external germinal layer is irreg-ular. F . The appearance of portion of the lobus centralis ventralis from a rat that survived to 30 days of age. The external germinal layer is no longer present at this age but there is a cell-sparse molecular layer (mo) and a granular layer (gl). The location of Purkinje cells in the granular layer is abnormal. Cresyl violet, X256. Slightly modified from Altman, Anderson, and Wright (16).

8 .0 -

CE

LL

T

HIC

KN

ES

S

b

O

1 1

~~~T

O k 2

30 d

8x200 R

,1mm,

FIGURE 1 4 . Tracings of midsagittal sections of the cerebellar vermis from 30-day-old control and irradiated rats. Compare with Figure 1 and note the graded reduction in cerebellar growth with increased successive daily exposures from birth on.

FIGURE 1 5 . Photomicrographs of the vermis (A) of a rat that received 8 doses of 1 5 0 - 2 0 0 R between days 0 and 1 3 . The cortex is composed essentially of the prenatally formed Purkinje cells with few postnatally forming elements being present. The cell bodies of Purkinje cells (B) look es-sentially normal in size or shape. From Altman and Anderson ( 1 0 ) .

CONTROL

5x200 R


FIGURE 16. The soma of a Purkinje cell from the degranulated pyramis of an irradiated rat. The asymmetrical accumulation of endoplasmic reticulum adjacent to the nucleus (upper left corner) is seen in the Purkinje cells of control animals, but is more prevalent in irradiated animals. From Altman and Anderson (10).

60 Joseph Altman

FIGURE 17. Camera lucida tracings of Purkinje cells impregnated with the Golgi technique. A. From the cerebellum of a normal adult rat (courtesy of Nancy Spivack). B . Purkinje cells at dif-ferent depth of the degranulated cerebellar cortex. Note the prevalence of massive primary and secondary dendrites with thorns, and the absence of spiny branchlets. The orientation of the dendrites is varied. Those near the surface are often deflected sideways or downward; those in the depth of the cortex may have dendrites penetrating the medullary layer (MED). The absence of tertiary dendrites is associated with the absence of parallel fibers. Modified after Altman and An-derson (10).

the cells of the regenera ted external ge rmina l layer fo rmed an " e c t o p i c " zone of granule cells in the molecular layer (Figure 20 ) . T h e morpho log ica l examina -t ion of the ce rebe l lum of these rats ind ica ted that the granule cells that we re arrested in the i r migra t ion th rough the molecular layer fo rmed synapses wi th m o s s y fibers that pene t ra ted into th is layer. T h e pos i t ion of the ectopic granule cell zone var ied in different lobules as a funct ion of the t ime of the c o m m e n c e m e n t of regenera t ion and the es t imated date of the descent of granule cells , and as a funct ion of reg ional differences in cort ical matura t ion and in the e s t ima ted upward g rowth of m o s s y f ibers. W i t h i n the same lobule , the longer regenera t ion w a s de layed the h i g h e r in the molecular layer the dif-ferent ia t ing and de scend ing granule cells we re " c a p t u r e d " b y the apparent ly au tonomous ly a scend ing m o s s y f ibers. If r egenera t ion started too early and a l lowed the descen t of granule cells in to the p re sumpt ive granular layer (early i r radia t ion) , or if r egenera t ion of g ranule cells w a s p reven ted a l together (late i r radia t ion) , an ec topic zone w a s no t formed. (This t e c h n i q u e w a s used for e s -t ima t ing the normal a u t o n o m o u s g rowth pat tern o f m o s s y fibers in the dif-ferent lobules . )

In the same and in s imi lar exper imen ta l mater ia l ano ther s tructural d isor-gan iza t ion was no ted (8) . In the molecu la r layer the parallel f ibers are normal ly or ien ted parallel to the long axis o f the fo l ium. Bu t in the regenera ted mo lecu -lar layer they t ended to b e o r ien ted r andomly in var ious p lanes . In the cere -

A B

FIGURE 18. A. A very long axon terminal (T) on the lateral aspect of the soma of a Purkinje cell (PC) with en passant synapses (arrows). This terminal has all the characteristics of a basket cell and is often seen in the degranulated cerebella that are largely devoid of basket and granule cells. (A few parallel fibers, PF, are occasionally seen.) B . A similar axon terminal (T) from another ir-radiated animal. But this terminal is clearly not that of a basket cell because it is myelinated. It is assumed to be the terminal of a Purkinje cell recurrent axon collateral. A, X 13,680; B , X 25,080. From Altman and Anderson (10,11).

61

FIGURE 19. A. Presumed mossy fiber terminal (MF?) with characteristic multiplying granular vesicles (MGV) making synaptic contact with the dendritic thorns (TH) of Purkinje cells. Post-synaptic membrane density is also seen where the dendritic thorn is contiguous with glial processes (GP). This is more clearly seen in B , where the glial process situated beneath the basal lamina (BL) probably represents the expansion of a Bergmann glia cell. PCD, Purkinje cell dendrite. In C the membrane density of the Purkinje cell dendritic thorn is contiguous with a fibrous astrocyte (FA). A, X 41,040; B and C, X 25,080. From Altman and Anderson (10).

62


FIGURE 2 0 . Anterior vermis of the cerebellum in a rat irradiated with 1 5 0 R on days 4 , 5 , 6 , and 7 and killed at day 3 0 . Note a wide ectopic zone of granule cells (arrow) dividing the molecular layer

into two halves. Hematoxylin-eosin, x 1 0 1 . From Altman ( 7 ) .

be l lum of s o m e cases , th is reor ien ta t ion w a s res t r ic ted to a s ingle lobule ,

f requent ly several lobules were affected, rarely the ent i re ve rmi s . Not in-

f requent ly the parallel f ibers we re o r ien ted in par ts or the ent i re molecu la r

layer at a r ight angle to the long axis of the fol ium (Figure 21 ) . In te res t ing ly ,

w h e r e parallel f ibers we re rota ted t ransverse ly , the axons of ba ske t cells and

the a rbor iz ing dendr i t es of Purk in je cells b e c a m e or ien ted longi tudina l ly . T h i s

and o the r cons ide ra t ions ind ica ted that parallel f ibers exer t a gu id ing influ-

ence on the pat tern of g rowth of the sp iny b ranch le t s of Purk in je cel ls .

T h e s e are examples of relat ively subt le morpho log ica l a l tera t ions in the

m o r p h o g e n e s i s and synap togenes i s of the cerebel la r cor tex w i t h u n k n o w n ,

poss ib ly sub t le , effects on cerebel lar func t ion ing . Bu t m o r e drast ic structural

a l terat ions can b e p roduced w h e n 2 - 3 success ive dai ly doses of 150 or 200 R

are de l ivered soon after b i r th . T h i s is the t ime w h e n the Purk in je cells b e c o m e

d i spersed in to a m o n o l a y e r and thei r dendr i tes b e c o m e o r i en ted radial ly

toward the surface of the cor tex. W i t h such an early i r radia t ion schedule s u b -

stant ial r egenera t ion can b e ach ieved , bu t the c rowded and d i sor ien ted Pur-

kin je cells never b e c o m e proper ly rea l igned . T h e drast ic a rch i tec tonic disor-

gan iza t ion p roduced resul ts (F igure 2 2 ) , as wil l b e desc r ibed later, in profound

cerebel lar mal func t ion ing . F igure 23 s u m m a r i z e s s o m e of the poss ib l e irradia-

FIGURE 21 . A. In two adjacent lobules the regenerated parallel fibers in the lower molecular layer are cut in cross section (which in sagittal sections is the normal longitudinal orientation) but in the upper molecular layer (arrows) they are cut parallel (which indicates rotation in the transverse direction). Bodian stain, X256. B . The orientation of parallel fibers in two directions is shown in this sagittally sectioned electron micrograph; R, rotated parallel fibers. X 13,680. From Altman (8).


FIGURE 22. A. Islands of the molecular layer embedded in the granular layer in the cerebellar cortex of 90-day-old rat that was irradiated with 5 successive doses of 200 R from birth on. Cresyl violet, X101. B. The disposition of Purkinje cells around such an abnormal island. X 2 5 6 . From Altman and Anderson (9).

t ion schedu les and the i r pos tu la ted deve lopmenta l effects, inc lud ing schedules w h i c h should produce (see be low) m i n i m a l structural d i so rgan iza t ion bu t m a x i m a l reduc t ion in the popu la t ion of granule cel ls .

Cell Reduction without Substantial Structural Disorganization

W i t h i r radia t ion started at b i r th (10) , i n d e e d even w h e n it i s s tarted at 4

days of age (11) , there is in ter ference w i t h the structural o rgan iza t ion of the

cerebel lar cor tex. T h i s w a s a t t r ibuted to the p reven t ion o f the d i spe r s ion and

a l i gnmen t o f Purk in je cel ls , the d i so r i en ta t ion of parallel f ibers , the penet ra -

t ion of m o s s y f ibers in to the molecu la r layer , and several o ther changes . In

v i e w of the fact that the format ion of granule cells does no t b e g i n in appre-

ciable n u m b e r s unt i l the s e c o n d w e e k and the i r bu lk is fo rmed dur ing the

1 D I S P E R S I O N OF

P - C E L L S

S T E L L A T E C E L L S

B A S K E T C E L L S

E A R L Y G R A N U L E C E L L S L A T E G R A N U L E C E L L S

1 • •

2 NO

N O N E

N O N E

N O N E N O N E

• • 1


P - C E L L S

N O N E

N O N E

N O N E NONE

1 • • 1


P - C E L L S

N O N E


N O N E


P - C E L L S

S T E L L A T E C E L L S


E A R L Y G R A N U L E C E L L S N O N E

6 NO S T E L L A T E C E L L S

N O N E

L A T E G R A N U L E C E L L S

I


P - C E L L S

N O N E B A S K E T C E L L S

L A T E G R A N U L E C E L L S

0 2 4 6 8 10 AGE IN

12 14 DAYS

16 18 20

FIGURE 2 3 . Diagram illustrating the postulated consequences of different schedules of X-ir-radiation on cell acquisition in the cerebellar cortex. Strips represent the cerebellar cortex; the thick black line the presence of an external germinal layer (original or regenerated); absence of black line the destruction of the external germinal layer. Notations within the strip indicate events in the development of the cerebellar cortex in normal (row 1 ) and irradiated cerebella (rows 2 - 7 ) with respect to age as indicated at the base of the diagram. Arrows above the strips show delivery of a dose of X-ray on the day indicated. Row 1. Sequential acquisition of different cell types in normal animals. Row 2 . Effects of repeated irradiations from birth on which destroy and prevent regeneration of the external germinal layer and produce a cortex which has no other elements but Purkinje cells that fail to disperse into a monolayer. Row 3 . Delaying the first irradiation to day 4 the Purkinje cells may become dispersed. [A recent study by Altman and Anderson ( 1 1 ) showed that this dispersion does not take place.] Rows 4 - 5 show schedules which allow the acquisition of the early forming cell types but prevent completion of cerebellar neurogenesis. Rows 6 - 7 show schedules in which only a few doses are delivered at selected intervals to permit regeneration of the external germinal layer and produce selective cell loss. Modified after Altman and Anderson ( 1 0 ) .


2 8 , 0 0 0

2 4 , 0 0 0

<s>

Zj 2 0 , 0 0 0 LU O ° 16,000

2 z 12 ,000

8 , 0 0 0

4 , 0 0 0

PYRAMIS

GRANULE CELLS

- 9 9 % -98V. - 9 2 % -75%

BASKET CELLS

H 0 0 % - 9 3 % - 3 0 % +12%

O d 4 d 8 d I2d DAY

Co WHEN

Od 4 d 8 d I2d Co IRRADIATION STARTED

2 2 0

180

O

4*

O

O

sii

3o

d

o uaaw

riN

6 0

2 0

FIGURE 24. Mean number of granule and basket cells in the pyramis in groups of rats in which ir-radiation was started on days 0, 4, 8, and 12 with a schedule that prevents regeneration (Co, con-trol). Note that basket cell reduction was obtained only when irradiation began on or before day 8. The reduction in granule cells was substantial even when irradiation was started on day 12. J . Altman and W. J . Anderson, unpublished results.

FIGURE 25. Purkinje cell dendritic arbors assuming the form of weeping willows in the cerebellar cortex of rats irradiated from day 8 onward. This irradiation schedule prevents the formation of the late forming granule cells which results in a truncated molecular layer devoid of its upper field of parallel fibers. Apparently the spiny branchlets of Purkinje cells turn downward and become contiguous with the early forming parallel fibers of the lower molecular layer. J. Altman, unpublished observations.

68 Joseph Altman

th i rd , it should b e poss ib l e to ob ta in a normal ly o rgan ized ce rebe l lum bu t

wi th a great ly reduced granule cell popula t ion b y s tar t ing i r radia t ion toward

the e n d of the second w e e k . B y con t inu ing the i r radia t ion for several days

thereafter to p reven t the mal format ion that is p roduced b y the regenera t ing

cel ls , the resul t should b e a normal ly o rgan ized bu t min ia tu re ce rebe l lum. F ig -

ure 24 s u m m a r i z e s the effects of p ro longed i r radia t ion star ted on days 0 , 4 , 8,

and 12 on the n u m b e r of baske t cells and granule cells in the py ramis of the

cerebel lar ve rmis . W i t h i r radia t ion star ted on day 8 there is a 9 2 % loss in

granule cells and a 3 0 % loss in ba ske t cells; w i th i r radia t ion started on day 12

there is no baske t cell loss in the molecu la r layer bu t there is a 7 5 % loss in

granule cells . S i n c e the py rami s ma tu res earl ier than m o s t of the lobu les of the

h e m i s p h e r e the total reduc t ion in cerebel lar granule cells m a y b e greater .

In an imals i r radia ted at 8 days the ce rebe l lum looks gross ly normal ,

bu t Golg i s tudies i nd ica t ed that the Purkin je cell dendr i t es , u p o n reach ing

the borde r of the arres ted molecu la r layer (where on ly the earl iest fo rming

parallel fibers are p resen t ) , turn backward and acqu i re a " w e e p i n g w i l l o w "

shape (Figure 2 5 ) . In those i r radia ted from 12 days onward , the ce rebe l lum ap-

pears morphologica l ly normal though min ia tu re in s ize .

T h e two types of ce rebe l lum produced , one of nea r no rmal s ize bu t disor-

gan ized and the o ther min i a tu re wi th essent ia l ly normal o rgan iza t ion , offer a

u n i q u e oppor tun i ty for the examina t ion of the differential s ignif icance of

normal cell n u m b e r and normal a rch i tec tonics in the func t ion ing of a b ra in

structure.

POSTNATAL DEVELOPMENT OF LOCOMOTION IN RATS

M a n y inves t iga tors have c o m m e n t e d on the co inc idence b e t w e e n the matu-

ra t ion of the cerebel la r cor tex in rats dur ing infancy and the acqu i s i t ion of

locomotor skil ls . T h e n e w b o r n rat has great difficulty in ambu la t ing over the

shor tes t d i s tance and is carr ied b y i ts m o t h e r to the nu r s ing si te . Bu t b y 21

days ( w h e n cerebel lar cy togenes i s has c o m e to an end) it can run and c l imb ef-

ficiently and has b e c o m e self-sufficient to the extent that it can b e w e a n e d .

Because of the k n o w n invo lvemen t of the ce rebe l lum in postural and m o t o r

coord ina t ion , w e unde r took to e x a m i n e in detai l the phase s in the deve lop-

m e n t o f l ocomot ion and re la ted skills in an a t tempt to correlate these , i f pos -

s ib le , w i t h s tages of cerebel lar deve lopment . Moreove r , these normat ive data,

it was felt, w o u l d a id us in a s sess ing the deficits p roduced in mo to r deve lop-

men t b y t rea tments that affect cerebel lar deve lopmen t , such as focal i r radia-

t ion of the ce rebe l lum, undernu t r i t ion , and h o r m o n a l t rea tments .

In an ex tens ive s tudy, the locomoto r act ivi ty of over 1200 rats, aged 1-21

days , was e i ther obse rved in the open field or tes ted exper imenta l ly b y u s ing

h o m i n g as a mo t iva t i on to i nduce l ocomot ion unde r different c i r cums tances

(19) . T h e " o p e n f ie ld" was an enc losed w o o d e n surface w h i c h w a s s u b d i v i d e d


in to smal l squa res for scor ing pu rposes . It w a s u sed to gauge the " s p o n t a n e -

o u s " e m e r g e n c e of different mo to r skills at dai ly in tervals over a s tandard ized

obse rva t ion pe r iod . In the exper imenta l s i tua t ion the pup w a s r e m o v e d from

its h o m e cage or w a s separa ted from its l i t te rmates and w a s cal led u p o n to

t raverse different pa thways and thus d isp lay i ts deve lop ing skil ls .

T h e ear ly ma tu ra t ion o f the infant rat 's t e n d e n c y to or ien t toward i ts

h o m e cage is s h o w n in F igure 2 6 . Ra t pups w e r e p laced dai ly from 1 day of age

on in to a circular f enced- in area w i t h the h o m e cage on o n e s ide and an e m p t y

control cage on the other . T h e an imal ' s o r ien ta t ion w a s scored over a 3 -minu te

per iod . It can b e seen from the resul ts that a tu rn ing t endency toward the

h o m e cage w a s ev iden t b y day 3 , and b y day 7 the major i ty of the an imals

tu rned toward the h o m e cage mos t of the t ime . H o w e v e r , if in s t ead of mere ly

tu rn ing t h e y w e r e expec ted to m o v e a shor t d i s t ance ove r a flat surface, no t a

s ingle an ima l 7 days of age succeeded in r each ing h o m e in the allotted t i m e

and on ly b y days 1 4 - 1 6 we re all an imals successful . T h e s e resul ts e s t ab l i shed

the early ma tu ra t ion of the t e n d e n c y to or ien t h o m e w a r d , w h i c h w a s overt ly

expressed due to the t endency of infant rats to " p i v o t . " Bu t they also s h o w e d

the re la t ively late ma tura t ion of ambu la to ry skil ls .

P ivo t ing (Figure 27) is the resul t of the earl iest l ocomoto r a t tempt . T h e

fo re l imbs m o v e separa te ly (as i n " p u n t i n g " ) o r toge ther (as in " t r e a d i n g " ; 19 ) ,

bu t m i n i m a l suppor t is p rov ided b y the h i n d l i m b s , and b e c a u s e the pe lv is

r e m a i n s anchored to the g round the fore l imb act ivi ty p roduces a circular

m o v e m e n t w i th little forward p ropu l s ion . M a x i m a l p ivo t ing is s een in the

open field b y day 7 (Figure 27) w h i c h then dec l ines as the an ima l b e g i n s to

crawl o r c reep . T h e lat ter resul ts f rom the coord ina ted ac t iv i ty o f the four ex-

t remi t ies w h i c h , h o w e v e r , are not able to suppor t the w e i g h t of the b o d y (ex-

cept for a few seconds ) unt i l abou t 1 1 - 1 2 days . T h e ra i sed q u a d r u p e d pos ture

b e c o m e s the p r e d o m i n a n t style of l ocomot ion at 1 2 - 1 3 days . T h e an ima l as yet

does not m o v e swift ly, the h i n d l i m b s often slip or are dragged. B y days 1 4 - 1 6

(by w h i c h t i m e the eyes have o p e n e d ) the an imal walks normal ly , t h o u g h the

expe r i enced eye m a y detect an exaggera ted lift ing of the h i n d l i m b s . T h e

e m e r g e n c e o f t rue wa lk ing b y the e n d o f the s econd w e e k m a y b e o n e o f the

factors in the sudden r ise in c ross ings in the o p e n field (Figure 28) and that all

the an imals reach h o m e w i t h i n the al lotted t ime (Figure 2 6 ) .

W h e n the rat acqu i r ed the capac i ty to wa lk over a w i d e pa th w i t h good trac-

t ion it m a y still lack the ba l anc ing skills r equ i r ed for nego t i a t ing a na r row

path . T h u s m a n y an ima l s could reach the i r l i t t e rmates b y day 15 over a 3 - c m

w i d e path; h o w e v e r , n o n e could do that at th is age , w h e n the pa th w a s on ly

0.5 c m w i d e (F igure 2 9 ) . Q u i t e late in d e v e l o p m e n t was the spon t aneous

e m e r g e n c e of rea r ing in the o p e n field (Figure 30) and the abi l i ty of c l i m b i n g

up on ropes or rods to a platform to avoid i m m e r s i o n in wate r (Figure 31) or

descend on s a m e to reach the g round (Figure 3 2 ) . T h e latter w a s par t icular ly

ins t ruct ive as it p rov ided an oppor tun i ty to analyze the e m e r g e n c e of the coor-

d ina t ion o f the fore- and h i n d l i m b s in the spec ies -spec i f ic m o d e of descen t in

70 Joseph Altman

3 5 7 9

AGE IN DAYS

FIGURE 26. A. Orientation of rats in a circular fenced-in area situated between an empty cage and the home cage. The direction of the head was tallied every 10 seconds over a 180-second period. B . Percentage of rats homing successfully when there is access to both the home cage and an empty cage. Estimate of speed of ambulation based on results from group of animals during the develop-mental period when all animals homed successfully. From Altman and Sudarshan, in press. Note that by day 7 the majority of the animals turned toward the home cage (in A) but none of the animals acquired the capacity to reach the home cage in the allotted time (in B ) .

ME

AN

O

RIE

NTA

TIO

N

SCO

RE


3 5 7 9 II 13 15 17 19 21

AGE IN DAYS

FIGURE 27. Mean percentage of time spent in pivoting (either "punting" or "treading"). The

decline in pivoting after day 7 is associated with the emergence of coordinated quadruped

progression with the trunk raised from the ground. Inset: tracings of three frames from a motion

picture showing the major events during "punting". A. The head is turned to the right. B . The

right arm is pulled out from under the head and placed to the right. C. The shoulder is pushed to

the right by the punting action of the left arm. From Altman and Sudarshan (19).

# 7 0 • SQUARES TRAVERSED

o o ROUGH SURFACE N = I 6

/ \ WALKING o

5 0 • a o SMOOTH SURFACE N = 4 8

3 0

C R A W L I N G ]

10

PIVOTING /

1 3 5 7 9 II 13

AGE IN DAYS

15 17 19 21

FIGURE 28. Mean number of squares (10 x 10 cm) traversed in an open field (50 x 50 cm). On a

rough (plywood) surface three stages are indicated (broken lines) which are associated with

pivoting, crawling, and walking (or running) as successive phases in the development of locomo-

tion. Exploratory activity is suppressed on a smooth (lucite) surface. From Altman and Sudarshan

(19).

PIVOTING

N-24

% 6 0 -

72 Joseph Altman

% T R A V E R S I N G N A R R O W P A T H S : WIDTH VARIED

3 5 7 9 II 13 15 17 19 21 AGE IN DAYS

FIGURE 29. Percentage of falls and percentage of animals successful in crossing a path leading to

a platform holding littermates. Path length, 60 cm; width 3.0 or 0.5 cm. Inset shows mean number

of fore- and hindlimb slips for the group traversing the 3.0-cm path. Slightly modified from

Altman and Sudarshan (19).

AGE IN DAYS

FIGURE 30. Mean frequency of rearing in the open field, with or without forelimb support. From

Altman and Sudarshan (19).


I 3 5 7 9 II 13 15 17 19 21 AGE IN DAYS

FIGURE 31 . Mean latency and percentage of animals successfully climbing a rod or a rope to avoid

fall into cold (15°C) water. From Altman and Sudarshan (19).

rats. Ini t ia l ly the an ima l s ju s t fell off, then sl id off w i t h the i r head in an

upward pos i t i on , and no t unt i l the e n d of the th i rd w e e k did the skill ma ture

of tu rn ing a round the rope to descend h e a d forward (Figure 3 2 B ) . T h e sys t em-

atic e m e r g e n c e o f va r ious m o t o r skills in the infant rat i s s u m m a r i z e d in F ig -

ure 3 3 .

EFFECTS OF FOCAL CEREBELLAR X-IRRADIATION ON LOCOMOTOR DEVELOPMENT

If cerebel lar ma tu ra t ion and the deve lopmen t of l ocomoto r skills are not

mere ly parallel even t s bu t are causal ly l inked p h e n o m e n a , t hen i r radia t ion

that leads to re tardat ion of cerebel lar g rowth should also resul t in deficits in

mo to r deve lopmen t . A n early ser ies of s tud ies (57,58) s h o w e d that focal ir-

radia t ion w i t h 8 - 1 0 doses of 200 R from b i r th (wh ich resul ts in cellular dec ima-

t ion coupled w i t h structural d i so rgan iza t ion) p roduced las t ing ataxia and

t remor (Figure 3 4 ) , the k ind that is s een fo l lowing ex tens ive cerebel la r abla-

t ion. Exposure to 3 doses or m o r e led to reduc t ion of spon taneous r u n n i n g in

an act ivi ty w h e e l (Figure 35) even t hough " g e n e r a l " or " r a n d o m " act iv i ty w a s

e levated , and as few as 2 doses (wh ich t end to p roduce structural d i sorgan-

iza t ion w i t h little cell loss) in terfered w i t h the speed of c l i m b i n g up on a rope

to avoid p u n i s h m e n t (Figure 36 ) .

S imi la r resul ts we re ob t a ined in a s u b s e q u e n t s tudy (12) in w h i c h 2 - 1 0

doses of 150 R were de l ivered from b i r th onward . Gene ra l and r a n d o m moto r

act ivi ty , as a s sessed b y the f requency of t ime spen t in ambu la t i on , w a s h ighe r

74 Joseph Altman

% 100

8 0

6 0

4 0

2 0

DESCENDING ON ROPE AND ROD (N=I6)

FALLS HEAD UP HEAD DOWN

• ROPE • ROD

7 100

8 0

6 0

4 0

2 0

12 15 12 15 18 AGE IN DAYS

12 15 18 21

FIGURE 32. A. Percentage of falls (a), sliding down with head pointing upward (b), and turning around and descending with head in a leading position (c) on a rope or rod (rope provides better traction). B . Sequence of movements (1-4) when a rat turns around to descend on a rope. From Altman and Sudarshan (19).

i n m o s t o f the i r radia ted an ima l s than in the controls for some t i m e after the

i r radia t ions . But the effective l ocomot ion , j u d g e d b y the n u m b e r o f squa res

t raversed b y crawl ing or wa lk ing , w a s no t h ighe r in the i r radia ted an ima l s ,

i n d e e d it w a s reduced in the h igh i r radia t ion groups (Figure 37 ) . T h i s

sugges t ed that cerebel lar i r radia t ion d id no t necessar i ly d i m i n i s h read iness for

b c


ambu la t ion bu t that it in ter fered , at least in the m o r e severe ly re tarded an-

imals , w i t h its e f fec t iveness . M a r k e d deficits w e r e seen in ma tu r ing i r radiated

an imals in a b a n d o n i n g such infant i le pa t terns o f l ocomot ion as p ivo t ing (Fig-

ure 38) and descend ing on a rope w i t h head upward (Figure 39) and in the

display of deve lop ing mo to r pat terns (Figure 38 and 39 ) . In s o m e of these

RIGHTING ON SURFACE

NEGATIVE GEOTAXIS ( I5° !NCLINE)

NEGATIVE GEOTAXIS ( 2 5 ° INCLINE)

PIVOTING

ORIENTATION WITH PIVOTING

CLIFF AVOIDANCE

PLACING (VIBRISSAE ELICITED)

PLACING (CHIN ELICITED)

ELEVATION OF SHOULDER

HOMING CON L E V E L SURFACE)

ELEVATION OF HEAD

QUADRUPED BALANCING (HIND LIMB)

ASCENDING ON WIRE MESH

HEAD POINTING (ROUGH SURFACE)

HEAD POINTING (SMOOTH SURFACE)

HINDLIMB SUPPORT (SUSPENDED)

ASCENDING ON LADDER

TRAVERSING 3 Cm PATH

RIGHTING IN MIDAIR

TRAVERSING 1.2cm PATH

DESCENDING ON WIRE MESH

ASCENDING ON ROPE

ASCENDING ON ROD

DESCENDING ON LADDER

REARING (WITHOUT SUPPORT)

DESCENDING ON ROPE (HEAD DOWN)

JUMPING (ACROSS CLIFF) j ~ J

1 3 5 7 9 i f 13 15 17 19 21

AGE IN DAYS

FIGURE 33. Summary diagram of the emergence of different postural, locomotor, and related

skills. In the majority of instances performance level (on the abscissa of the strips) refers to percent-

age of animals (0, 25, 50, 75, and 100) successfully displaying the motor act indicated. In some in-

stances the reference is to the development of asymptotic response frequency. From Altman and

Sudarshan (19).

100

TREMOR-"HIGH*

• CONTROL • I x A 2x • 3x

1-5 6-10 11-15 16-20 21-25 2 6 - 3 0

DAYS AFTER LAST IRRADIATION SESSION

FIGURE 3 4 . Percentage of animals showing high degree of intention tremor. After Wallace and Altman ( 5 7 ) .

750

6 6 0

570

480

> 390

>-, 3 0 0

210

120

30

CO CO CO

>: >- >- >-< < < < Q Q Q o

O O O o m O m m

CONTROL 1x200 3 x 2 0 0 5 x 2 0 0 8 x 2 0 0 1 0 x 2 0 0

TREATMENT

FIGURE 3 5 . Median daily revolution in activity wheels as a function of number of radiation doses received during infancy and age of animals. After Wallace and Altman ( 5 8 ) .

s e c .

110

8 0

50

20

A. RUNNING TIME • l O O g m

• 200 g m

• 3 0 0 g m

B. CLIMBING TIME

CONTROL 1x200 2 x 2 0 0 3 x 2 0 0 1x200 2x200 3x200 4 x 2 0 0 CO.

TREATMENT

FIGURE 3 6 . A. Running time in a straight alley where harnessed adult rats were pulling weights for food reward at the end of the alley. B. Climbing time of adult rats on ropes of different width to a platform to escape shock. Note the minimal effect of 1-A X 2 0 0 R X-irradiation on weight pulling and the graded effect on climbing latency. After Wallace and Altman ( 5 8 ) .

ME

DIA

N

DA

ILY

R

EV

OL

UT

ION

S

IUU

UAT j

|

150

DA

YS

~[~

50

D

AY

S

[

100

D

AY

S

1

| 15

0

DA

YS

50

D

AY

S]

1 10

0

DA

YS

1 15

0

DA

YS

| 5

0

DA

YS

1 10

0

DA

YS

I 15

0

DA

YS

~]

50

D

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S

1 10

0

DA

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| 15

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s e c .

2nd R o p e

3 r d R o p e

to _J < 1 <

o I-2 UJ o cr Ld CL

+ 4x A 5x • 8x o 10 x


CRAWLING AND WALKING

1

8 0

70

6 0

5 0

4 0

3 0

2 0

10

"EFFECTIVE" LOCOMOTION

<N = 4 2 )

CONTROL - o 2 X150 R

R R R R

o o 4 X 1 5 0 • • 6X150 A- -a 8 X 1 5 0 + + 10X150

11-14 15-18 - L .

2 3 - 2 6

B - L .

2 7 - 3 0 19-22

AGE IN DAYS (BLOCKS)

FIGURE 37. A. Time spent in the open field in crawling and walking (interpreted as random locomotion). B. Number of squares traversed in the open field (interpreted as effective locomo-tion). Note the increase in the random activity level of the irradiated rats, combined in the 8-10 x 200 R groups with a reduction in the number of squares crossed. After Altman, Anderson, and Strop (12).

motor tasks on ly the h igh i r radia t ion groups we re affected ( those in w h i c h

there was cerebel lar cell r educ t ion coup led w i t h structural d i so rgan iza t ion) ; in

o thers the in t e rmed ia t e group w a s also affected; and on ly in o n e of the tes ts

( rear ing) we re also the l o w i r radia t ion an ima l s h a n d i c a p p e d , the an ima l s in

w h i c h s o m e structural d i so rgan iza t ion w a s p resen t bu t m i n i m a l cell loss . G e n -

eral obse rva t ion sugges ted that o n e of the m o s t p r o n o u n c e d deficits in the ir-

radia ted an imals w a s the incoord ina t ion of the h i n d l i m b s .

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FIGURE 3 8 . A. Persistence of the infantile trait of pivoting in the open field in normal and irradiated rats. B . Frequency of rearing without

forelimb support. C. Incidence of falling during locomotion. After Altman, Anderson, and Strop ( 1 2 ) .

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FIGURE 39. Mean frequency of falls (A), sliding down with head pointing upward (B), and turning around and descending with head in a leading position (C) when placed on a vertical rope. Note the longer persistence of falls and sliding in the groups that received 6-10 X 200 R and the slower acquisition of the mature descending posture. After Altman, Anderson, and Strop (12).

2. Interference w

ith Cerebellar M

aturation 79

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o u. z <t

UJ S

FIGURE 40. Low power photomicrographs of midsagittal cerebellar sections from 60-day-old rats that received 4 doses of X-ray (A), 2 doses (B), and of a control rat (C). Apart from the greater reduction in cerebellar growth there is evidence of laminar disorganization (double arrows) and ectopia (single arrows) in A; in B there is only mild ectopia (single arrows) present. From Brunner and Altman (24).

80

A

C

2. Interference with Cerebellar Maturation 8 1

W h i l e these s tud ies e s t ab l i shed a causal l ink b e t w e e n the d i so rgan iza t ion

and re tardat ion of cerebel lar deve lopmen t , on the o n e h a n d , and the matura-

t ion of m o t o r ski l ls , on the o ther , the exact role o f the ce rebe l lum w a s not

clearly ind ica ted . In a n e w ser ies of s tud ies , the first of w h i c h w a s recent ly

p u b l i s h e d (24) , we e x a m i n e d the locomoto r capaci ty of adult rats that had

rece ived e i ther two (2 X r) or four doses (4 X r) of cerebel lar X - r a y from day 4

on. T h e s e schedu les p roduced , respec t ive ly , modera te or drast ic in ter ference

w i th cerebel la r deve lopmen t (Figure 40) w i th an e s t ima ted 49 and 7 2 % reduc-

t ion in granule cells in the ve rmis . T h r e e c lasses of m o t o r funct ions were

e x a m i n e d in these rats w h e n they b e c a m e mature : va r ious forms of l ocomo-

t ion ( s w i m m i n g , r u n n i n g on a t readmil l , and t ravers ing ro ta t ing rods) , one

type of j u m p i n g , and two types of c l imb ing . B o t h i r radia ted g roups equa led

controls in s w i m m i n g speed . S w i m m i n g , l ike r u n n i n g , r equ i re s the coordi -

na ted m o v e m e n t of fo re l imbs , h i n d l i m b s , h e a d , and tail for effective forward

propuls ion . It differs from locomot ion on land in that the ant igravi ty reac t ions

are essen t ia l ly s u s p e n d e d because rats t end to float in water . M o r e o v e r , b e -

cause of the re la t ively s table h y d r o d y n a m i c cond i t ions on the surface of calm

water , the n e e d to ut i l ize ex terocept ive and propr iocep t ive f eedback cues for

adjus t ing the e n d o g e n o u s rhy thmic i ty o f s w i m m i n g m o v e m e n t s to c h a n g i n g

external cond i t i ons m a y b e m i n i m a l . W e conc luded tenta t ively , in l ine w i th an

ear l ier obse rva t ion in decerebel la te dogs (44) , that no rma l cerebel lar funct ion-

ing is not essent ia l for coord ina ted s w i m m i n g on the surface of water . F r o m th is

the in ference fol lows that a pat tern of rhy thmic p rogress ion w h i c h requ i res

m i n i m a l concur ren t postural ad jus tments and ex terocept ive and p ropr iocep-

t ive f eedback m a y b e execu ted successful ly b y an an imal w i th an underde -

ve loped ce rebe l lum.

T h e task of r unn ing on a t readmil l on a w i d e and level surface w i t h good

T a b l e I E x t e n d e d T r e a d m i l l R u n n i n g Tes t s

Control 2 X r 4 X r n = 18 n = 18 n = 18

Experiment 2A (1000 ft) Horizontal best speed (ft/min) 286 272 256" Uphill best speed (ft/min) 170 159 122" Downhill best speed (ft/min) 353 364 275" Distance (feet) endurance 5806 9389 5284

Control 2 X r 4 X r

n = 12 n = 12 n = 6

Experiment 2B (500 ft)

Horizontal best speed (ft/min) 271 273 2i7«

" Refers to significance (t test) < 0.05.

82 Joseph Altman

t ract ion (Table I) invo lves an addi t iona l per formance var iab le , namely , the ac-

t iva t ion of ant igravi ty r e sponses to sus ta in the quadruped s tance dur ing

ambula t ion . T h i s did no t hand icap the 2 X r group e i ther in te rms of r unn ing

speed or endurance . Even w h e n ano ther per formance var iab le w a s added,

name ly , r unn ing on inc l ines , the per formance of 2 X r group w a s no t affected.

T h e 4 X r group was h a n d i c a p p e d min ima l ly on the level r u n n i n g task, m o d -

erately on uphi l l and downhi l l max ima l r unn ing speeds . T h e s e expe r imen t s

sugges ted that the postural and feedback r equ i r emen t s in a s imple runn ing

s i tua t ion place m i n i m a l d e m a n d s on cerebel lar con t r ibu t ion and , therefore,

modera te cerebel lar re tardat ion has no effect on runn ing per formance , and

severe re tardat ion has only a s l ight effect.

Accord ing ly , in the next ser ies of tests (Table I I ) , the d e m a n d s for sensory

and postural control we re progress ive ly inc reased and the deficits of the 4 X r

an imals and then also of the 2 x r g roup b e c a m e apparent . Ra t s were requ i red

to cross rods of different w id th , texture, and speed of ro ta t ion to reach a goal

b o x for food reward . O n the rough-sur faced , s ta t ionary rods there were e s sen -

tially no falls in any of the exper imen ta l groups at any of the wid ths tes ted ,

and only in te rms of c ross ing speed (Figure 41) did the hand icaps of the 4 X r

group and then of the 2 X r group b e c o m e evident . T h e c o m b i n a t i o n of

reduced wid th and s l ippery surface led to occas iona l falls and disqual i f ica t ions

in the 2 X r an imals , to m o r e in the 4 X r g roup . O n the nar rowes t s l ippery

rod the 2 X r group w a s as bad ly affected as the 4 x r an imals in te rms of

speed of c ross ing . B e c a u s e of the poor per formance of b o t h exper imenta l

g roups on the nar rowes t s l ippery rod wh i l e s ta t ionary , ro ta t ion w a s not at-

t empted .

In the o ther exper imenta l s i tua t ions , such as j u m p i n g and c l i m b i n g , we did

not use e n o u g h var iab les to a t tempt th is type of an analys is . In general ,

Table II Proportion of Falls and Disqualifications When Crossing on Rods of Different Texture, Width, and Speed of Rotation

Falls (%) Disqualified (%)

Texture Width

(inches) Rotation (ft/min) Co 2 X r 4 X r Co 2 X r 4 x r

Rough 4.25 Stationary 0 0 0 0 0 0

Rough 4.25 7.5 0 0 0 0 0 0 Rough 4.25 18.5 0 8 17 0 0 0 Rough 1.25 Stationary 0 0 0 0 0 0

Rough 1.25 7.5 0 0 0 0 0 0

Smooth 1.25 Stationary 14 16 39 0 10 40 Smooth 1.25 7.5 1 7 33 0 10 40 Rough 0.87 Stationary 0 0 4 0 0 0

Smooth 0.87 Stationary 5 7 20 0 10 40

4.25" Dia. 1.87" Dia. 1.25" Dia. 1.25" Dia. .87" Dia. .87" Dia. 4.25" Dia. 4.25" Dia. 4.25" Dia. 1.25" Dia. 1.25" Dia.

ROUGH ROUGH ROUGH SMOOTH ROUGH SMOOTH 7.5'/min. 18.5'/»in. 28.4'/min. 7.5'/»in. 7.5'/»in.

ROUGH ROUGH ROUGH ROUGH SMOOTH

FIGURE 41 . Average rod crossing latency for the control group (unshaded), group 2 x r (stippled), and group 4 X r (shaded). Texture and

width of the rods and rotational speeds are indicated. From Brunner and Altman (24).

2. Interference w

ith C

erebellar Maturation

83

LA

TE

NC

Y

84 Joseph Altman

j u m p i n g , even in the s imple form tes ted , appeared to b e qu i te sens i t ive to

cerebel lar re tardat ion. T h i s m a y b e due to the major role p layed b y the

h i n d l i m b s w h i c h t end to b e m o r e severe ly affected than the fo re l imbs . T h e

c l i m b i n g test w a s less sens i t ive and only on the m a c h i n e d rod (and not on the

sc reen-covered rod w h i c h offers sufficient foothold) were the i r radia ted an-

imals deficient.

In summary , the locomotor tests u sed in th is s tudy sugges ted that full

cerebel lar i nvo lvemen t is not essent ia l for successful p rogress ion if there is n o

n e e d for p rec i se concur ren t p ropr iocep t ive and ex te rocept ive control . As the

n e e d for th is sensory control inc reases , the per formance deficits of an imals

wi th severe and then modera te cerebel la r re tardat ion b e c o m e s manifes t .

T h e s tudy conce rned w i t h spon taneous runn ing in ac t iv i ty whee l s (Figure

35) ind ica ted a complex in te rac t ion wi th age , as the control rats reduced their

act ivi ty level w h i l e the severe ly affected an imals inc reased it. W e began

recent ly (25) to s tudy sys temat ica l ly t he effects of age on the infant i le treat-

m e n t used in order to es tab l i sh the extent of " r ecove ry of f u n c t i o n / ' T h e

an imals rece ived seven X- ray doses b e t w e e n days 4 and 15 , and in s o m e

expe r imen t s the per formance of j uven i l e s ( 1 7 - 2 1 days) and adults ( 3 - 4 m o n t h s )

was compared , in o thers that o f y o u n g adults (2 m o n t h s ) wi th older adults (8

m o n t h s ) .

CEREBELLUM AND OPEN FIELD ACTIVITY

JUVENILES (17-21 DAYS)

ADULTS (3-4 MONTHS)

JUVENILES (17-21 DAYS)

ADULTS (3-4 MONTHS)

FIGURE 42. A. Mean number of squares traversed in the open field by nonirradiated control and irradiated rats of two ages. B . Mean frequency of rearing in the open field by the same group of animals. From Brunner and Altman (25).

A

CEREBELLUM AND REARING

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YOUNG

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ADULT

(8 MONTHS)

FIGURE 43. Maximal rotational speeds that control and irradiated rats of two ages could negotiate

in crossing for food reward. This is a task which shows decrement in performance with age in

normal animals, and there was no recovery of function in the irradiated animals. From Brunner

and Altman (25).

In c ross ings in the open field (Figure 42A) there w a s a dec l ine in the act ivi ty

level of no rma l adults w i th respect to j uven i l e s . In the i r radia ted an imals th is

dec l ine did not occur such that the per formance o f control and i r radia ted

adults b e c a m e s imi lar . In the f requency of rear ing (Figure 4 2 B ) there w a s an

increase w i t h age in b o t h groups bu t the difference b e t w e e n the control and

expe r imen ta l an ima l s r e m a i n e d subs tant ia l . F ina l ly , n o i m p r o v e m e n t w a s seen

w h e n 2 - and 8 -month -o ld an imals we re c o m p a r e d in the i r abi l i ty to t raverse

rough or s m o o t h ro ta t ing rods (Figure 43 ) . In s u m m a r y , in s o m e tes ts no rma l -

iza t ion of funct ion d id occur , in o thers there w a s s o m e i m p r o v e m e n t , wh i l e

still in o thers no recovery at all could b e demons t ra ted . T h e ten ta t ive in terpre-

ta t ion is that cons ide rab le recovery m a y occur in easy and genera l funct ions

(the an imal can traverse the open field in m a n y ways ) b u t little or n o recovery

in difficult and specific skills (such as ba l anc ing on a ro ta t ing rod) .

PREWEANING TREATMENTS THAT INTERFERE WITH CEREBELLAR AND LOCOMOTOR DEVELOPMENT

T h e foregoing resul ts e s t ab l i shed that in ter ference w i t h cerebe l la r deve lop-

m e n t in rats b y focal X- i r rad ia t ion dur ing the suckl ing pe r iod p roduces loco-

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86 Joseph Altman

moto r defici ts , s o m e of w h i c h m a y s h o w no amel iora t ion or recovery w i t h

t ime . In the context of th is p resen ta t ion , X- i r rad ia t ion of the ce rebe l lum can b e

v i e w e d as an exper imen ta l tool that a l lows us to e x a m i n e the structural and

funct ional c o n s e q u e n c e s of se lect ive in ter ference wi th the deve lopmen t of a

part icular b ra in reg ion . It should b e ev iden t that the X- ray doses used in these

expe r imen t s are far above the level that the g rowing o rgan i sm, an imal or

h u m a n , is exposed to unde r o ther than except iona l c i rcumstances . H o w e v e r ,

there are m a n y prevalent env i ronmen ta l s i tua t ions w h i c h can lead to subs tan-

tial in ter ference w i t h cerebel lar deve lopment . For example , cell loss in the

deve lop ing cerebel lar cor tex was repor ted b y m a n y inves t iga tors fol lowing

severe prenata l undernu t r i t ion ( 2 6 - 2 9 , 3 1 , 3 9 , 4 9 ) . T h e fo l lowing are examples of

t rea tments that w e have b e e n s tudy ing in our laboratory .

In a recent s tudy (22) w e found that severe undernu t r i t ion of lactat ing rat

mothe r s ( 5 0 % diet res t r ic t ion) p roduces in the re tarded pups a severe loss in

the cell popula t ion of the external ge rmina l layer of the cerebel lar cor tex,

w h i c h then results in a decrease in the n u m b e r of different iated granule cells

(Figure 4 4 ) . If the mo the r s are unde rnou r i shed b y the t ime they b e g i n to

suckle the neona t e s (wh ich is ach ieved b y foster ing the n e w b o r n of normal

mothe r s to mo the r s w h o s e diet res t r ic t ion w a s b e g u n wh i l e they were preg-

nan t ) , the effect on the y o u n g is comparab le to that ach ieved wi th c o m b i n e d

ges ta t iona l - lac ta t iona l undernu t r i t ion . T h e loss in the different iated granule

cell popula t ion pers i s t s after the rats have b e e n rehabi l i t a ted dur ing the

p o s t w e a n i n g per iod , bu t there is recovery in the area occup ied b y the granular

layer (Figure 45 ) . T h e latter f inding, w h i c h is tenta t ive ly in terpre ted as a c o m -

- D A Y S 2 0 - DAYS

FIGURE 4 4 . Effects of severe undernutrition of rat mothers during lactation or during gestation

and lactation combined on the cell population of the external germinal layer in the pyramis at 1 0

days of age (A) and on the number of differentiated granule cells at 2 0 days (B). After Barnes and

Altman ( 2 2 ) .

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FIGURE 45. Effect of age on the area and cell population of the granular layer following rehabili-

tation of rats whose mothers were mildly (75%) or severely (50%) undernourished during the

suckling period. After Barnes and Altman (23).

pensa tory g rowth of the neurop i l (perhaps inc rease in the s ize of m o s s y fiber

t e rmina ls and granule cell dendr i t e s ) , conf i rms a result that w e o b t a i n e d in an

earl ier s tudy (18) . T h e behav io ra l examina t i on of t hese an imals is still in

progress . T h e resul ts o f an ear l ier s tudy (20) ind ica ted t rans ien t re tardat ion in

the deve lopmen t of different locomotor pat terns (such as rear ing , c l i m b i n g ,

and descend ing ) w h i c h d i sappeared after rehabi l i t a t ion . P re l imina ry results of

n e w e r s tud ies sugges t that las t ing deficits prevai l w h e n adult rats under -

nou r i shed dur ing infancy are tes ted on such ski l led tasks as t ravers ing s m o o t h

rota t ing rods for food reward .

A n o t h e r example of a t rea tment that affects cerebel lar cy togenes i s is interfer-

ence w i th thyro id h o r m o n e secre t ion (21 ,42) . In our labora tory the s tudies of

AGE (days) ( d " s >

FIGURE 46. A. Developmental increases in the mean number of granule cells in matched sections

of the pyramis in hypothyroid (Ho), hyperthyroid (Hr), and control (Co) rats. In inset mean

number of basket cells in the three groups. From Nicholson and Altman (51). B. Estimated

number of synaptic profiles in 96 fjum2 samples of the molecular layer in the three groups. From

Nicholson and Altman (50).

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TA

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88 Joseph Altman

Dr. J ean Lauder N icho l son ( 5 0 - 5 2 ) e s t ab l i shed that hype r thy ro id i sm as well as

hypo thy ro id i sm have an effect not only on the n u m b e r of cells in the cerebel lar

cortex bu t also on synap togenes i s . In hyper thyro id rats there w a s an early ter-

m i n a t i o n of cell prol i ferat ion in the external ge rmina l layer , coupled wi th

p recoc ious onse t of cell d i f ferent ia t ion and a t e rmina l decrease in granule cells

(Figure 4 6 A ) . The re w a s also a loss in o ther cell types as wel l as a reduc t ion in

the e s t ima ted n u m b e r of synapt ic profiles (Figure 4 6 B ) . In contras t , h y p o -

thy ro id i sm caused pro longa t ion of cell prol i ferat ion in the ge rmina l layer , retar-

dat ion o f cell d i f ferent ia t ion and , as in hyper thyro ids , a reduc t ion in the

n u m b e r of synapt ic profiles. B o t h t rea tments , though for different reasons

resul ted in a cerebel lar cor tex in w h i c h synapse format ion w a s marked ly inter-

fered wi th .

CONCLUSION

It is h o p e d that these resul ts suppor t our be l i e f that a c o m b i n e d analysis of

the deve lopmen t of the ce rebe l lum and locomot ion unde r different rear ing

cond i t ions can provide useful data abou t op t imal cond i t ions of n e u r o b e h a v -

ioral matura t ion . Not on ly is the g rowing ce rebe l lum an ex t remely vu lnerab le

b ra in s tructure, bu t harmful effects on its g rowth are relat ively easy to assess .

The re is a large b o d y of in format ion at our d isposa l regard ing the structural

and funct ional o rgan iza t ion of the ce rebe l lum, and its direct l ink w i th clearly

d i sce rn ib le overt m o t o r ac t iv i t ies offers the p romise that s o m e in format ion

will also b e ga ined abou t cond i t ions that adverse ly affect behav io ra l matura-

t ion.

ACKNOWLEDGMENT

I wish to thank all the individuals who cooperated in this research program over the last half-dozen years, in particular William J . Anderson, David Barnes, Robert L. Brunner, Gopal D. Das, Kiran Sudarshan, Robert B. Wallace, and Kenneth A. Wright. I am also thankful to Kunda Das, Zeynep Kurgun, and Donna Whitehurst for technical assistance. Support was provided by the National Institute of Mental Health and the U.S. Atomic Energy Commission.

REFERENCES

1. Altman, J . Autoradiographic and histological studies of postnatal neurogenesis. II. A longi-tudinal investigation of the kinetics, migration and transformation of cells incorporating trit-iated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. / . Comp. Neurol, 1966, 128: 431-474.

2. Altman, J . Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. / . Comp. Neurol, 1969, 136: 269-294.


3. Altman, J . Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brain Res., 1971, 30: 311-322.

4. Altman, J . Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. / . Comp. Neurol., 1972, 145: 353-398.

5. Altman, J. Postnatal development of the cerebellar cortex in the rat. II. Phases in the matura-tion of Purkinje cells and of the molecular layer. / . Comp. Neurol., 1972, 145: 399-464.

6. Altman, J . Postnatal development of the cerebellar cortex in the rat. III. Maturation of the com-ponents of the granular layer. / . Comp. Neurol., 1972, 145: 465-514.

7. Altman, J . Experimental reorganization of the cerebellar cortex. III. Regeneration of the ex-ternal germinal layer and granule cell ectopia. / . Comp. Neurol., 1973, 149: 153-180.

8. Altman, J . Experimental reorganization of the cerebellar cortex. IV. Parallel fiber reorientation following regeneration of the external germinal layer. / . Comp. Neurol., 1973, 149: 181-192.

9. Altman, J . , and Anderson, W. J. Irradiation of the cerebellum in infant rats with low-level X-ray. Histological and cytological effects during infancy and adulthood. Exp. Neurol., 1971, 30: 492-509.

10. Altman, J . , and Anderson, W. J. Experimental reorganization of the cerebellar cortex. I. Morphological effects of elimination of all microneurons with prolonged X-irradiation started at birth. / . Comp. Neurol, 1972, 146: 355-406.

11. Altman, J . , and Anderson, W. J . Experimental reorganization of the cerebellar cortex. II. Effects of elimination of most microneurons with prolonged X-irradiation started at four days. / . Comp. Neurol, 1973, 149: 123-152.

12. Altman, J . , Anderson, W. J . , and Strop, M. Retardation of cerebellar and motor development by focal X-irradiation during infancy. Physiol. Behav., 1971, 7: 143-150.

13. Altman, J . , Anderson, W. J . , and Wright, K. A. Selective destruction of precursors of micro-neurons of the cerebellar cortex with fractionated low-dose X-rays. Exp. Neurol, 1967, 17: 481-197.

14. Altman, J . , Anderson, W. J . , and Wright, K. A. Gross morphological consequences of irradia-tion of the cerebellum in infant rats with repeated doses of low-level X-ray. Exp. Neurol, 1968, 21: 69 -91 .

15. Altman, J . , Anderson, W. J . , and Wright, K. A. Early effects of X-irradiation of the cerebellum in infant rats: Decimation and reconstitution of the external granular layer. Exp. Neurol, 1969, 24: 196-216.

16. Altman, J . , Anderson, W. J . , and Wright, K. A. Reconstitution of the external granular layer of the cerebellar cortex in infant rats after low-level X-irradiation. Anat. Rec, 1969, 163: 453-172.

17. Altman, J . , and Das, G. D. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions. /. Comp. Neurol, 1966, 126: 337-390.

18. Altman, J . , and McCrady, B . The influence of nutrition on neural and behavioral development. IV. Effects of infantile undernutrition on the growth of the cerebellum. Develop. Psychobiol, 1972, 5: 111-122.

19. Altman, J . , and Sudarshan, K. Postnatal development of locomotion in rats. Anim. Behav., (submitted for publication).

20. Altman, J . , Sudarshan, K., Das, G. D., McCormick, N. , and Barnes, D. The influence of nutri-tion on neural and behavioral development. III. Development of some motor, particularly locomotor patterns during infancy. Dev. Psychobiol, 1971, 4: 97-114.

21. Balazs, R., Kovacs, S., Cocks, W. A., Johnson, A. L. , and Eayrs, J . T. Effect of thyroid hor-mone on the biochemical maturation of rat brain: Postnatal cell formation. Brain Res., 1971, 25: 555-570.

22. Barnes, D. , and Altman, J . Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neurol, 1973, 38: 406-419.

90 Joseph Altman

23. Barnes, D. , and Altman, J . Effects of two levels of gestational-lactational undernutrition on the postweaning growth of the rat cerebellum. Exp. Neurol, 1973, 38: 420-428.

24. Brunner, R. L. , and Altman, J . Locomotor deficits in adult rats with moderate to massive re-tardation of cerebellar development during infancy. Behav. Biol, 1973, 9: 169-188.

25. Brunner, R. L. , and Altman, J . The effects of interference with the maturation of the cerebel-lum and hippocampus on the development of adult behavior. In: Plasticity and Recovery of Function in the Central Nervous System. (D. G. Stein, J . J . Rosen, and N. Butters, Eds.), Aca-demic Press, New York, 1974: 129-148.

26. Chase, H. P., Lindsley, W. F. B . , and O'Brien, D. Undernutrition and cerebellar development. Nature {Lond.), 1969, 2 2 1 : 554-555.

27. Cheek, D. B . , Graystone, J . E . , and Rowe, R. D. Hypoxia and malnutrition in newborn rats: Effects on RNA, DNA and protein in tissues. Am. J. Physiol, 1969, 217: 642-645.

28. Culley, W. J . , and Lineberger, R. O. Effect of undernutrition on the size and composition of the rat brain. / . Nutr., 1968, 96 : 375-381.

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30. Ebels, E. J . The influence of age upon the effect of early postnatal X-irradiation on the devel-opment of the cerebellar cortex in rats. Acta Neuropathol, 1970, 15: 298-307.

31. Fish, I., and Winick, M. Effect of malnutrition on regional growth of the developing rat brain. Exp. Neurol, 1969, 25: 534-540.

32. Fujita, S. Quantitative analysis of cell proliferation and differentiation in the cortex of the postnatal mouse cerebellum. / . Cell Biol, 1967, 32: 277-288.

33. Fujita, S., Shimada, M., and Nakamura, T. H 3 -Thymidine autoradiographic studies on the cell proliferation and differentiation in the external and internal granular layers of the mouse cerebellum. / . Comp. Neurol, 1966, 128: 191-208.

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35. Hicks, S. P. Radiation as an experimental tool in mammalian developmental neurology. Physiol Rev., 1958, 38: 337-356.

36. Hicks, S. P., and D'Amato, C. J . Effects of ionizing radiations on mammalian development. In: Advances in Teratology. (D. H. Woollam, Ed.), Vol. 1. Academic Press, New York, 1966: 195-250.

37. Hirano, A., and Dembitzer, H. Cerebellar alterations in the weaver mouse. / . Cell Biol, 1973, 56: 478-186.

38. Hirano, A., Dembitzer, H., and Jones, M. An electron microscopic study of cycasin-induced cerebellar alterations. /. Neuropathol. Exp. Neurol, 1972, 3 1 : 113-125.

39. Howard, E . , and Granoff, D. M. Effect of neonatal food restriction in mice on brain growth, DNA and cholesterol, and on adult delayed response learning. / . Nutr., 1968, 95: 111-121.

40. Kornguth, S. E . , Anderson, J . W., and Scott, G. The development of synaptic contacts in the cerebellum of Macaca mulatta. J . Comp. Neurol, 1968, 132: 531-546.

41. Larramendi, L. M. H. Analysis of synaptogenesis in the cerebellum of the mouse. In: Neurobiology of Cerebellar Evolution and Development. (R. Llinas, Ed.), Amer. Med. Ass., Chicago, Illinois, 1969: 803-843.

41a. Larsell, O. The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat. / . Comp. Neurol, 1952, 97: 281-356.

42. Legrand, J . Comparative effects of thyroid deficiency and undernutrition on maturation of the nervous system and particularly on myelination in the young rat. In: Hormones in Develop-ment. (M. Hamburgh and E. J . W. Barrington, Eds.). Appleton, New York, 1971: 381-390.

2 . Interference with Cerebellar Maturation 9 1

43. Llinas, R., Hillman, D. E. , and Precht, W. Neuronal circuit reorganization in mammalian agranular cerebellar cortex. / . Neurobiol., 1973, 4 : 69-94.

44. Luciani, L. II Cervelletto: Nuovi Studi di Fisiologia Normale e Patologica. Le Monnier, Florence, 1891; quoted from The Physiology and Pathology of the Cerebellum. (R. S. Dow and G. Moruzzi). Univ. of Minnesota Press, Minneapolis, 1958, 25: 101-102 .

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47. Mugnaini, E. Ultrastructural studies on the cerebellar histogenesis. II. In: Neurobiology of Cerebellar Evolution and Development. (R. Llinas, Ed.). Amer. Med. Ass., Chicago, Illinois, 1969: 749-782.

48. Mugnaini, E . , and Forstronen, P. F. Ultrastructural studies on the cerebellar histogenesis. I. Differentiation of granule cells and development of glomeruli in the chick embryo. Z. Zell-forsch., Mikrosk. Anat., 1967, 77: 115-143.

49. Neville, H. W., and Chase, H. P. Undernutrition and cerebellar development. Exp. Neurol, 1971, 33: 485-497.

50. Nicholson, J . L . , and Altman, J . Synaptogenesis in the rat cerebellum: Effects of early hypo-and hyperthyroidism. Science, 1972, 176: 530-531.

51. Nicholson, J . L . , and Altman, J . The effects of early hypo- and hyperthyroidism on the devel-opment of rat cerebellar cortex. I. Cell proliferation and differentiation. Brain Res., 1972, 44: 13-23.

52. Nicholson, J . L. , and Altman, J . The effects of early hypo- and hyperthyroidism on the devel-opment of rat cerebellar cortex. II. Synaptogenesis in the molecular layer. Brain Res., 1972, 44: 25-36.

53. Rakic, P. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electromicroscopic study in Macacus rhesus. / . Comp. Neurol, 1971, 1 4 1 : 283-312.

54. Rakic, P. Kinetics of proliferation and latency between final cell division and onset of dif-ferentiation of cerebellar stellate and basket neurons. /. Comp. Neurol, 1973, 147: 523-546.

55. Rakic, P., and Sidman, R. L. Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice. / . Comp. Neurol, 1973, 152: 133-162.

56. Ramon y Cajal, S. Studies on Vertebrate Neurogenesis, (trans by L. Guth). Thomas, Springfield, Illinois, 1960.

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Qualitative observations in infant and adolescent rats. Dev. Psychobiol, 1969, 2: 257-265.

58. Wallace, R. B . , and Altman, J . Behavioral effects of neonatal irradiation of the cerebellum. II. Quantitative studies in young-adult and adult rats. Dev. Psychobiol, 1969, 2 : 266-272.

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Nutr., 1966, 89: 300-306.

3 Neuronal Sprouting after

Hippocampal Lesions

G A R Y L Y N C H

Department of Psychobiology, University of California Irvine,

Irvine, California

B y us ing a c o m b i n a t i o n of neu roana tomica l tools , D r . Rak ic has p rov ided

s o m e clarif icat ion of the classic ques t i on of h o w n e w l y fo rmed n e u r o n s reach

the i r u l t imate loci in the cor tex, that i s , b y free migra t ion or b y process ex ten-

s ion and t rans loca t ion of the per ikaryon . H e has demons t r a t ed that the m o d e

of migra t ion is d e p e n d e n t u p o n the b i r thda te of the cell popula t ion in q u e s -

t ion; early forming cells follow the pat tern desc r ibed recent ly b y Mores t (16)

and o thers , w h i l e cells u n d e r g o i n g final d iv i s ion later in deve lopmen t t rack

a long the p rocesses of radial gl ia . I was in t r igued b y h i s sugges t ion that this

m e c h a n i s m is necess i t a t ed b y the cons ide rab le d i s tances that late fo rming

cells m u s t t ravel to reach the i r u l t imate loca t ions .

T h e q u e s t i o n that natural ly ar i ses is w h e t h e r gl ia play such a cr i t ical devel-

opmen ta l funct ion at s i t es o ther than cerebra l and cerebel lar cor t ices . At th is

po in t w e can on ly b e g i n to apprec ia te the n u m b e r of structural features of

b ra in o rgan iza t ion that m i g h t b e accoun ted for b y a m e c h a n i s m such as glial

gu idance of mig ra t ing neu rons .

Dr . R a k i c ' s sugges t ion that the format ion o f cort ical gyri is no t direct ly

related to the n u m b e r of deve lop ing cells shou ld b e of in teres t no t on ly to

deve lopmenta l neu rob io log i s t s bu t to workers a t t empt ing to formulate

theor ies of b r a in evo lu t ion as wel l (7) .

Dr . A l tman ' s p resen ta t ion s u m m a r i z e d h i s efforts to relate ana tomica l devel-o p m e n t to the ma tu ra t ion of behav io ra l r e sponse s . H e uses a ba t te ry o f tech-n i q u e s to arr ive at a comple te p ic ture o f the deve lop ing ce rebe l lum and then employs a k i n d of mic rosu rge ry (X-rays) to e l imina te cer ta in cell popula t ions . As s o m e o n e w h o h a s t r ied to quant i fy the effects of early cerebel lar damage on

93

9 4 Gary Lynch

l ocomotor behav io r s in rodents , I can assure y o u that Dr . Al ta ian ' s scrut iny of

the behav iora l c o n s e q u e n c e s of h i s man ipu la t ions represen ts a n e w level of

ach ievemen t . H i s expe r imen t s hopeful ly wi l l p rov ide ins igh t s in to the func-

t ions per formed b y the var ious cerebel lar cell g roups w h i c h w h e n c o m b i n e d

w i t h neurophys io log ica l mode l s o f cerebel lar opera t ion (2) should result in a

mos t exc i t ing story.

A p r o b l e m c o m m o n to all s tudies involv ing des t ruc t ion of b ra in r eg ions or

remova l of part icular cell popula t ions is w h e t h e r the resul t ing behav io ra l

changes are due to loss of e l emen t s or are caused b y the reorganiza t ion of

r ema in ing , intact sys tems . W e k n o w that n e w circui ts are fo rmed in areas par-

tially denerva ted b y l e s ions , and Dr . A l tman has s h o w n that a var ie ty of

abnormal i t i es a c c o m p a n y the e l imina t ion of cerebel la r m ic roneu rons . T h e

p rob lem of ass ign ing behav io ra l deficits to lost e l ements or to abno rma l orga-

n iza t ion has n o easy solut ion and mus t b e r ecogn ized as a factor in the proper

evaluat ion of the behav io ra l resul ts of l es ion s tudies . A long these l ines I

wonde r h o w rats w i thou t the ce rebe l lum perform on Dr . Al ta ian ' s ba t tery of

tests .

A final po in t that I w o u l d l ike to ra ise conce rns the radical ly different devel-

opmenta l p rograms desc r ibed b y Dr . A l tman and Dr . Rak ic . Dr . A l tman shows

that m u c h of the migra to ry and different ia t ion pe r iod of rat cerebel lar devel-

o p m e n t occurs postnata l ly wh i l e Dr . Rak ic has found that m a n y of these

events h a p p e n prenatal ly i n the p r ima te cerebra l cor tex. S o m e of th i s dif-

ference is undoub ted ly due to the different b ra in reg ions these sc ien t i s t s have

chosen for s tudy, bu t the poss ib i l i ty o f var ia t ion b e t w e e n spec ies s e e m s to b e

very real. S u c h differences w o u l d p re sumab ly b e of interest , no t on ly to devel-

opmen ta l neurob io log i s t s bu t to workers c o n c e r n e d w i t h effects of early expe-

r ience on the matura t ion of b ra in and behav io r .

To return n o w to a d i scuss ion of the abnorma l c i rcui t ry w h i c h forms after

b ra in l e s ions and its pos s ib l e s ignif icance in the deve lopmen t of behav iora l

deficits, the idea that in tact axons m i g h t g row n e w collaterals to replace inputs

lost because of l es ions goes back to the late n ine t een th century [6; and see

Edds (3) for an excel lent r ev i ew] and expe r imen t s in the 1940 ' s and 1950 ' s

p rov ided sat isfactory demons t r a t ions of the effect in the per iphera l ne rvous

sys tem (3 ,5 ,6 ) . W o r k on sprout ing in the central ne rvous sys tem w a s in i t ia ted

b y L iu and C h a m b e r s (9) s ince w h i c h there have b e e n dozens of repor ts . T h e

format ion of aber ran t c o n n e c t i o n s represen ts a t empt ing explana t ion for a

var ie ty o f p sychob io log ica l p h e n o m e n a inc lud ing recovery from b ra in damage

and perhaps in s o m e cases men ta l re tardat ion. But a hos t of u n a n s w e r e d ques -

t ions m a k e s it difficult to evaluate these sugges t ions realist ical ly. I wou ld l ike

briefly to relate the resul ts o f expe r imen t s done in m y labora tory and that of

Carl C o t m a n w h i c h we re d i rec ted at cer ta in of t hese ques t ions . W e have s tud-

ied the effects o f r e m o v i n g the major afferent to the denta te gyrus of the h ip -

p o c a m p u s on the d i s t r ibu t ion of i ts r e m a i n i n g inputs . In do ing th is , w e h o p e d

3 . Neuronal Sprouting after Hippocampal Lesions 95

to p rov ide data pe r t inen t to the fo l lowing i s sues :

1. Is sp rou t ing un iversa l or is the effect res t r ic ted to cer ta in popu la t ions of in tact afferents?

2. Are there major deve lopmenta l differences in pos t l es ion axonal g rowth?

3. D o e s sp rou t ing result in funct ional c o n n e c t i o n s and i f so ,

4 . H o w long does the p rocess r equ i re?

W e chose the denta te gyrus to inves t iga te these ques t i ons b e c a u s e of its

unusua l ana tomica l s impl ic i ty and l amina t ion of afferents. T h e s e po in t s are

i l lustrated in F igu re s 1 and 2 . No te that the denta te gyrus cons i s t s of two

layers , a layer of g ranule cell b o d i e s and a molecu la r layer w h i c h is c o m p o s e d

of the dendr i t ic p rocesses of these cells (Figure 1) . T h e molecu la r layer can b e

subd iv ided in to an i n n e r and outer z o n e , the first of w h i c h rece ives inpu t s

from the pyramida l cells of the ipsi lateral ( "assoc ia t iona l " ) and contralateral

( " commis su ra l " ) s ides . T h e outer zone o f the molecu la r layer is mass ive ly in-

nerva ted b y the en to rh ina l cor tex (" ipsi la teral t e m p o r o - a m m o n i c t r a c t / ' TA)

and to a m u c h lesser extent b y a g roup of ace ty lchol ines te rase ( A C h E ) - c o n -

ta in ing f ibers w h i c h in all l ike l ihood or ig ina te in the s e p t u m (Figure 2 ) . In addi-

t ion to these p ro jec t ions , the contralateral en torh ina l cor tex g ives r ise to a tract

( the " c r o s s e d t e m p o r o - a m m o n i c t rac t" or C T A ) w h i c h e n d s o n the apical t ips

of the pyramida l cells o f the regio super ior i m m e d i a t e l y adjacent to bu t not

in contac t w i t h the distal ends of the granule cell dendr i tes (Figure 2 ) . The re

are then four inpu t s to the denta te gyrus w i th a fifth p ro jec t ion end ing

nea rby . Fo l lowing remova l of the en to rh ina l cor tex the four r e m a i n i n g af-

FIGURE 1. Summary of the origins of extrinsic projections to the hippocampal formation and den-tate gyrus. Four systems are shown: (1) the septal projections (dotted lines) which are thought to contain acetylcholinesterase and (2) commissural from contralateral pyramidal cells, (3) crossed temporo-ammonic (CTA) from contralateral entorhinal cortex, and (4) the uncrossed temporo-am-monic fibers (TA) from the ipsilateral entorhinal cortex.

96 Gary Lynch

Crossed Temporo-Ammonic (5)

Entorhinal (4)

(Septal) (3 )

Commissural ( 2 )

Associational (I)

FIGURE 2. Schematic of the distribution of five afferent projections of the dentate gyrus and ad-

jacent stratum moleeulare of the regio superior. Note that the commissural (2) and associational (1)

inputs are restricted to the inner molecular layer and do not overlap with the septal (3) and ipsilat-

eral entorhinal (4) projections to the outer molecular layer. Thus the afferents to the molecular

layer of the dentate form two nonoverlapping lamina (the inner and outer molecular layers). The

fibers from the contralateral entorhinal cortex (5) end on the apical tips of the pyramidal cells and

do not make contact with the granule cells.

ferents " s p r o u t " and invade the deafferentated outer molecu la r layer . Specif i -

cally, the assoc ia t iona l and commissu ra l p ro jec t ions spread ou tward , the

A C h E - c o n t a i n i n g e n d i n g s normal ly loca ted in the outer molecu la r layer prolif-

erate , and the axons from the contralateral en torh ina l cor tex es tab l i sh con-

tacts th roughou t the en t i re medio la te ra l extent o f the ou ter molecular layer

(Figure 3 ) . [Deta i led descr ip t ions of these f indings can b e found in ( 1 , 1 0 - 1 3 ,

17 ,18 ,20 ) . ]

In the adult rat, these four sys t ems do not g row randomly into the outer

molecular layer bu t ins tead re lamina te themse lves ; that i s , C T A inputs do no t

overlap wi th the expanded commissu ra l and assoc ia t iona l sys tem (Figure 3 ) .

Fur the rmore , there are impor tan t deve lopmenta l differences in the degree of

g rowth bu t these vary accord ing to the afferent unde r cons idera t ion . T h e c o m -

missura l and assoc ia t iona l p ro jec t ions ex tend further into outer molecu la r

layer after en torh ina l l e s ions in y o u n g rats than after comparab le l e s ions in

adults (14,20) bu t the inc rease in A C h E act ivi ty is m o r e res t r ic ted. In contrast ,

w e could find little deve lopmenta l difference in the pos t l es ion changes o f the

crossed en torh ina l p ro jec t ions .

Tu rn ing n o w to the specif ic ques t ions w h i c h i n part p rompted these exper i -

men t s , w e can n o w conc lude that l e s ions of the ipsi la teral t e m p o r o - a m m o n i c

tract cause r e sponses in each of the afferents r e m a i n i n g to the denta te


gyrus. H o w e v e r , there is cons ide rab le ev idence that sp rou t ing is no t o b -

ta ined in every s i tua t ion [for examples , see G o o d m a n and Hore l (4) and

Kerr (8)] . In o ther s tudies w e have found that r emova l of the afferents to the

i n n e r molecular layer (that i s , the commissu ra l and assoc ia t iona l sys tems)

does not cause any major changes in the d i s t r ibu t ion of the en to rh ina l in-

puts in the den ta te gyrus (15) . C o t m a n and I h a v e h y p o t h e s i z e d that cer ta in

les ions create s i tua t ions appropr ia te for pos t l es ion g rowth and in these

cases sp rou t ing can occur in all appropr ia te ly p laced intact afferents. O t h e r

l es ions apparent ly do no t p roduce cond i t ions su i tab le for genera l ized

sprout ing .

It is also ev iden t from the resul ts o b t a i n e d in the denta te gyrus that

sprou t ing in adults is no t a r a n d o m process bu t ins tead is b o t h regula ted and

gu ided . Tha t i s , the i nvad ing afferents form n e w layers w i th sharp ly defined

b o u n d a r i e s b e t w e e n t h e m . T h e s i tua t ion after neona ta l l e s ions is less clear.

T h e c h a n g e s in the A C h E - c o n t a i n i n g afferents do not s e e m to over lap w i t h the

expanded commis su ra l - a s soc i a t i ona l sys t ems , bu t the i nvad ing c rossed en-

torhinal p ro jec t ions apparent ly do share dendr i t ic space w i t h these two projec-

t ions . Re levan t to th is is the obse rva t ion m e n t i o n e d above that deve lopmenta l

FIGURE 3. The distribution of remaining afferents to dentate gyrus after an ipsilateral entorhinal cortical lesion in an adult rat. The associational (1) and commissural (2) fibers sprout into the outer molecular layer (right panel) while the inputs from the contralateral entorhinal cortex (5) establish contacts throughout the outer molecular layer. The changes in the septal projections described in the text are not illustrated in this figure. The left-hand panel shows that despite extensive sprout-ing of its afferents the dentate gyrus retains a laminated organization; that is, the extended commissural and associational projections do not overlap with the sprouted crossed temporo-ammonic fibers. The arrow in the left-hand panel indicates that the boundary separating the afferent fields moves "out" the dendrites of the granules cells after the entorhinal lesion.

Crossed Temporo-Ammonic (5)

Crossed Temporo-\^

Ammonic (5) l

Entorhinal ( 4 ) \

(Septal) (3)

Commissural ( 2 ) Associational (1)

98 Gary Lynch

differences in the degree of pos t les ion growth s e e m to b e different in the

var ious afferents.

Final ly , these results afforded us the oppor tun i ty to test the funct ional status

of the aberrant ly located t e rmina l popula t ions . W e have found that electr ical

s t imula t ion of e i ther the C T A (17,18) or commissu ra l p ro jec t ions (14 ,19) pro-

duce m o n o s y n a p t i c extracellular potent ia ls in those outer molecular layer

reg ions they invade after the en torh ina l l es ion . T h e s e phys io log ica l results

were ob t a ined in rats l e s ioned e i ther as neona te s or as adults .

T h e al tered d i s t r ibu t ion of potent ia ls in adults w a s first obse rved at 9 days

pos t les ion ind ica t ing that th is a m o u n t of t ime is r equ i red for pos t les ion

growth to form effective synapses .

SUMMARY

It appears that aber ran t bu t funct ional c i rcui t ry can rapidly develop after

removal o f part icular afferent p ro jec t ions . T h e parameters desc r ib ing th is

process appear to vary accord ing to (1) the age at w h i c h the exper imenta l ma -

n ipu la t ion was per formed, (2) the specific afferent b e i n g s tudied , and (3) the

part icular input w h i c h is e l imina ted or pos s ib ly the b ra in reg ion w h i c h is

deafferented. It m u s t also b e e m p h a s i z e d that abno rma l g rowth is not a lways

found after l es ions even in s i tua t ions w h i c h a pr ior i appear appropr ia te .

Hopeful ly , these ana tomica l and phys io log ica l results o b t a i n e d in a relat ively

" s i m p l e " b ra in sys tem wil l b e of he lp in evalua t ing the role p layed b y abnorma l

wi r ing in the deve lopmen t of behav iora l deficits .

REFERENCES

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2. Eccles, J. C., Ito, M. , and Szentagothai, J . The Cerebellum as a Neuronal Machine. Springer-Verlag, New York, 1967.

3. Edds, M. V. Collateral nerve regeneration. Quart. Rev. Biol, 1953, 28: 260-276. 4. Goodman, D. C , and Horel, J . A. Sprouting of optic tract projections in the brain stem of the

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Aust. J. Exp. Biol. Med. Sci., 1950, 28: 383-397. 7. Jerison, H. J . Evolution of the Brain and Intelligence. Academic Press, New York, 1973. 8. Kerr, F. W. L. The potential of cervical primary afferents to sprout in the spinal nucleus of V

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10. Lynch, G., Deadwyler, S., and Cotman, C. W. Postlesion axonal growth produces permanent functional connections. Science, 1973, 180: 1364-1366.

11. Lynch, G., Matthews, D., Mosko, S., Parks, T., and Cotman, C. Induced acetylcholinesterase-rich layer in rat dentate gyrus following entorhinal lesions. Brain Res., 1972, 42: 311-318.

12. Lynch, G., Mosko, S., Parks, T. , and Cotman, C. W. Relocation and hyperdevelopment of the dentate gyrus commissural system after entorhinal lesions in immature rats. Brain Res., 1973, 50: 174-178.

13. Lynch, G. , Rose, G. , Gall, C , Stanfield, B . , and Cotman, C. Sprouting by the dentate gyrus associational system: A horseradish peroxidase histochemical study. (Submitted for publication.)

14. Lynch, G., Stanfield, B . , and Cotman, C. W. Developmental differences in post-lesion axonal growth in the hippocampus. Brain Res., 1973, 59: 155-168.

15. Lynch, G., Stanfield, B . , Parks, T., and Cotman, C. W. Evidence for selective post-lesion ax-onal growth in the dentate gyrus of the rat. Brain Res., 1974, 69: 1-11.

16. Morest, D. K. A study of neurogenesis in the forebrain of opossum pouch young. Z. Anat. Entwicklungs. Gesch., 1970, 130: 265-305.

17. Steward, O., Cotman, C. W., and Lynch, G. Re-establishment of electrophysiologically func-tional entorhinal cortical input to the dentate gyrus deafferented by ipsilateral entorhinal lesions: Innervation by the contralateral entorhinal cortex. Exp. Brain Res., 1973, 18: 306-414.

18. Steward, O., Cotman, C. W., and Lynch, G. Growth of a new fiber projection in the brain of adult rats: Re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions. Exp. Brain Res., 1974, 20: 45-66.

19. West, J . , Deadwyler, S., Cotman, C. W. , and Lynch, G. Time course for changes in the dis-tribution of commissural responses in the dentate gyrus following lesions of the entorhinal cortex in adult rats. (Submitted for publication.)

20. Zimmer, J . Extended commissural and ipsilateral projections in post-natally de-entorhinated hippocampus and fascia dentata demonstrated in rats by silver impregnation. Brain Res., 1973,64: 293-311.

4 Physiological Properties of Vertebrate Nerve Cells in

Tissue Culture

M A R C A . D I C H T E R

Department of Medicine, Beth Israel Hospital, Boston, Massachusetts*

T h e ver tebra te centra l ne rvous sys t em is a large, compl ica ted col lec t ion of

in t imate ly i n t e r connec t ed sys t ems . It has b e e n s tud ied at all levels—from the

vantage of the w h o l e sy s t em, as a " b l a c k b o x , " to the van tage of ind iv idua l

molecu la r reac t ions w i t h i n ind iv idua l cells . E a c h of these approaches has

added s o m e t h i n g to our unde r s t and ing o f ba s i c m e c h a n i s m s o f m e n t a l retar-

dat ion. Recen t ly , advances in t i ssue culture t echno logy have suppor t ed the

deve lopmen t of a m o d e l s y s t e m d e s i g n e d to facil i tate the s tudy of the deve lop-

m e n t and funct ion of smal l por t ions of the ver tebra te ne rvous sys t em at the

cellular and synap t ic level .

Ve r t eb ra t e ne rvous t i s sue can b e r e m o v e d from a deve lop ing e m b r y o and

m a i n t a i n e d in vitro for w e e k s or m o n t h s . T h e n e u r o n s wi l l g r o w and differen-

tiate and form n e w c o n n e c t i o n s in vitro. S u c h p repara t ions are d e s i g n e d to ac-

compl i sh two goals : (1) to s impl i fy a h igh ly c o m p l e x sys t em b y separa t ing it

in to smal ler c o m p o n e n t par ts , and (2) to a l low m o r e specific cont ro l ove r the

e n v i r o n m e n t in w h i c h the ne rvous t i s sue is deve lop ing and func t ion ing .

The re are three bas i c m e t h o d s that are current ly e m p l o y e d for m a i n t a i n i n g p r imary cul tures o f n e u r o n s :

1. Explan t cul ture. Smal l s l ices of ne rvous sys t em are o b t a i n e d from the

reg ion o f in teres t ( i . e . , cor tex , sp ina l cord , e tc . ) and are p laced in a t i ssue cul-

ture vesse l in nu t r i en t b ro th . T h e local in terna l s t ructure of the explant is

und i s tu rbed wh i l e m a n y neu rons are d i s connec t ed from dis tant areas of

* Present address: Department of Neurology, Beth Israel Hospital, Boston, Massachusetts.

1 0 2 Marc A. Dichter

nervous sys tem. H e n c e , neu rons have s o m e degree of p reex is t ing f ramework

in w h i c h to deve lop and form n e w c o n n e c t i o n s (5 ,17) .

2 . Reaggrega te cul tures . Cel ls from the relevant area of the ne rvous sys tem

are d i ssoc ia ted from o n e ano the r b y gent le enzymat i c t rea tment or b y m e c h a n -

ical m e a n s . A suspens ion of re lat ively spher ica l s ingle cells is o b t a i n e d w h i c h

con ta ins b o t h n e u r o n s and n o n n e u r o n a l e l emen t s . T h e s e cells are t ransferred

to a nu t r ien t b ro th in a flask and m a i n t a i n e d on a shaker platform. T h e cells

r andomly col l ide in the vesse l and appear to adhere preferent ial ly to o ther cells

in a relat ively specific pat tern . After several days of deve lopment , the cy-

toarchi tec ture of the explant m a y remarkab ly r e s e m b l e that o f the or iginal in-

tact ne rvous sys tem. S u c h reaggregate cul tures have b e e n prepared from

normal ver tebra te ne rvous t i s sue and from an ima l s w i th gene t ic defects w h i c h

are mani fes t b y abno rma l ne rvous sys t em deve lopmen t ( 7 , 8 , 1 4 , 1 7 ) .

3. D i s soc i a t ed cell cul tures g rown as modi f ied mono laye r s . Cells from

nervous t i ssue are d i s soc ia ted as in reaggregate cul tures , bu t are g rown at rela-

t ively low dens i ty on cover s l ips or plast ic petr i d i shes in such w a y as to keep

the neu rons separate from one another . In th is sys tem, the neu rons g row and

differentiate w i thou t phys ica l contac t w i t h o n e ano ther . T h e y ex tend p rocesses

w h i c h form de novo connec t i ons . T h e culture g rows up as a modif ied m o n o -

layer wi th the neu rons often s i tuated on top of a s ingle layer of n o n n e u r o n a l

cells. In these d i s soc ia ted cell cul tures the or ig ina l cy toarchi tec ture of the

nervous sys tem is totally d i s rupted , the cells are all freely exposed to the cul-

ture env i ronmen t , and ind iv idua l cel ls , or small g roups of cel ls , can b e o b -

served as they deve lop . M o r e o v e r , in the mature cul ture , ind iv idua l neu rons

can b e v i sua l ized in the l iv ing state wh i l e deta i led phys io log ica l and phar-

macologica l expe r imen t s can b e per formed ( 2 , 6 , 9 - 1 2 , 1 5 , 1 6 , 1 8 , 2 1 ) . T e c h n i q u e s

are b e i n g deve loped to separa te the in i t ia l popula t ion of d i ssoc ia ted cells into

var ious c o m p o n e n t s b y e i the r phys ica l , or p o s s i b l y funct ional , m e a n s (1) . T h i s

m a y al low the c o m p a r i s o n of the deve lopmen t and funct ion of different c lasses

of neu rons separate ly in these cul tures .

All of the t i ssue culture sys tems in p resen t use are mode l s w h i c h , at bes t ,

are approx imat ions of p h e n o m e n a in the intact ne rvous sys tem. T h e s e sys t ems

can also b e cons ide red as two different k inds of mode l s . In one case , these are

mode l s of the process of development o f the ne rvous sys tem. Tha t i s , the devel-

o p m e n t o f neurona l s tructure and funct ion in vitro, the format ion of synapses

in vitro, and the deve lopmen t of a neurona l o rganiza t ion in vitro are though t

to b e ana logous to s imi lar even t s in vivo, or at least the two wil l share s o m e

genera l p r inc ip les . In the s econd case , the sys tem can b e cons ide red as a

s implif ied m o d e l of the function o f ver tebra te centra l neu rons . S tud ies of the

phys io logy , pha rmaco logy , and pa thophys io logy of the ind iv idua l n e u r o n s in

culture wou ld b e cons ide red to apply to s imi lar funct ions of the neu rons in situ.

4. Physiological Properties of Vertebrate Nerve Cells 103

THE MODEL SYSTEM

Culturing Conditions and Cell Morphology

W e have b e e n g rowing a var ie ty of ver tebra te n e u r o n s and musc l e cells in

the d i s soc ia ted cell sys t em. N e u r o n s from rat and m o u s e cor tex, rat, h u m a n ,

and ch i ck dorsal root (or sensory) gangl ia , and ch ick sp ina l cord , and , in addi-

t ion , skeletal musc l e cells from rat and ch ick have b e e n kept alive for as long

as 4 m o n t h s in vitro. M o s t of the w o r k to b e desc r ibed dea l ing w i t h ch i ck sp i -

nal cord and dorsal root gangl ion cells w a s d o n e in co l labora t ion w i t h Dr .

Gera ld F i s c h b a c h ( 9 - 1 2 ) . T h e cells are d i s soc ia ted w i t h t ryps in and gent le

m e c h a n i c a l ag i ta t ion and are plated onto plast ic petr i d i shes or glass cover

sl ips w h i c h are covered wi th col lagen or n o n n e u r o n a l cel ls . T h e y are g r o w n in

Eagle ' s M i n i m u m Essen t ia l M e d i u m wi th Ear le ' s salts ( G i b c o ) , 5 % fetal calf

s e rum (for rat ce l ls ) , or 1 0 % horse s e rum plus 2 % ch ick e m b r y o extract (for

ch ick neu rons ) and kept at p H 7.4 in a 5 % C 0 2 a tmosphe re . T h e ind iv idua l

cells settle on to the b a c k g r o u n d w i t h i n hours of p la t ing (F igures 1 A and B ) ,

and the n e u r o n s b e g i n to ex tend p rocesses shor t ly thereafter (F igure 1C) .

W i t h i n days the n e u r o n s have formed ex tens ive ne tworks of neur i t e s . T h e

neu rons also differentiate from thei r in i t ia l ly spher ica l form to m o r e character-

is t ical ly " n e u r o n a l " m o r p h o l o g y (Figure I D and 2 ) . T h e r e are also several

types of n o n n e u r o n a l cells w h i c h develop in these cul tures inc lud ing cells

w h i c h r e s e m b l e S c h w a n n cel ls , glial cel ls , and e p e n d y m a l cells .

F igure 2 i l lustrates the var ie ty o f morpho log i e s w h i c h rat cor t ical n e u r o n s

can deve lop . All of these cells we re l iv ing at the t ime they were p h o t o g r a p h e d

us ing phase contras t m ic roscopy . Cel ls s h o w n in A - C are genera l ly s y m m e t -

rical w i t h p rocesses rad ia t ing in all d i rec t ions . T h e cells in D - F have a m o r e

l inear conf igurat ion. T h e cells in G - I have a pyramida l appea rance w i t h one

long apical dendr i te and shor ter , b r a n c h e d basa l dendr i tes . In te res t ing ly , the

three cells in I are a l igned wi th the i r apical and basa l dendr i t es o r i en ted in

parallel .

It can b e s h o w n that the neu rons in these cul tures can b e s t a ined w i t h si lver

s imi lar to n e u r o n s in situ. F igure 3 i l lustrates four cells f rom ch ick sp ina l cord

and o n e from ch ick dorsal root gangl ion cultures s t a ined w i t h si lver . T h e spi-

nal cord cells are o f va r i ab le s i ze a n d h a v e mul t ip le p rocesses e m a n a t i n g from

their somata . T h e dorsal root gang l ion cell in cul ture re ta ins its character is t ic

appearance as a spher ica l cell w i t h one or a few p rocesses c o m i n g abrupt ly out

of the soma . Dorsa l root gang l ion cel ls , b o t h f rom the ch i ck and the rat, a lways

ma in t a in the i r charac ter is t ic m o r p h o l o g y in cul ture and n e v e r differentiate

into cells w h i c h r e s e m b l e the sp ina l cord or cort ical neu rons . T h e y m a y g row

s ingly or in small g roups , as i l lustrated in the phase contras t pho-

tomicrographs of F igu re 4 , and m a y b e ove rg rown b y the b a c k g r o u n d non-


FIGURE 1. Rat cortical neurons in cell culture. A . Freshly dissociated cortical cells from 18-day rat embryo settling on collagen-coated plastic petri dish. B . Same as A but petri dish covered with flat, nonneuronal cells. C. Three hours after plating. Note several neurons have already begun sending out processes. D. Rat cortical neurons approximately 1 week in culture. Background non-neuronal cells have covered the surface of the dish.

4. Physiological P

roperties of Vertebrate N

erve C

ells 105

FIGURE 2. Living rat and mouse cortical neurons. A - E , H. Rat. F , G, I. Mouse. Ages in vitro: A, 19 days; B , 21 days; C, 15 days; D, 28 days; E, 28 days; F , 15 days; G, 15 days; H, 28 days; I, 12 days. Calibration: 100 fim.

106 Marc A. Dichter

FIGURE 3. A - D . Silver stains of chick spinal cord. E . Dorsal root ganglion cell grown in cell cul-

ture. From Fischbach and Dichter (12).

neurona l cells . Occas iona l ly , the gang l ion cells appear to b e su r rounded b y a

capsule o f satel l i te cells . F requen t ly , the axons of the gangl ion cells are covered

b y a type of cell w h i c h r e sembles a S c h w a n n cell.

Elec t ron mic roscop ic e x a m i n a t i o n of these d i ssoc ia ted cell cul tures ind ica tes

that m a n y of the n e u r o n s lie uncove red b y glial e l emen t s and are in direct con-

tact w i th the b a t h i n g solu t ion . T h e neu rons con ta in the usual c o m p l e m e n t of

intracellular organel les , i nc lud ing neuro tubu les and neurof i laments and small

synapt ic ves ic les . S y n a p s e s can b e seen w h i c h r e semble synapses in the intact

ne rvous sys t em, w i t h the i r pre- and pos t synap t ic m e m b r a n e modi f ica t ions

and col lec t ions of synapt ic ves ic les nea r the presynapt ic m e m b r a n e (Fig-

ure 5 ) .

Physiological Studies

T h e neu rons in these cul tures can b e pene t ra ted unde r direct v isua l control

w i th mic roe lec t rodes . T h e cells have res t ing potent ia ls r ang ing from approxi -

ma te ly - 4 0 to —75 m V , h a v e i npu t res i s tances of 2 to 60 m i l , and can

4 . Physiological Properties of Vertebrate Nerve Cells 1 0 7

FIGURE 4. Living rat dorsal root ganglion cells. A and B . Isolated, ovoid neurons with one process seen emanating from soma. C. Two neurons lying opposed to one another. D. Two neurons (arrows) enmeshed in background cells and partially buried. E. Dorsal root ganglion cell buried and flattened (arrow). Processes of this cell and those in D not visible. F . Group of approxi-mately six neurons in a cluster with numerous neuronal processes projecting in several directions. The relatively spherical neurons become bright in the phase contrast microscope and when pho-tographed appear to have a halo.

genera te a l l -or -noth ing ove r shoo t ing ac t ion poten t ia l s . Different cells p roduce

ac t ion poten t ia l s w i t h different p roper t ies . Fo r example , F igures 6 A and B

il lustrate two ac t ion potent ia l s from two ch ick spinal cord cells w h i c h b o t h h a d

res t ing potent ia l s of —53 m V . T h e ac t ion po ten t ia l in 6 A has a m o r e rap id rate

of r i se , larger overshoo t , a n d m o r e rap id fall t han the sp ike in 6 B . A n o t h e r

character is t ic w h i c h differs from cell to cell is the degree of repe t i t ive firing

p roduced b y a long pu lse o f current . T h e cell in 6 C p roduced a t ra in o f sp ikes

wh i l e the cell in F igure 6 D could on ly p roduce o n e ac t ion po ten t ia l before

a c c o m m o d a t i n g . A c t i o n potent ia l s from all the cells s tud ied in c h i c k sp ina l

cord cul tures or rat cor t ical cul tures w e r e ve ry sens i t ive to te t rodotoxin , an

agent k n o w n to b lock the act ive s o d i u m sp ike m e c h a n i s m .

W h e n n e u r o n s f rom ch ick sp ina l cord or rat cor tex cul tures are impa led ,

m a n y of the cells e x h i b i t smal l s p o n t a n e o u s depola r iz ing or hyperpo la r i z ing

potent ia ls . T h e s e po ten t ia l s have the appearance of typical exci ta tory post -

synapt ic potent ia l s (EPSPs ) or i nh ib i t o ry pos t synap t i c po ten t ia l s ( IPSPs) seen

108 Marc A. Dichter

FIGURE 5. Electron microscopy of chick spinal cord neurons. A. View of portion of cell surface.

Note lack of glial covering for much of the surface. B . Two neuronal somata and contacting

processes. Note that at the junction of the two cells a process forms a synapse with the cell on the

right (arrow) and contacts the other cell also (although without a synaptic ending). C. Neuropil of

culture. The process running vertically across the figure receives several synaptic inputs from

other processes (arrows). Fixed in O s 0 4 . Electron micrographs kindly provided by Dr. John

Heuser.

JJJM

FIGURE 6. Action potentials in chick spinal cord cells. A and B. Two cells with steady resting po-

tentials of 53 mV. The action potential in B has a slower rise and is of longer duration than that in

A. C and D. Response to prolonged depolarizing current pulses in two cells. C. Repetitive firing

for as long as the current persists. D. Cell fires only one action potential despite continued

suprathreshold stimulation. E. Abolition of action potential following addition of tetrodotoxin

(10~ 7 gm/ml) to the media. The bump in the depolarizing potential is due to delayed rectification

and is not an active response. Calibration: A - D , 50 mV; A, B , 5 msec; C, D, 50 msec; E, 10 mV, 5 msec.

From Fischbach and Dichter (12).

A

c D E

4. Physiological Properties of Vertebrate Nerve Cells 109

in situ. F igure 7 A i l lustrates spon taneous ly occurr ing E P S P s in ch ick spinal

cord n e u r o n s . T h e E P S P s occas iona l ly reach th resho ld and p roduce an ac t ion

potent ia l . F igure 7B s h o w s record ings from two cells w h i c h had spon taneous ly

occurr ing IPSPs (one of w h i c h also had spon taneous E P S P s ) . T h e P S P s we re of

va ry ing s izes and t ime courses . T h e larger P S P s d i sappear w h e n te t rodotoxin

is added to the m e d i u m ind ica t ing that they p r o b a b l y arise as a result o f spon-

taneous ac t ion potent ia l act ivi ty in the p resynap t ic cell . T h e smal ler P S P s

r ema in even in the p resence of te t rodotoxin and are p robab ly due to spon tane -

ous re lease of t ransmi t te r from the presynapt ic cell , that i s , t hey are min ia tu re

M o s t pos t synap t i c potent ia l s in the ver tebra te central ne rvous sys t em are a

result o f a pe rmeab i l i t y change to specific an ions or ca t ions in the pos t -

synapt ic m e m b r a n e . O n e of the cri t ical charac ter is t ics of such a change in per-

meab i l i t y is that the s ize of the pos t synapt ic potent ia l is p ropor t iona l to the

difference b e t w e e n the res t ing potent ia l of the cell and the e q u i l i b r i u m po ten-

tial of the ion(s) w h o s e pe rmeab i l i t y has jus t b e e n inc reased . There fo re , if the

res t ing poten t ia l of the cell is inc reased , that i s , if it is m o v e d further from the

E P S P e q u i l i b r i u m potent ia l , the s ize of an E P S P should inc rease . O n the o ther

h a n d , s ince the e q u i l i b r i u m potent ia l of the IPSP is m o r e nega t ive than the

res t ing poten t ia l , hyperpo la r iza t ion of the cell wil l cause the IPSP to decrease

in s ize . In e i ther case , i f the res t ing potent ia l is c h a n g e d sufficiently to go

b e y o n d the P S P e q u i l i b r i u m poten t ia l , the E P S P or IPSP wou ld invert . F igure

8 i l lustrates two cells w i th spon taneous E P S P s and IPSPs . In F igure 8 A the

E P S P s get smal ler as the cell is depolar ized and inver t to nega t ive potent ia l s

as the cell is depolar ized b e y o n d —5 m V . F igure 8B i l lustrates spon taneous

IPSPs w h i c h inver t w h e n the cell is hyperpo la r ized b e y o n d the IPSP e q u i l i b -

r ium potent ia l . T h u s , the spon taneous potent ia ls w h i c h r e s e m b l e P S P s also

demons t ra te phys io log ica l character is t ics cons i s t en t w i th chemica l synapt ic

potent ia ls .

U s i n g o n e intracel lular microe lec t rode and a m o b i l e extracellular s t imula t ing

microe lec t rode or two intracel lular mic roe lec t rodes , the ne twork proper t ies of

FIGURE 7. Spontaneous postsynaptic potentials in chick spinal cord cells. A. Recurrent, high gain oscilloscope sweeps showing spontaneous EPSPs which occasionally reach threshold and trigger action potentials (off screen). B . Recurrent oscilloscope sweeps illustrating spontaneous IPSPs (top) in one cell and both spontaneous EPSPs and IPSPs in another cell (bottom). Calibration: Vertical bar = A, 10 mV; B , 12 mV. Horizontal bar = A, 5 msec; B , 10 msec. From Fischbach and Dichter (12).

P S P s .

A B

110 Marc A. Dichter

A

B

FIGURE 8. Inversion of synaptic potentials. A. Repetitive oscilloscope sweeps showing decrease

in EPSP size with cell depolarization from Vm = - 4 4 mV (bottom) t o - 2 2 mV (middle) and in-

version with depolarization beyond the EPSP equilibrium potential (VM = + 4 mV - top trace).

A constant pulse of current was injected in the middle of each sweep. B. Inversion of an IPSP with

cell hyperpolarization from Vm = - 2 8 mV (top) to Vm = - 4 0 mV (bottom). From Fischbach and

Dichter (12).

the neu rons in vitro can b e s tudied . S o m e n e u r o n s in sp ina l cord or cor tex cul-

tures form synapt ic connec t i ons b a c k on themse lves—ei the r direct ly or

th rough an in te rneuron . Recur ren t c o n n e c t i o n s , b o t h exci ta tory and i n h i b i -

tory, are fundamenta l bu i ld ing b locks in the ver tebra te C N S and can b e seen

from spinal cord to cor tex in the in tact an imal .

A l though the n e u r o n s can connec t w i t h one ano ther in cul ture they do not

form funct ional synapses wi th every o ther neu ron they contact . In fact, prox-

imi ty is not a neces sa ry r e q u i r e m e n t for format ion of connec t ions . S o m e cells

have synapt ic connec t i ons w i th ano ther cell hundreds of mic romete r s away in

the d ish bu t not w i t h several in a n e a r b y cluster . S o m e of the n e u r o n pairs in

the cul tures are reciprocal ly connec t ed wh i l e m a n y are not . T h e r e are f requent

examples o f several neu rons b e i n g inne rva ted b y o n e c o m m o n neuron .

S e n s o r y gang l ion cells in culture form synapses wi th spinal cord neu rons

bu t only rarely have synapses b e e n found b e t w e e n two sensory gangl ion cells.

Sp ina l cord neu rons form funct ional synapses w i th skeletal musc le cells in cul-

ture. Dorsa l root gang l ion cells have neve r b e e n seen to form funct ional

synapses w i th musc le cel ls . It is no t k n o w n i f the pat terns of synapse format ion

seen in these cul tures are due to the preserva t ion of in t r ins ic specif ici ty in the

cul tures or to nonspec i f ic factors.

4 . Physiological Properties of Vertebrate Nerve Cells 111

USE OF THE CULTURE SYSTEM AS A MODEL OF NEURONAL FUNCTION

Dorsa l root gang l ion cells from b o t h ch i ck and rat ma in t a in the i r m o r p h o -

logical d i f ferent ia t ion in the cul ture sys tem. T h i s is no t a funct ion of the cul-

ture cond i t i ons b u t ra ther appears as a m a i n t e n a n c e of the dif ferent ia t ion

w h i c h takes place in vivo. T h e r e are also phys io log ica l funct ions w h i c h serve

to d i s t ingu i sh sensory gangl ion cells f rom central neu rons . D e s p i t e the fact

that these cells are large, are fairly access ib le to phys io log i s t s , and are ra ther

impor tan t as the neu rons w h i c h are in te rposed b e t w e e n the external wor ld

and the central ne rvous sys tem, thei r cellular p roper t ies have b e e n little s tud-

ied b y phys io log i s t s .

C h i c k dorsal root gangl ion cells in cul ture have an unusua l ac t ion potent ia l

( 3 , 4 , 9 , 1 8 , 2 1 ) . F igure 9 compare s a typical ch ick sp ina l cord cell ac t ion po ten-

tial w i t h that from a ch i ck senso ry gang l ion cell . No te the s l ight ly s lower rate

of r i se and , m o r e s ignif icant ly , the b reak in the falling phase of the ac t ion po-

tent ial . T h i s is s een in the major i ty of ch i ck senso ry gang l ion cells s tud ied and

is no t corre la ted w i t h cell injury. S u c h a sp ike is neve r s een in a ch ick spinal

cord n e u r o n or in a rat cort ical neu ron . O n further ana lys i s , th is ac t ion po ten-

tial is gene ra ted b y a different m e c h a n i s m than that of spinal cord neu rons .

Te t rodo tox in does not abo l i sh th is sp ike , bu t does lower the rate of r i se .

R e m o v a l of all external N a ions s imi lar ly does no t abo l i sh the sp ike . H o w e v e r ,

if all external Ca is also r emoved , or i f an i n h i b i t o r of Ca sp ikes ( i . e . , C o C l 2 ) is

added to the Na-free or te t rodotoxin m e d i u m , the sp ike is abo l i shed . R e m o v a l

of Ca a lone or add i t ion of C o C l 2 a lone causes the d i sappearance of the b reak in

the falling phase and m a k e s the sp ike th inne r ( r e sembl ing n o w a sp ina l cord

5 mV, 5 msec

FIGURE 9. Chick spinal cord (SC) and dorsal root ganglion ( D R G ) cell action potentials illustrated at two sweep speeds.

sc

drg


neuron sp ike) . T h e r e m a i n i n g th in sp ike is t hen sens i t ive to te t rodotoxin . T h u s , the ch ick dorsal root gang l ion cell soma act ion poten t ia l is p roduced b y bo th a N a and Ca m e c h a n i s m together . Fur ther conf i rmat ion o f th i s o b t a i n e d b y m e a s u r e m e n t s of ac t ion potent ia l proper t ies in so lu t ions con ta in ing dif-ferent concen t ra t ions of o n e of the act ive i o n s , in the a b s e n c e of the o ther (Figure 10 ) .

In teres t ingly , it can b e s h o w n that a l though the sensory gang l ion cell s o m a

sp ike -gene ra t ing m e c h a n i s m cons i s t s o f b o t h a N a and C a c o m p o n e n t , the

axons of these cells ut i l ize exc lus ive ly , or p r edominan t ly , a N a m e c h a n i s m .

T h e axon is capab le o f firing h i g h f requency , repet i t ive d i scharges of th in

sp ikes wh i l e the soma can on ly fire at lower f r equenc ies and wi th fatter

sp ikes . S i n c e the s o m a is s i tua ted in such a pos i t ion in th is p seudoun ipo la r

cell that impu l se s do no t have to pass t h rough it, the re levance of such an

a r r angemen t in the in tact an ima l is no t k n o w n .

A n o t h e r in te res t ing , and pe rhaps u n i q u e , proper ty of these cells is that in

s o m e , bu t no t all, ch ick s enso ry gang l ion cells the s o m a act ion potent ia l is

fol lowed b y a p ro longed large depolar iz ing poten t ia l (4 ,9 ) . T h i s potent ia l is

genera ted b y a large conduc tance inc rease and d i sappears w h e n C a is r e m o v e d

from the m e d i u m . It is a ssoc ia ted on ly w i th the C a c o m p o n e n t o f the s o m a

ac t ion potent ia l . It r e m a i n s in Na-free m e d i u m o r te t rodotoxin m e d i u m even

w h e n the axon i s u n a b l e to genera te an ac t ion potent ia l . T h e exact o r ig in of th i s

potent ia l is not k n o w n , b u t it does no t appear to b e a synapt ic potent ia l . Its

occur rence in the in tac t adult gang l ion is no t k n o w n .

T h e p re sence of b o t h a re la t ively p ro longed act ion potent ia l and a p ro longed

late depolar iz ing w a v e in the somata of ch ick dorsal root gang l ion cells ha s the

potent ia l for s ignif icant ly affecting the t r ansmis s ion o f impu l se s a long the

Na a Co Ha-Free C a - F r e e

Abol ished by C o C L Abo l ished by T T X

i lOmV 5msec No change with T T X No change with C o C L

FIGURE 10. Chick dorsal root ganglion cell soma action mechanism. Representative action poten-tials in control medium (145 mM Na, 1.8 mM Ca), Na-free medium (1.8 mM Ca), and Ca-free medium (145 mM Na). Note the overshooting action potential in all three media but the marked differences in threshold, rate of rise, and duration.

4 . Physiological Properties of Vertebrate Nerve Cells 113

gangl ion cell axon from the pe r iphe ry to the sp ina l cord . In adult dorsal root

gangl ia the s o m a g ives off o n e axon w h i c h then b r a n c h e s in to a central ly and

per iphera l ly d i rec ted b r a n c h (pseudounipo la r ) . A s impu l se s t ravel up the

axon from the pe r iphe ry at re la t ively h i g h f r equenc ies , there is a var iab i l i ty in

the conduc t ion o f these impu l se s past the b r a n c h po in t such that , a b o v e cer-

tain f r equenc ies , the impu l se s do not invade the b r a n c h to the cell b o d y and at

o ther f r equenc ies t r ansmis s ion to the sp ina l cord is b locked ( 1 3 , 1 9 , 2 0 ) . T h e

character is t ics o f t r ansmis s ion th rough th is b r a n c h po in t are d e p e n d e n t on the

phys ica l charac ter is t ics of the axon , the e lec t rophys io log ica l charac ter i s t ics of

the axon sp ike ( i . e . , dura t ion of sp ike , dura t ion of refractory pe r iod , sp ike af-

te rpolar iza t ions , e tc . ) and t r ansmis s ion in n e a r b y f ibers . A long- las t ing depo-

lar iz ing current o r ig ina t ing from the s o m a late depolar iza t ion could o b -

v ious ly affect such a m e c h a n i s m . T h e adult ch i cken dorsal root gang l ion cell

has no t b e e n s tud ied and it is no t k n o w n i f the ac t ion po ten t ia l charac ter i s t ics

of these cells in vitro wil l b e found in the cells in the adult.

SUMMARY

Ver teb ra t e n e u r o n s in t i ssue culture are p rov id ing us w i th a n e w m o d e l

sys t em for s tudy ing the complex even ts w h i c h occur dur ing neu rona l differen-

t ia t ion , synap togenes i s , and neura l ne twork format ion . It is a l ready apparen t

that d i s soc ia ted e m b r y o n e u r o n s are capab le o f dif ferent ia t ing b o t h m o r p h o -

logical ly and phys io log ica l ly a long p r ede t e rmined l ines in the a b s e n c e of ex-

ternal in f luences . T h e s e n e u r o n s can form n e w c o n n e c t i o n s w i t h o n e ano the r

bu t re ta in s o m e specif ic i ty in the i r se lec t ions . B o t h s imple and c o m p l e x neura l

ne tworks can b e seen .

At the p resen t t i m e , the deve lopmen t o f the in vitro m o d e l sy s t em is jus t

b e i n g explored . T h e potent ia l va lue of a sys t em of th i s k i n d at a var ie ty of in-

ves t iga t ive levels shou ld b e apprec ia ted . Q u e s t i o n s of a fundamenta l na ture in

neu rob io logy , such as h o w synapses fo rm, w h a t rules govern such in te rac t ion ,

h o w cells r ecogn ize o n e another , and the na ture of the bas i c two- , th ree- , or

four-cell c i rcui ts that compr i s e the m o r e complex ne rvous t i s sue can b e ap-

p roached w i t h th i s sys t em. S tud ies of the n e u r o n s and synapses t hemse lve s can

lead to a m o r e bas i c unde r s t and ing o f ve r tebra te ne rvous sys t em func t ion ing .

T h e deve lopmen t o f cer ta in pa thophys io log ica l p roces se s and the effects of

neuroac t ive drugs on ver tebra te n e u r o n s m a y b e s tud ied at the cellular level .

F inal ly , the bas ic m e c h a n i s m of s o m e gene t ic abnorma l i t i e s w h i c h p roduce

a b n o r m a l ne rvous s t ructure and funct ion m a y b e m o r e eas i ly d e t e r m i n e d in a

s impli f ied in vitro m o d e l than in the intact centra l ne rvous sys t em.

T h e value of any m o d e l is no t i nhe ren t in the e legance of the m o d e l itself,

bu t on ly in its ab i l i ty to sugges t answers to fundamenta l q u e s t i o n s abou t the

sys tem b e i n g m o d e l e d . M a n y fundamenta l ques t i ons abou t b r a in m e c h a n i s m s

114 Marc A. Dichter

in men ta l re tardat ion r ema in u n a n s w e r e d . Pe rhaps s o m e day the mode l of

nerve cells in t i ssue cul ture wil l b r ing us c loser to the answers to these q u e s -

t ions .

REFERENCES

1. Barkley, D. , Rakic, L. , Chaffee, J . , and Wong, D. Cell separation by velocity sedimentation of postnatal mouse cerebellum. /. Cell. Physiol, 1973, 18: 271-280.

2. Cavanaugh, M. W. Neuron development from trypsin dissociated cells of differentiated spinal cord of the chick embryo. Exp. Cell Res., 1955, 9: 4 2 ^ 8 .

3. Crain, S. Resting and action potentials of cultured chick embryo spinal ganglion cells. /. Comp. Neurol, 1956, 104: 285-329.

4. Crain, S. Intracellular recordings suggesting synaptic functions in chick embryo spinal sen-sory ganglion cells isolated in vitro. Brain Res., 1971, 26: 188-191.

5. Crain, S. Tissue culture models of developing brain functions. In: Studies on the Development of Behavior and the Nervous System. Vol. 2. (G. Gottlieb, Ed.). Academic Press, New York, 1974: 69-114.

6. Crain, S., and Bornstein, M. Organotypic bioelectric activity in cultural reaggregates of disso-ciated rodent brain cells. Science, 1972, 176: 182-184.

7. De Long, G. R. Histogenesis of fetal mouse isocortex and hippocampus in reaggregating cell cultures. Dev. Biol, 1970, 22 : 563-583.

8. De Long, G. R. and Sidman, R. Alignment defect of reaggregating cells in cultures of devel-oping brains of reeler mutant mice. Dev. Biol, 1970, 22: 584-600 .

9. Dichter, M. , and Fischbach, G. Physiological properties of chick dorsal root ganglion cells grown in dissociated cell cultures. (In preparation.)

10. Fischbach, G. Synaptic potentials recorded in cell cultures of nerve and muscle. Science, 1970, 169: 1331-1333.

11. Fischbach, G. Synapse formation between dissociated nerve and muscle cells in low density cultures. Dev. Biol, 1972, 28: 407-429.

12. Fischbach, G., and Dichter, M. Electrophysiologic and morphologic properties of neurons in dissociated chick spinal cord cells cultures. Dev. Biol, 1974, 37: 100-116.

13. Ito, M., and Saiga, M. The mode of impulse conduction through the spinal ganglion. Jap. J . Physiol, 1959, 9: 3 3 ^ 2 .

14. Moscona, A. Recombination of dissociated cells and the development of cell aggregates. In: Cells and Tissue in Culture. Vol. 1, (E. N. Willmer, Ed.). Academic Press, New York, 1965: 489-529.

15. Peacock, J . , and Nelson, P. Synaptogenesis in cell cultures of neurones and myotubes from chickens with muscular dystrophy. / . Neurol. Neurosurg. Psychiatry, 1973. 36: 3 8 9 - 398.

16. Peacock, J . , Nelson, P., and Goldstone, M. Electrophysiologic study of cultured neurons dis-sociated from spinal cords and dorsal root ganglia of fetal mice. Dev. Biol, 1973, 30: 137-152.

17. Sato, G. (Ed.) Tissue Culture of the Nervous System. Plenum, New York, 1973. 18. Scott, B . , Engelbert, V. , and Fisher, K. Morphological and electrophysiological characteristics

of dissociated chick embryonic spinal ganglion cells in culture. Exp. Neurol, 1969, 23: 230-248.

19. Tagini, G. Aspetti del potenziale di azione nelle cellule dei gangli spinali di rana. Boll Soc. Hal Biol. Sper., 1971, 47: 130-132.

20. Tagini, G. Risposta delle cellule di gangli spinali di rana a stimoli di diversa frequenza. Boll Soc. Ital. Biol. Sper., 1971, 47: 132-133.

21. Varon, S., and Raiborn, C. Excitability and conduction in neurons of dissociated ganglionic cell cultures. Brain Res., 1971, 30: 83-98.

4A Discussion: Biochemical Studies

in Various Culture Systems of Neural Tissues

J. de VELLIS, G. A. M. BREEN,* and J. F. McGINNIS

Mental Retardation Research Center and Department of Anatomy,

University of California Los Angeles,


T h e e lec t rophys io logica l s tudies p resen ted b y Dr . D ich t e r p rov ide clear ev i -

dence that d i s soc ia ted neu rons from a var ie ty of e m b r y o n i c ne rvous t i ssues

survive for several w e e k s in l o w dens i ty cul tures and form funct ional exci ta-

tory and i nh ib i t o ry synapses . T h e s imilar i ty often obse rved b e t w e e n the elec-

t rophys io logica l p roper t ies of neu rons in cul ture and in vivo va l ida tes t i ssue

culture as a mode l to s tudy synapse format ion and different iated b ioe lec t r ic

proper t ies . Before p r o c e e d i n g to the d i scuss ion of b i o c h e m i c a l p roper t ies of

neura l t i s sues in cul ture it i s w o r t h w h i l e to desc r ibe br ief ly the va r ious m e t h o d s

of t i s sue cul ture ava i lab le and then the advantages and l imi ta t ions of each.

The re are four bas i c types , classif ied accord ing to h o w the t i s sue is pro-

cessed and m a i n t a i n e d in culture. Firs t , an explant culture cons i s t s of small

p i eces of t i ssue (0.5 to 1 m m 3 ) d i ssec ted from the organ. N u m e r o u s t e c h n i q u e s

have b e e n used to g row t h e m in cul ture. Recen t ly w e dev i sed a s imple m e t h o d

to g row large n u m b e r s of explants b e t w e e n str ips of perforated ce l lophane

(7 ,9) . T h e second sys t em is cal led d i s soc ia ted cell cul ture b e c a u s e the t i ssue is

d i ssoc ia ted enzymat ica l ly or mechan ica l ly to a s u s p e n s i o n of s ingle cells. T h e

d i spersed cells are cul tured on the pre t reated surface of a d i sh e i the r at l ow

dens i ty o r at h i g h dens i ty . At l ow dens i ty the cell b o d i e s will r e m a i n sepa-

rated from each o the r as s een in Dr . D ich te r ' s cul tures . O n the o ther h a n d , at

* Present address: Molecular Biology Department, Roswell Park Memorial Institute, Buffalo, New York.

116 /. de Vellis, G. A. M. Breen, and J. F. McGinnis

h igh dens i ty , cells will form small clusters on top of a b a c k g r o u n d layer. T h e

third type of t i s sue culture p rov ides cond i t ions that wi l l p romote the forma-

t ion of aggregates . D i s soc i a t ed cells are p laced in a ro ta t ing flask. As a result ,

w i t h i n a few hours small round aggregates (0 .6 to 2 m m ) are formed ( 1 4 , 2 2 , 2 3 ,

32 ) . T h e fourth sys t em is c lonal cell l ines e s t ab l i shed b y cul tur ing the p rogeny

of a s ingle cell. T h e cul ture, therefore , cons i s t s o f cells genet ica l ly h o m o g e -

neous . T h e cell l ine does no t n e e d to b e subcul tured con t inuous ly s ince it can

b e kept frozen for long pe r iods o f t i m e , thus preserv ing the or ig ina l gene t ic

m a k e u p . Dur ing con t inuous cul ture, mu ta t ions and c h r o m o s o m a l loss m a y

occur w h i c h s o m e t i m e s can p rov ide useful mutan t s . Un l ike connec t ive t i ssue

from w h i c h f ibroblast cell l ines have b e e n e s t ab l i shed , it has no t ye t b e e n pos-

s ib le to ob ta in cell l ines from normal neura l t i s sues bu t only from tumors [for

r ev iew, see M c M o r r i s et al. (26) ] .

Each of the sys t ems desc r ibed has the advantage of p rov id ing the exper i -

m e n t e r w i t h a b roader range of control o f the phys ica l and chemica l env i ron-

men t of the cells than is poss ib l e w i th the intact an imal . S u b s t a n c e s can b e

qu ick ly added or w i t h d r a w n from the culture m e d i u m a l lowing a prec ise t em-

poral analys is of the s e q u e n c e of events w h i c h for ins t ance occur in h o r m o n e

act ion or cell d i f ferent ia t ion. T i s s u e culture also c i r cumven t s the p r o b l e m of

the b l o o d - b r a i n barr ier . E a c h of the types of culture sys tems offers part icular

advantages . D i s soc i a t ed cell cul tures al low the v i sua l iza t ion of ind iv idua l l iv-

ing cells w h i c h can b e mon i to r ed dur ing cul ture morpholog ica l ly and elec-

t rophysiological ly . A l though no t ye t ach i eved it shou ld also b e pos s ib l e to o b -

ta in and correlate b i o c h e m i c a l , morpho log ica l , and e lec t rophys io logica l data

from a s ingle cell. A n al ternat ive to obse rv ing and quan t i t a t ing va r i ab les in a

s ingle cell i s to separate in to pure fract ions the cell types presen t in the sus-

p e n s i o n of cells and t hen cul tur ing each fraction separate ly o r in va r ious c o m -

b ina t i ons (2) . T h i s wou ld great ly faci l i tate , for in s t ance , the s tudy of the role of

he te ro typic ce l l -ce l l contac ts in cell d i f ferent ia t ion (36) and of the specif ici ty of

synapse format ion. E n r i c h e d bu t not totally pure cell cul tures can b e ob t a ined

b y select ive pla t ing (38 ,39) or se lect ive m e d i a (6 ,24) . O n e of the advantages of

aggregate cul tures over d i s soc ia ted cell cul tures is to p rov ide t r id imens iona l

g rowth . T h e cells i n s ide the aggregate are first d i s t r ibu ted at r a n d o m then

they sort out in to pat terns often r e s e m b l i n g the organiza t ion seen in vivo

( 9 , 1 4 , 1 5 , 3 2 , 3 6 ) . In aggregates the v i sua l iza t ion o f ind iv idua l cells is lost. T h i s

prepara t ion does no t p resen t real advantages for the e lec t rophysio logis t . As

for d i ssoc ia ted cul tures , cell types can b e separa ted before aggregat ion . Un l ike

the two types of t i ssue culture jus t desc r ibed , explants conserve the or ig ina l

t i ssue organiza t ion . It is a s s u m e d that no rmal cel l -cel l contacts are m a i n -

ta ined . O f course , the o rgan iza t ion can b e d i s tu rbed b y death of cells and pos -

s ib ly b y n e w cell contac ts a r i s ing in long- te rm cul tures . T h e impor t ance of

t r id imens iona l t i ssue o rgan iza t ion is i l lustrated b y the obse rva t ion that glu-

t amine syn the tase , a marker o f ret inal different ia t ion, is i nduc ib le b y hydro-

4A. Biochemical Studies in Various Culture Systems 117

cor t i sone in aggregates bu t no t in d i s soc ia ted cell cul tures (27 ,28) . A n d finally

c lonal cell l ines p rov ide h o m o g e n e o u s cell popu la t ions in large quan t i t i e s in a

very reproduc ib le m a n n e r . T h e s e cul tures are the eas ies t for the b i o c h e m i s t to

s tudy w i t h s tandard b i o c h e m i c a l assay w i t h o u t resor t ing to s emiquan t i t a t i ve

h i s tochemica l or microanaly t ica l t e c h n i q u e s . T h i s p repara t ion is idea l to relate

structure and funct ions at the cellular and molecular level b y an in te rd is -

c ip l inary s tudy a m o n g ana tomis t s , b i o c h e m i s t s , e lec t rophys io log is t s , and

pharmaco log i s t s . A l though tumor in o r ig in , clonal neura l cell l ines have con-

t r ibu ted useful knowledge in n e u r o b i o l o g y dur ing the last 6 years (26) .

Av ian and m a m m a l i a n fetal neura l t i s sues different iate wel l in the three

types of p r imary cul ture. Func t iona l synapse s have b e e n demons t r a t ed in

explants ( 1 0 , 1 1 , 1 3 , 3 7 ) and d i s soc ia ted cell cul tures ( 3 , 4 , 1 2 , 2 0 , 2 1 , 3 0 ) . Ag-

gregates in rotary cul tures have morpho log ica l ly different ia ted synapses (34)

bu t the i r e l ec t rophys io logy is not ye t k n o w n . Ultrastructural s tudies have also

revealed mye l ina t i on in all three sys tems . T h e specif ic i ty of synapse format ion

in cell cul tures has often b e e n ques t i oned . H o w e v e r , recent ly O l s o n and

B u n g e (29) s h o w e d that synapses in explants o f fetal super io r cervica l gangl ia

fo rmed w h e n the gangl ia we re cul tured w i t h sp ina l cord , bu t no t w h e n cul-

tured wi th o ther super io r cervical gangl ia or cerebra l explants . B i o c h e m i c a l

s tudies of p r imary cul tures have dealt largely w i th e n z y m e s of neuro t rans -

mi t te r syn thes i s and degrada t ion such as cho l ine acetyl t ransferase , ace-

ty lchol ines te rase , and g lu tamate decarboxylase w h i c h have b e e n s h o w n to

increase w i t h t ime in cul ture in c lose re la t ion to the in vivo d eve lopmen t

( 3 1 - 3 3 , 3 5 , 3 7 , 4 0 , 4 1 ) . H o w e v e r , the level o f these e n z y m e s , par t icular ly in d i s so-

cia ted cul tures , is m u c h lower than in vivo ( 3 1 , 3 5 , 4 0 , 4 1 ) . A m o n g several poss i -

b i l i t ies (8) is the lack of appropr ia te s t imulus . For th is reason , w e have focused

on the s tudy of pa ramete rs of neural dif ferent ia t ion w h i c h are control led b y

h o r m o n e s (16 ,19) and s o m e of our s tudies wil l b e brief ly ou t l ined to i l lustrate

the use of the va r ious cul ture m e t h o d s .

T h e e n z y m e g lycero lphospha te dehyd rogenase (E .C . 1 .1 .1 .8 , G P D H ) is spe-

cifically i nduc ib l e b y g lucocor t ico ids in the va r ious areas of the ne rvous

sys t em that have b e e n tes ted (17) . T h i s i nduc t ion is not found in o ther o rgans .

A l though the r eason for th i s phys io log ica l g lucocor t ico id ac t ion h a s no t ye t

b e e n clarified, it e s t ab l i shes the b ra in as a major target o rgan for g lucocor-

t icoids . U s i n g the four types of t i ssue cul ture, w e have a t t empted to define the

factors w h i c h regulate the express ion of G P D H in bra in . In explants (Figure 1 ) ,

d i s soc ia ted cell cul tures (Figure 2 ) , and aggregates (9) , the level o f the e n z y m e

r ema ins ex t remely low in the a b s e n c e of g lucocor t ico ids even after several

w e e k s in cul ture ( 7 - 9 ) . W h e n hydrocor t i sone is added to the cul ture m e d i u m

G P D H act iv i ty r i ses to levels w h i c h approx ima te those in vivo in less than 5

days b y w h i c h t ime it has r eached a n e w s teady state. T h e s e resul ts i l lustrate

that w i thou t the k n o w l e d g e of the h o r m o n a l r e q u i r e m e n t w e w o u l d have

conc luded that p r imary rat cerebra l cul tures do no t different iate w i th respect


Days of HC

FIGURE 1. Time course of GPDH induction in rat cerebral explants. Hydrocortisone 0.5 /xg/ml (HC) was added to the media for the number of days indicated. Each point represents the average specific activity of GPDH of 4 cultures ± S.D. From Breen and de Vellis (7).

TIME COURSE OF GPDH I N D U C T I O N BY HC

, HC

C o n t r o l

Days of HC

FIGURE 2. Time course of GPDH induction in rat cerebral dissociated cell cultures. Same condi-tions as in Figure 1. From Breen and de Vellis (8).

GP

DH

(u

nit

s/m

g

pro

tein

)

GP

DH

(u

nits

/m

g p

rote

in)

INDUCTION OF GPDH

4A. Biochemical Studies in Various Culture Systems 1 1 9

to G P D H s ince the basa l level is low and r ema ins cons tan t t h roughou t the cul-

ture per iod . G P D H is also i nduc ib l e in a c lonal glial t u m o r cell l ine , C6 (16 ,19) .

T h e t ime course of the induc t ion in s ta t ionary phase mono laye r C6 cells (Fig-

ure 3) is s imi lar to that o b s e r v e d in p r imary cul tures (F igures 1 and 2 ) . H o w -

ever , the basa l and i nduced levels o f G P D H are h i g h e r in the C6 cell l ine than

in p r imary cul tures and in vivo, pe rhaps ref lect ing d i lu t ions of G P D H specific

act ivi ty b y G P D H deficient cell types . O t h e r character is t ics o f G P D H induc-

t ion such as sens i t iv i ty to i nh ib i to r s of R N A and pro te in syn theses are very

s imi lar in all four cul ture sys tems .

F r o m these resul ts w e can reach two conc lus ions . Firs t , G P D H induc t ion ,

unl ike g lu tamine syn the tase in the re t ina (27 ,28 ) , does no t r equ i re the ce l l -ce l l

contac t p resen t in explant or aggregates s ince it is fully i nduc ib l e in d i ssoc i -

ated cell cul tures . S e c o n d , the ex tens ive s imi lar i ty of the induc t ion character-

is t ics in all types of culture sys t ems sugges ts that the t u m o r cell l ine , C 6 , is a

val id m o d e l to s tudy s o m e aspects o f G P D H induc t ion . H o w e v e r , reg iona l

s tudies of the ne rvous sys t em could on ly b e done us ing p r imary cul tures .

500

5 0 H

1 2 3 4 5

Days of HC

FIGURE 3. Time course of GPDH induction in C6 cells. Same conditions as in Figure 1 except that

HC concentration was 0.2 /xg/ml. From de Vellis and Inglish (18).

GP

DH

,! U

nit

s/m

g p

rote

in )


6

o 1

0 8 0 0.4 1.2 1.6 2.0

ul A b

FIGURE 4. Immunological titration of GPDH from control and hydrocortisone-induced cells. Con-

stant amounts of GPDH from induced ( • ) and uninduced cells ( O ) were reacted with increasing

amounts of GPDH antisera. The antibody-GPDH complex was enzymatically inactive. The

amount of enzyme remaining after incubation with antiserum was substracted from that in the

absence of antiserum and the results were plotted as the number of units of enzyme removed

against the microliters of antiserum present. From McGinnis and de Vellis (25).

The m e c h a n i s m s of G P D H induc t ion b y hydrocor t i sone at the molecu la r and

cellular level can bes t b e inves t iga ted in the clonal C6 cell l ine s ince i ts cul tures

are h o m o g e n e o u s d ip lo id cel ls . The re exis ts a s ingle molecular form of G P D H

for b o t h i nduced and basa l s tates (25) . T h e inc reased G P D H act ivi ty found in

the cul tures after addi t ion of C o r t i s o l could result from an increase in the

n u m b e r of G P D H molecu les or in an inc rease in the catalytic act ivi ty of each

G P D H molecule . U s i n g G P D H ant i sera w e s h o w e d that the enzymat i c act ivi ty

per G P D H molecu le (an t igen) r ema ins the s ame in induced and n o n i n d u c e d

cells s ince a g iven a m o u n t o f an t i se ra inac t iva tes the s ame a m o u n t o f e n z y m e

act ivi ty in each (Figure 4 ) . S imi la r ly , the decrease in b ra in G P D H act ivi ty fol-

l owing h y p o p h y s e c t o m y in vivo was s h o w n to b e due to fewer G P D H mole -

cules .

In conc lus ion , w e can state that recent advances are m a k i n g t i ssue cul ture a

useful tool for mos t b io log ica l d i sc ip l ines dea l ing w i t h the ne rvous sys tem.

T h e demons t ra t ion that cul tured cells are able to express different iated proper-

t ies is at tract ing m a n y inves t iga tors to the analys is o f the p rocess of neura l dif-

ferent ia t ion and of the factors that can inf luence it. T i s sue culture is po ten-

tially a sens i t ive and qu ick test for sc reen ing subs tances potent ia l ly harmful to

the ne rvous sys t em, part icularly to the deve lop ing ne rvous sys tem. T i s s u e cul-

ture of b ra in has b e e n used to s tudy demye l ina t ing d i seases (5) and malnut r i -

GPDH

Unit

s In

acti

vate

d

4A. Biochemical Studies in Various Culture Systems 121

t ion effects (1) . Hopeful ly , t i ssue culture m o d e l s of specific men ta l re tardat ion

dysfunct ions wi l l b e deve loped in the future.

ACKNOWLEDGMENTS

Work from the authors' laboratory was supported by grants from NICHD and USAEC.

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4. Bornstein, M. R., and Model, P. G. Development of synapses and myelin in cultures of disso-ciated embryonic mouse spinal cord, medulla and cerebrum. Brain Res., 1972, 37: 287-293.

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6. Bray, D. Branching patterns of individual sympathetic neurons in culture. / . Cell Biol, 1973,

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hydrocortisone in primary rat brain cultures. Trans. Soc. Neuroscl, 1973, 3: 427. 10. Bunge, M. B . , Bunge, R. P., and Peterson, E. R. The onset of synapse formation in spinal cord

cultures as studied by electron microscopy. Brain Res., 1967, 6: 728-749. 11. Crain, S. M. Development of "organotypic" bioelectric activities in central nervous tissues

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dissociated rodent brain cells. Science, 1972, 176: 182-184. 13. Crain, S. M., and Peterson, E. R. Onset and development of functional interneuronal connec-

tions in explants of rat spinal cord-ganglia during maturation in culture. Brain Res., 1 9 6 7 6 : 750-762.

14. De Long, G. R. Histogenesis of fetal mouse isocortex and hippocampus in reaggregating cell cultures. Dev. Biol, 1970, 22: 563 -583 .

15. De Long, G. R. , and Sidman, R. L. Alignment defect of reaggregating cells in cultures of devel-oping brains of reeler mutant mice. Dev. Biol, 1970, 22: 584-600.

16. de Vellis, J . , and Brooker, G. Induction of enzymes by glucocorticoids and catecholamines in a rat glial cell line. In: Tissue Culture of the Nervous System, Chapter 10. (G. Sato, Ed.). Plenum, New York, 1973: 231-245.

17. de Vellis, J . , and Inglish, D. Hormonal control of glycerol phosphate dehydrogenase in the rat brain. / . Neurochem., 1968, 15: 1061-1070.

18. de Vellis, J . , and Inglish, D. Age-dependent changes in the regulation of glycerol phosphate dehydrogenase in the rat brain and in a glial cell line. Progr. Brain Res., 1973, 40: 321-330.

19. de Vellis, J . , Inglish, D. , Cole, R., and Molson, J. Effects of hormones on the differentiation of


cloned lines of neurons and glial cells. In: Influence of Hormones on the Nervous System. (D. Ford, Ed.). Karger, Basel, 1971: 25-39.

20. Fischbach. G. D. Synaptic potentials recorded in cell cultures of nerve and muscle. Science, 1970, 169: 1331-1333.

21. Fischbach, G. D. Synapse formation between dissociated nerve and muscle cells in low den-sity cell cultures. Dev. Biol, 1972, 28: 407-429.

22. Garber, B. B . , and Moscona, A. A. Reconstruction of brain tissue from cell suspensions. I. Aggregation patterns of cells dissociated from different regions of the developing brain. Dev. Biol, 1972, 27: 217-234.

23. Garber, B. B . , and Moscona, A. A. Reconstruction of brain tissue from cell suspensions. II. Specific enhancement of aggregation of embryonic cerebral cells by supernatant from homol-ogous cell cultures. Dev. Biol, 1972, 27: 235-243.

24. Mains, R. E . , and Patterson, P. H. Primary cultures of dissociated sympathetic neurons. I. Es-tablishment of long-term growth in culture and studies of differentiated properties. /. Cell Biol, 1973, 59: 329-345.

25. McGinnis, J . , and de Vellis, J . Cortisol induction of glycerol phosphate dehydrogenase in a rat brain tumor cell line. Nature (Lond.), 1974, 250: 422-424.

26. McMorris, F. A., Nelson, P. G., and Ruddle, F. H. Contributions of clonal systems to neurobiology. Neuroscience Res. Bull, 1973, 11: 412-536.

27. Morris, J . E . , and Moscona, A. A. Induction of glutamine synthetase in embryonic retina: Its dependence on cell interactions. Science, 1970, 167: 1736-1738.

28. Morris, J. E . , and Moscona, A. A. The induction of glutamine synthetase in cell aggregates of embryonic neural retina: Correlations with differentiation and multicellular organization. Dev. Biol, 1971, 25: 420-444.

29. Olson, M. I. , and Bunge, R. P. Anatomical observations on the specificity of synapse forma-tion in tissue culture. Brain Res., 1973, 59: 19-33.

30. Peacock, J . H., Nelson, P. G., and Goldstone, M. W. Electrophysiological study of cultured neurons dissociated from spinal cord and dorsal root ganglia of fetal mice. Dev. Biol, 1973, 30: 137-152.

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33. Seeds, N. W. Differentiation of aggregating brain cell cultures. In: Tissue Culture of the Nervous System. (G. Sato, Ed.). Plenum, New York, 1973: 35-53.

34. Seeds, N. W., and Vatter, A. E. Synaptogenesis in reaggregating brain cell culture. Proc. Natl. Acad. Sci. USA, 1971, 68: 3219-3222.

35. Shapiro, D. L. , and Schrier, B . K. Cell cultures of fetal rat brain. Growth and marker enzyme development. Exp. Cell Res., 1973, 77: 239-247.

36. Sidman, R. L. Cell-cell recognition in the developing CNS. In: The Neurosciences: Third Study Program. (F. O. Schmitt, Ed.). MIT Press, Cambridge, Massachusetts, 1974: 743-758 .

37. Tunnicliff, G., and Kim, S. U. Synaptogenesis and the development of neurotransmitter enzymes in organotypic cultures of chick spinal cord. Brain Res., 1973, 49: 410-416.

38. Varon, S., and Raiborn, C. W., Jr. Dissociation, fractionation and culture of chick embryo sympa-thetic ganglionic cells. /. Neurocytol, 1972, 1: 2 1 1 - 2 2 1 .

39. Varon, S., and Raiborn, C. W. , Jr. Dissociation, fractionation and culture of embryonic brain cells. Brain Res., 1969, 12: 180-199.

40. Werner, I., Peterson, G. R., and Shuster, L. Choline acetyltransferase and acetylcholinesterase in cultured brain cells from chick embryos. / . Neurochem., 1971, 18: 141-151.

41. Wilson, S. H., Schrier, B . K., Farber, J . L. , Thompson, E. J . , Rosenberg, R. N., Blume, A. J . , and Nirenberg, M. W. Markers for gene expression in cultured cells from the nervous system. /. Biol. Chem., 1972, 247: 3159-3169.

5 Synaptic and Dendritic Development

and Mental Defect

P E T E R R . H U T T E N L O C H E R

Department of Pediatrics, The University of Chicago,

Chicago, Illinois

T h e pa thologica l s tudy of b r a in s from the re tarded is often d i sappo in t ing

in that it m a y fail to provide a clear exp lana t ion for the d isorder of funct ion

obse rved dur ing life. A b o u t 1 5 % of such b ra in s appear en t i re ly no rma l on stan-

dard gross and mic roscop ic e x a m i n a t i o n , and a cons ide rab ly larger p ropor t ion

s h o w s only s l ight c h a n g e s , such as b e l o w no rma l total b r a in w e i g h t or poor

different ia t ion of cort ical layers , w h i c h bea r little re la t ionsh ip to t he sever i ty

of the re tardat ion (1 ,10 ) .

T h e s e are frustrat ing cases to the pa thologis t and also to the c l in ic ian w h o is

sea rch ing for an exp lana t ion for a g iven cl inical s tate. It has b e e n ques t i oned

w h e t h e r such cases m i g h t represen t abnorma l i t i e s in the fine s t ructure of cere-

bral cor tex, inc lud ing te rmina l axons , synapses , and dendr i tes , w h i c h are no t

adequa te ly demons t r a t ed b y s tandard neuropa tho log ica l m e t h o d s . T h e r e has

b e e n little direct pa thologica l ev idence for th is hypo thes i s . H o w e v e r , exper i -

men ta l ev idence w o u l d t end to suppor t the v i e w that axonal and dendr i t ic

growth in deve lop ing b ra in is part icular ly vu lnerab le , and that it is readi ly af-

fected b y tox ic , m e t a b o l i c , and nut r i t iona l d i s tu rbances . T h e sens i t ive pe r iod

appears to b e that of m o s t rapid g rowth , w h i c h is the per ina ta l and early post-

natal pe r iod in m o s t m a m m a l i a n spec ies (5 ,29) . Examples inc lude decreased

dendr i t ic and synapt ic deve lopmen t in exper imen ta l pos tnata l hypo thy ro id -

i sm (8 ,25 ) , decreased axonal g rowth in postnata l malnu t r i t ion (19) , decreased

deve lopmen t of the neurop i l after neona ta l anox ia (18) and after pos tna ta l l ow

dose i on i z ing i r radia t ion (16 ,17 ) , and decreased dendr i t ic g rowth after ex-

posure to h igh doses of cor t icos te ro id h o r m o n e s (26 ,30) . Dendr i t i c deve lop-

m e n t has also b e e n s h o w n to b e inf luenced b y m o r e subt le env i ronmen ta l

changes . V i sua l depr iva t ion from b i r th has b e e n found to decrease dendr i t ic

123

124 Peter R. Huttenlocher

growth in calcar ine cor tex, especia l ly the deve lopmen t o f dendr i t ic sp ines on

apical dendr i tes of pyramida l cells (4 ,11 ,33 ) .

In the h u m a n , in format ion regarding the effects of var ious per ina ta l d is -

orders on dendr i t ic and synapt ic deve lopmen t is pract ical ly nonex i s t en t . T h e

FIGURE 1. Large axonal swelling containing irregularly arranged tubular structures (lead citrate, X 17,400). This figure and Figures 2 and 3 originally appeared in a report of a cortical biopsy from a child with progressive dementia [Herman, Huttenlocher, and Bensch (15)].

5. Synaptic and Dendritic Development 125

two t e c h n i q u e s used m o s t successful ly in the exper imen ta l work—the Golg i

m e t h o d and electron m i c r o s c o p y — h a v e rarely b e e n appl ied to the s tudy of

defects in cerebra l deve lopmen t in m a n . Elec t ron m i c r o s c o p y has en joyed

s o m e use in the s tudy of cort ical b i o p s i e s from severe ly re tarded ch i ld ren , and

a few examples o f synapt ic c h a n g e s in cort ical samples from such pa t ien ts

have b e e n repor ted . In two cases desc r ibed b y Gona ta s and co-workers in 1965

and 1967 ( 1 2 - 1 4 ) , ce rebra l cor tex appeared no rma l on l ight mic roscopy , bu t on

electron m i c r o s c o p y there were s t r ik ing changes in t e rmina l axons and in pre-

synapt ic t e rmina l s , w i th abundan t prol i ferat ion of s m o o t h m e m b r a n e s ar-

ranged as i r regular tubular s t ructures . W i t h H e r m a n and B e n s c h (15) we

repor ted a s imi lar case in 1969 and have seen two s ib l ings w i t h the s a m e cor-

tical abnormal i t i e s s ince then . T h e s e three ch i ldren had a h i s to ry o f normal

deve lopmen t up to abou t age 9 m o n t h s , w i t h gradual loss of m o t o r and menta l

funct ion s u b s e q u e n t l y . F igure 1 s h o w s a huge ly en larged te rmina l axon , filled

wi th i rregular ly a r ranged tubules in a cort ical sample o b t a i n e d from a frontal

b i o p s y in such a case . In F igure 2 an axonal swel l ing i s s h o w n unde r greater

magni f ica t ion ; th i s figure also i l lustrates synapses of the abno rma l axonal end-

ing w i t h two smal l adjacent n o n m y e l i n a t e d axons or dendr i t i c sp ines . At

t imes , the a b n o r m a l tubular s t ructures w e r e found adjacent to synapt ic ve s -

ic les , sugges t ing a pos s ib l e re la t ionsh ip b e t w e e n format ion of synapt ic ve s -

icles and the prol i ferat ion of these tubu les (Figure 3 ) . T h e ques t i on h a s b e e n

ra i sed w h e t h e r th is smoo th m e m b r a n e prol i ferat ion m a y represen t a mal-

deve lopmen t or overproduc t ion of synapt ic ves ic les . T h i s as wel l as o ther

h y p o t h e s e s regard ing pa thogenes i s are d i scussed in m o r e detai l e l sewhere

(15) . It i s our i m p r e s s i o n that the pa thologica l changes repor ted b y G o n a t a s

and co-workers and b y us p r o b a b l y are u n c o m m o n and that these cases rep-

resent examples of o n e or several rare, s lowly p rogress ive degenera t ive dis-

orders of cerebra l cor tex w h i c h have the i r onse t in early infancy . Q u i t e as ide

from the final noso logy , these cases are of in teres t in that t hey p rov ide infor-

ma t ion regard ing the type of i m p a i r m e n t in funct ion that occurs in an i sola ted

l e s ion of cort ical p resynapt ic t e rmina ls . All these ch i ldren h a d profound defi-

ci ts in men ta l funct ion. In addi t ion , s o m e of t h e m have had f requent se izures

inc lud ing infant i le spa sms and a " h y p s a r r h y t h m i c " pat tern in the E E G .

Elec t ron mic roscopy , wh i l e an excel lent t e c h n i q u e for de tec t ion of fine struc-

tural changes in ne rve t e rmina l s , is of little value in the s tudy o f the spat ial

a r r angement or o rgan iza t ion of these s t ructures in cerebra l cor tex. T h e Go lg i

s ta in and its var ious modi f ica t ions are the on ly m e t h o d s current ly avai lable for

such inves t iga t ions . T h e dendr i t ic t rees of ind iv idua l cort ical n e u r o n s are

demons t r a t ed in the i r en t i re ty b y th is m e t h o d , and quant i t a t ive measu re s of

dendr i t ic spread can b e o b t a i n e d for s ingle cells . W e have recent ly u t i l ized the

G o l g i - C o x t e c h n i q u e in s tudy ing cerebral cor tex from a group of severe ly re-

tarded ind iv idua ls in an a t tempt to test the h y p o t h e s i s , also advanced b y Mar in -

Padi l la , that d isorders i n g rowth and in spat ia l a r r angemen t of dendr i t es migh t


FIGURE 2. Portion of an axonal swelling which makes synaptic contact with two neighboring

small nonmyelinated axons or dendritic spines (lead citrate, x 42,900).

under l ie s o m e cases of men ta l re tardat ion ( 2 0 , 2 1 , 2 3 , 2 4 ) . W e chose to e x a m i n e

neurons in the midd le frontal gyrus . The re were two reasons for th is ; one was

the pr ior avai labi l i ty of excel lent s tandards of no rmal in th is area for the h u m a n

(29) , the o ther the access ib i l i ty of th is r eg ion for cort ical b iopsy . T o date w e

have e x a m i n e d six au topsy and five b i o p s y cases . T h e b i ops i e s we re carr ied out

in profoundly re tarded chi ldren wi th men ta l age b e l o w 6 m o n t h s , in an a t tempt

to rule out genet ica l ly d e t e r m i n e d degenera t ive disorders of bra in . Severa l b ra ins

from infants , ch i ldren , and adults w h o died of o ther than neurologica l d i seases

served as controls . In each case a m i n i m u m of 20 pyramida l cells in cort ical

layer 5 and layer 3 we re pro jec ted on a flat surface. A n analys is o f basa l

dendri t ic spread w a s carr ied out b y the m e t h o d in t roduced b y Shol l (32) ,

5 . Synaptic and Dendritic Development 127

FIGURE 3. Terminal axons containing normal appearing synaptic vesicles (top and lower left)

adjacent to abnormal interconnected tubules (lead citrate, x60,000) .


w h i c h depends on enumera t i on of in te r sec t ions of basa l dendr i tes wi th con-

centr ic c i rc les d rawn a round the cen te r of the cell b o d y , in our s tudy at in -

tervals of 25 fim (Figure 4 ) . W e have ex tended the m e t h o d s o m e w h a t b y also

enumera t i ng in te r sec t ions w i th l ines d rawn parallel to the apical dendr i te to

ob ta in a measu re of b r a n c h i n g s ar i s ing from the apical dendr i te (Figure 4 ) .

T h e results can b e brief ly s u m m a r i z e d as fol lows. A group of five cases wi th

modera te to severe re tardat ion, r ang ing in age from 14 years to adult , s h o w e d

only ques t i onab le dev ia t ions from the normal or ab sence of any def inable

abnormal i t i e s . O n e of these w a s a pat ient w i th m o n g o l i s m ; the e t io logy of the

re tardat ion was u n k n o w n in the o ther four. In contras t , marked dendr i t ic

defects were p resen t in all s ix of a group of young , profoundly re tarded

chi ldren w h o ranged in age from 1 to 10 years . T h e e t io logy of the re tardat ion

was u n k n o w n in four of these cases ; in one the d iagnos i s w a s that of familial ,

recess ive ly inher i t ed mic rocepha ly , in ano ther a congeni ta l mal format ion of

b ra in w a s found. T h e f indings in two of these cases wil l b e desc r ibed in some

detail .

FIGURE 4. Drawing of a pyramidal neuron with superimposed grid of concentric circles for quan-

titation of basal dendritic spread, and rectangular grid for quantitation of branches arising from

the apical dendrites. Concentric circles and parallel lines are at 25-/xm intervals; the length of the

rectangular grid is 300 ,am.

4 K


FIGURE 5 . A. Sparse horizontal and tangential dendritic branches in layers 2 and 3 of middle

frontal gyrus from a severely retarded 10-year-old child (case 1 ) . Golgi-Cox method, X 8 0 . B .

Middle frontal gyrus from a 6-year-old child without known neurological disease. Golgi-Cox

method, X 8 0 .

T h e oldest in the group , a 10-year -o ld girl w i th amen t i a and te t rapares is ,

had a m i n o r deve lopmenta l anoma ly of b ra in cons i s t ing of Ch ia r i T y p e I mal -

format ion o f the ce rebe l lum wi th assoc ia ted mi ld hydrocepha lus . T h e b ra in

w e i g h e d 1080 gm. T h e s tandard neuropa tho logy did no t p rov ide an explana-

t ion for the p rofound defects in menta l and m o t o r funct ions . E x a m i n a t i o n of

cerebra l cor tex s ta ined b y the G o l g i - C o x m e t h o d s h o w e d a diffuse defect in

dendr i t ic a rbor iza t ion of cort ical neu rons . T h i s is v i s ib l e on in spec t ion of

frontal cor tex unde r low magni f ica t ion , w h i c h s h o w s no rma l neu rona l per i -

karya and p r imary apical dendr i tes , bu t def ic iency o f hor izon ta l and t angen-

tial b r a n c h i n g s , especia l ly in the uppe r cort ical layers (Figures 5 A and B ) . T h e

abnormal i ty is m o r e ev iden t unde r h i g h e r magni f ica t ion (Figures 6 A and B ) .

Here the spars i ty of b r a n c h e s ar i s ing from apical dendr i tes is s t r ik ing , and

there also appears to b e a decrease in n u m b e r of sp ines on the apical dendr i t ic

shafts. Quan t i t a t ive analys is o f b r a n c h e s ar is ing from the first 300 j i i m l ength

of the apical dendr i te conf i rms this deficit (Figures 7 A and B ) . T h e abnorma l i t y

appears to b e s o m e w h a t m o r e m a r k e d in the apical dendr i t ic b r a n c h e s of layer

3 cells. In contras t , quant i t a t ive ana lys is of basa l dendr i t ic spread s h o w s little

ev idence of abnormal i t i e s (Figure 8 ) . T h e r e appears to b e a s l ight decrease in

b r anches ex tend ing a d i s tance greater than 100 j L i m from the per ikaryon .


FIGURE 6. A. Apical dendrites in layer 2 of middle frontal gyrus, case 1. Golgi-Cox method, X360. Sparsity of tangential branches and of dendritic spines is evident. B. Layer 2 of middle frontal gyrus, 6-year-old normal brain. The apical dendrite on the right has numerous spines and several tangential branches.


50 100 150 200 250

A DISTANCE FROM APICAL DENDRITE ( /O

12 h

FIGURE 7. A. Mean number and length of branches arising from the first 300 fjum of the apical

dendrite in layer 3, middle frontal gyrus, case 1 and control. Each point represents the mean for 12

pyramidal cells. B. Same as Figure 7A, but for pyramidal cells in layer 5. The difference from the

normal is somewhat less marked than in layer 3.

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B DISTANCE FROM APICAL DENDRITE (/i)


BASAL DENDRITE BRANCHES, LAYER 5

DISTANCE FROM PERIKARYON U)

FIGURE 8. Spread and branching of basal dendrites in layer 5 pyramidal cells, as determined by

enumeration of intersections with concentric circles. Each point represents the mean for 20 cells.

There appears to be a slight decrease in very long basal dendritic branches in the abnormal brain.

Pr imary basa l dendr i tes and shor ter b r a n c h e s are p resen t in no rma l n u m b e r s .

T h e fairly se lect ive i n v o l v e m e n t of apical dendr i t ic deve lopmen t is o f s o m e

interes t in v i e w of expe r imen ta l f indings w h i c h sugges t that deve lopmen t of

apical dendr i tes m a y b e especia l ly vu lnerab le to abnorma l inf luences (33) .

H o w e v e r , h is to logica l c h a n g e s w e r e no t ed in basa l dendr i tes as wel l . T h e s e

were ra ther smoo th and del icate , w i th less ev idence of sp ine format ion than in

the normal (Figures 9 A and B ) .

M o r e s t r ik ing dev ia t ions from normal were obse rved in cerebral cort ical

samples from a 2 Vi-year-old chi ld w h o had amen t i a and infant i le myoc lon ic

se izures , of u n k n o w n e t io logy. His se izures started in the n e w b o r n per iod

and he r e m a i n e d comple te ly he lp less , w i th f requent myoc lon ic j e rks unt i l he

d ied of asp i ra t ion p n e u m o n i a . T h e b ra in w a s small , w e i g h i n g 760 g m , and

there w a s s l ight vent r icu lar en la rgement . Mic roscop ic examina t ion of mul t ip le

b ra in sec t ions s ta ined wi th hematoxy l in and eos in , Niss l s ta in , and mye l in

s tain w a s unreward ing . T h e G o l g i - C o x m e t h o d s h o w e d diffuse, severe defects

in dendr i t ic deve lopmen t o f cort ical neu rons . B o t h basa l and apical dendr i tes

of pyramida l cells we re marked ly s tunted . F igure 10 s h o w s a typical pyramida l

neu ron in layer 5 of midd le frontal gyrus . T h e basa l dendr i tes are short , sec-

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FIGURE 9 . A . A large pyramidal cell in layer 5 of middle frontal gyrus, case 1 . The basal dendrites

are smooth and delicate, with few visible spines. B . A large pyramidal cell in layer 5 from a

normal brain, showing extensive dendritic spine formations. Golgi-Cox method, X 360.

ondary b r a n c h i n g s are inf requent , and dendr i t ic sp ines are sparse . Quan t i t a -

t ive ana lys is of basa l dendr i te spread of pyramida l neu rons in b o t h layer 3 and

layer 5 s h o w e d marked differences from the age -ma tched control , w i th den-

drit ic deve lopmen t ra ther s imi lar to that of a 3 -mon th control case (Figures

11A and B ) . S imi la r abnormal i t i e s were no ted in med ia l frontal gyrus of four

o ther profoundly re tarded ch i ldren at ages 1 to 4 years . F igures 12A and B

s h o w examples of pyramida l n e u r o n s in midd le frontal gyrus o b t a i n e d b y

b ra in b i o p s y in a 4 -year -o ld b o y w i t h amen t i a and myoc lon i c se izures . Deve l -

o p m e n t of basa l dendr i tes is no more complex than that seen in the normal

neona t e (Figure 1 2 C ) .

N o dendr i t ic abnormal i t i e s were found in a group of older pa t ien ts w h i c h

inc luded several p rofoundly re tarded ind iv idua ls . It i s qu i t e p o s s i b l e that

o ther , as ye t undef ined abnormal i t i e s in cerebra l cor tex under l ie the men ta l

defect in these cases . Fu r the rmore , l imi ta t ions of the Golg i m e t h o d m a y ac-

count for nega t ive f indings . T h i s m e t h o d wou ld b e expec ted to s h o w on ly dif-

fuse, p rofound defects re l iably . M o r e subt le or local ized changes in dendr i t ic

a rbor iza t ion w o u l d e lude the s tudy of on ly a very few cells in a few small cor-

tical areas . O n e also has to cons ide r the poss ib i l i t y that dendr i t ic g rowth in

the b ra in s o f the re tarded m a y con t inue at a s low rate th roughou t the late


FIGURE 10. Stunted pyramidal cell in layer 5, middle frontal gyrus, from a 2£-year-old child with amentia and infantile myoclonic seizures (case 2).

ch i ldhood years , w i t h gradual na r rowing o f the gap from normal . T h e latter

poss ib i l i ty rece ives s o m e suppor t from s tudies in exper imenta l neuropathol -

ogy , w h e r e ca tch-up g rowth in deve lop ing bra in has b e e n found unde r sev-

eral c i rcumstances . Examples inc lude ca tch-up mye l ina t ion and axonal g rowth

in ma lnour i shed y o u n g an ima l s after refeeding (2 ,19 ) , and accelera ted axonal

g rowth in the rat w i th exper imenta l c re t in i sm after thyro id r ep lacement ther-

apy (19) . If such late ca tch-up g rowth occurs in the h u m a n , it does no t appear

to b e correlated w i th i m p r o v e d funct ion. In the cret in , cer ta inly , thyro id

rep lacement does no t improve already es tab l i shed menta l defect.

Four of the six ch i ldren in w h o m w e found marked dendr i t ic abnormal i t i e s

had a h i s to ry of infant i le myoc lon ic se izures ( infanti le spasms) and a " h y p s -

a r r h y t h m i c " pat tern in the E E G in addi t ion to ament ia . Infanti le myoc lon ic

se izures and hypsa r rhy thmia were also p resen t in a chi ld wi th fine structural

abnormal i t i e s in p resynapt ic areas repor ted b y Gona ta s and G o l d e n s o h n (14) .


FIGURE 11. Basal dendritic spread of pyramidal cells from layer 3 ( A ) and layer 5(B), case 2. Each

point represents the mean for 20 cells. The complexity of basal dendrites in the abnormal brain at

age 2 i years resembles that of a normal 3-month-old brain.

T h e s e obse rva t ions ra ise the ques t i on w h e t h e r synapt ic and dendr i t ic defects in cerebra l cor tex p rov ide an ana tomica l subs t ra te for th i s type of se izure . A few s tudies on the neu ropa tho logy of cases wi th men ta l defect and infant i le spasms are ava i lab le ; the type of changes seen are the s ame as those found in a group of the severe ly re tarded in genera l . Occas iona l ly , these b ra in s have appeared no rma l , usual ly they are s o m e w h a t r educed in s ize . C h r i s t e n s e n and M e l c h i o r r emarked that the m o s t c o m m o n f inding w a s an i m m a t u r e appear-ance of cerebra l cor tex, w i t h incomple t e strat if ication of cort ical n e u r o n s (3).

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B D I S T A N C E FROM P E R I K A R Y O N ( / t )


FIGURE 12. A and B. Pyramidal cells in layer 3, middle frontal gyrus, from a cortical biopsy in a

4-year-old profoundly retarded child with myoclonic seizures. C. Pyramidal cell in layer 3, middle

frontal gyrus, of a full-term newborn without known brain disease. Golgi-Cox method, X360.

A

G


T h i s is the type o f change o n e w o u l d expec t to see on cell s ta ins o f cerebra l

cortex that is def ic ient in dendr i t ic and axona l b r a n c h i n g s .

T h e m o s t s t r ik ing aspect of e lectrocort ical ac t iv i ty , as j u d g e d from E E G , in

ch i ldren w i t h infant i le m y o c l o n i c se izures i s i ts total d i so rgan iza t ion . S p i k e

wave and s low w a v e d i scharges in the E E G occur i ndependen t l y from mul t ip le

foci th roughout the b ra in . S u c h i n d e p e n d e n t foci wou ld b e m o s t l ikely to

occur in a s i tua t ion w h e r e in t racor t ica l—primar i ly axodendr i t i c—pathways

have failed to form or have b e e n des t royed . T h e demons t r a t i on of decreased

hor izonta l and tangent ia l b r a n c h i n g s on Golg i s ta in therefore p rov ides a

p laus ib le ana tomica l b a s i s for the o b s e r v e d abnorma l i ty in cort ical funct ion.

T h e occur rence of se izures and dendr i t ic defect in these ch i ldren also is of

in teres t in v i e w of w h a t is k n o w n conce rn ing the fine structural l e s ion in the

se izure focus. W e s t r u m , W h i t e , and W a r d (34) have s h o w n decrease in

n u m b e r of dendr i t ic sp ines and in length of basa l dendr i tes in n e u r o n s in

exper imen ta l a lumina -c ream se izure foci . S imi la r dendr i t ic abnorma l i t i e s have

recent ly b e e n d e s c r i b e d in h i p p o c a m p a l n e u r o n s from pa t ien t s w i t h t empora l

lobe ep i l epsy (31) .

It is n o w wel l k n o w n that the syndrome of infant i le myoc lon ic se izures ,

hypsa r rhy thmia , and men ta l defect ha s a n u m b e r of d is t inc t e t io logies . T h e s e

inc lude neona ta l anox ia , encepha l i t i s in early infancy , severe head t rauma in

the infant , and a n u m b e r of genet ica l ly de t e rmined me tabo l i c d i seases of in -

fancy inc lud ing un t rea ted pheny lke tonur i a and py r idox ine d e p e n d e n c y (22) .

All of these d isorders occur in the per ina ta l and early pos tna ta l pe r iods . T h i s

is the pe r iod of b r a in g rowth in w h i c h dendr i t ic and synapt ic d e v e l o p m e n t in

h u m a n cerebra l cor tex normal ly progress at the m o s t rap id rate (5 ,29 ) . W e m a y

therefore b e dea l ing wi th specific ins t ances of the theory d i scussed b y D o b -

b i n g (6 ,7) of specia l vu lnerab i l i ty in rapidly deve lop ing neura l sy s t ems .

T h i s concep t sugges t s a th i rd ca tegory of neuropa tho log ica l c h a n g e un-

der ly ing menta l defect and se izures , in add i t ion to gross structural mal -

deve lopmen t s and frank des t ruc t ive l e s ions . In th is ca tegory the defect ap-

pears to b e o n e of in te r fe rence w i t h normal deve lopmenta l even t s in the fine

structure of the b ra in . T h i s type of pa tho logy has a l ready b e e n c o m m e n t e d

u p o n in the h u m a n wi th regard to mye l ina t ion , w h i c h is the o ther major

deve lopmen ta l even t that occurs in ear ly pos tna ta l b ra in . A lag in mye l ina t i on

of the subcor t ica l w h i t e mat te r has b e e n found in a n u m b e r of pos tnata l en-

cepha lopa th ie s , inc lud ing several of the i n b o r n errors o f a m i n o acid m e t a b o -

l i sm (27) , malnu t r i t ion dur ing early in fancy (35) , and congen i t a l hypo thy ro id -

i s m (28) . It is qu i t e l ike ly that abnormal i t i e s in axonal , synapt ic , or dendr i t ic

deve lopmen t wil l b e found in several o r all o f these d i sorders , w h e n appropr i -

ate s tudy of the cerebra l cor tex is car r ied out . A b n o r m a l i t i e s in fine structure

of cerebra l cor tex w o u l d p rov ide a m o r e p laus ib le subs t ra te for the obse rved

deficits in intel lectual func t ion ing than do the p rev ious ly desc r ibed defects in

mye l ina t ion .


M u c h rema ins to b e learned regard ing the control of axonal and dendr i t ic

g rowth in deve lop ing bra in . K n o w l e d g e regard ing the factors that i n h i b i t and

those that s t imulate g rowth of neurona l p rocesses in the b ra in of the infant

m a y well p rov ide n e w ins igh t s in to the pa thogenes i s of men ta l re tardat ion.

SUMMARY

T h e hypo thes i s that specific defects in synapt ic and dendr i t ic deve lopmen t

of cerebra l cor tex m a y form the ana tomica l bas i s in some cases of menta l

defect ha s b e e n e x a m i n e d b y electron mic roscopy and b y use of the Go lg i -Cox

m e t h o d . T w o types of abnormal i ty have b e e n ident i f ied to date. O n e is a spe-

cific l es ion of p resynapt ic t e rmina l s , first repor ted b y Gona t a s and Go lden -

sohn (14) in a chi ld w i th men ta l re tardat ion and myoc lon ic se izures . Th i s

l es ion , cons i s t ing of m a s s i v e prol i ferat ion of m e m b r a n o u s s t ructures in te rmi-

nal axons , appears to b e rare and m a y b e the ana tomica l subs t ra te of one or

more genet ical ly de t e rmined d e m e n t i n g i l lnesses in infancy. M o r e c o m m o n l y ,

cerebra l cor tex from the severe ly re tarded shows defects in n u m b e r , l ength ,

and spat ia l a r r angement of dendr i tes and synapses , b e s t demons t ra t ed b y the

Golg i m e t h o d . S u c h abnormal i t i e s have b e e n found in s ix out of e leven

b ra ins from severe ly re tarded ind iv idua l s e x a m i n e d b y us . T h e e t io logy of the

re tardat ion was u n k n o w n in the major i ty ; two had o ther r ecogn izab le devel-

opmen ta l mal format ions of b ra in . It is sugges ted that a n u m b e r of different

e t iological factors, if ac t ive dur ing the pe r iod of rapid synapt ic and dendr i t ic

g rowth in cerebra l cor tex ( i . e . , from the last t r imes te r o f p r egnancy to the end

of the first pos tnata l year) m a y result in s tun ted deve lopmen t of these struc-

tures .

ACKNOWLEDGMENT

This work was supported by USPHS Grant No. NB 07105.

REFERENCES

1. Benda, C. E. Structural cerebral histopathology of mental deficiencies. Proc. Int. Congr. Neuro-pathol., 1st, 1952; 2: 7-50.

2. Benton, J. W., Moser, H. W. , and Dodge, P. Modification of the schedule of myelination in the rat by early nutritional deprivation. Pediatrics, 1966, 38: 801-807.

3. Christensen, E . , and Melchior, J . C. Neuropathologic findings in children with infantile spasms and hypsarrhythmia. Dan. Med. Bull, 1960, 7: 121-127.

4. Coleman, P. D. , and Riesen, A. H. Environmental effects on cortical dendritic fields. 1. Rearing in the dark. /. Anat., 1968, 102: 363-374.

5. Conel, J . L. The Postnatal Development of the Human Cerebral Cortex. Vols. 1-8. Harvard Univ. Press, Cambridge, Massachusetts, 1939-1967.


6. Dobbing, J. Undernutrition and the developing brain. In: Developmental Neurobiology. (W. A. Himwich, Ed.). Thomas, Springfield, Illinois, 1970: 260-261.

7. Dobbing, J . Vulnerable periods of brain development. In: Lipids, Malnutrition and the Develop-ing Brain. Elsevier, Amsterdam, 1972: 9.

8. Eayrs, J . T. The cerebral cortex of normal and hypothyroid rats. Acta Anat., 1955, 25: 160-183. 9. Fox, } . H., Fishman, M. A., Dodge, P. R., and Prensky, A. L. The effect of malnutrition on

human central nervous system myelin. Neurology, 1972, 22: 1213-1216. 10. Freytag, E . , and Lindenberg, R. Neuropathologic findings in patients of a hospital for the

mentally deficient. A survey of 359 cases. Johns Hopkins Med. J . , 1967, 121: 379-392. 11. Globus, A. , and Scheibel, A. B. The effect of visual deprivation on cortical neurons: A Golgi

study. Exp. Neurol, 1967, 19: 331-345. 12. Gonatas, N. K. Axonal and synaptic lesions in neuropsychiatric disorders. Nature {Lond.),

1967, 214: 352-355. 13. Gonatas, N. K., Evangelista, I., and Walsh, G. O. Axonal and synaptic changes in a case of

psychomotor retardation: An electron microscopic study. / . Neuropathol. Exp. Neurol, 1967, 26: 179-199.

14. Gonatas, N. K., and Goldensohn, E. S. Unusual neocortical presynaptic terminals in a patient with convulsions, mental retardation and cortical blindness: An electron microscopic study. /. Neuropathol Exp. Neurol, 1965, 24: 539-562.

15. Herman, M. K. , Huttenlocher, P. R., and Bensch, K. G. Electron microscopic observations in infantile neuroaxonal dystrophy. Arch. Neurol. {Chicago), 1969, 20: 19-34.

16. Hicks, S. P., and D'Amato, C. J. Low-dose irradiation of developing brain. Science, 1963, 141: 903-905.

17. Hicks, S. P. , and D'Amato, C. J . Effects of ionizing radiations on mammalian development. Adv. Teratol, 1966, 1: 195-250.

18. Hicks, S. P., Cavanaugh, M. C., and O'Brien, E. D. Effects of anoxia on the developing cere-bral cortex of the rat. Am. J. Pathol, 1962, 40: 615-635.

19. Horn, G. Thyroid deficiency and inanition: Effects of replacement therapy on development of the cerebral cortex in young albino rats. Anat. Rec, 1955, 121: 63-79.

20. Huttenlocher, P. R. Dendritic development and mental defect. Neurology, 1970, 20: 381 (abstr.).

21. Huttenlocher, P. R. Dendritic development in neocortex of children with mental defect and with infantile spasms. Neurology, 197A, 24: 203-210.

22. Jeavons, P. M., and Bower, B. D. Infantile spasms. In: Clinics in Developmental Medicine No. 15. Heinemann, London, 1964: 14-18.

23. Marin-Padilla, M. Structural abnormalities of the cerebral cortex in human chromosomal aber-rations. A Golgi study. Brain Res., 1972, 44: 625-629.

24. Marin-Padilla, M. Structural organization of the cerebral cortex (motor area) in human chro-mosomal aberrations. A Golgi study 1. D1 (13-15) trisomy, Patau syndrome. Brain Res., 1974, 66: 375-391.

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27. Prensky, A. L. , Carr, S., and Moser, H. W. Development of myelin in inherited disorders of amino acid metabolism. Arch. Neurol. {Chicago), 1968, 19: 552-558.

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6 Normal and Aberrant Neuronal Development

in the Cerebral Cortex of Human Fetus and Young Infant

D O M I N I C K P . P U R P U R A

Rose F. Kennedy Center for Research in Mental

Retardation and Human Development,

Albert Einstein College of Medicine,

Bronx, New York

INTRODUCTION

Eluc ida t ion of the neurob io log ica l m e c h a n i s m s under ly ing men ta l s u b -

normal i ty wi l l r equ i re acqu i s i t i on of a vas t a m o u n t o f fundamenta l data o n

normal and aber ran t deve lopmenta l p rocesses in the h u m a n bra in . Da ta col lec-

t ion for th i s purpose is l ikely to b e facil i tated w h e n opera t iona l approaches

have a l ready y ie lded s ignif icant in format ion on on togene t i c s tud ies invo lv ing

labora tory an ima l s . A n approach that has b e e n part icular ly useful in our

p rev ious on togene t i c s tud ies o f the immatu re fel ine b ra in has evolved from at-

t empts to ident i fy tempora l s e q u e n c e s in morphophys io log i ca l features of

neu rona l and synapt ic deve lopmen t ( 3 5 , 3 8 - 4 7 , 4 9 , 5 0 , 5 5 , 5 6 , 6 2 ) . S u c h an ap-

proach has genera ted sufficient data over the past decade to encourage further

pursui t o f th i s bas i c l ine of i n q u i r y in labora tory an ima l s ( 2 6 , 4 8 , 5 7 , 6 0 ) . In the

presen t repor t an a t tempt is m a d e to i l lustrate h o w several q u e s t i o n s ra ised in

ear l ier expe r imen ta l i nves t iga t ions m a y b e profi tably explored i n m o r p h o -

logical s tud ies of the immatu re h u m a n b ra in .

T h e first ques t i on cons ide red he re ar ises out of our long-s tand ing in teres t in

the morphophys io log i ca l pa t tern of dendr i t ic deve lopmen t of cort ical n e u r o n s

( 4 5 , 4 6 , 4 9 , 5 0 ) . Its g e n e s i s m a y b e t raced to the v i e w that the per iod of max ima l

dendr i t ic g rowth and different ia t ion in the cerebra l cortex is cri t ical for the

141

142 Dominick P. Purpura

e labora t ion of the major recep t ive surface of n e u r o n s for d iverse synapt ic

inpu ts (9 ,32 ,37 ) . It fol lows from th is that even t s w h i c h interfere w i th dendr i t ic

different iat ion dur ing th is "cr i t ica l p e r i o d " migh t resul t in less than op t ima l

dendr i t ic surface area for synapt ic in te rac t ion in cort ical ne tworks . T h e q u e s -

t ion p o sed here is : W h e n does th is pe r iod occur in the h u m a n cerebra l cortex

and wha t morpho log ica l features of dendr i tes character ize i t?

T h e second ques t i on of conce rn der ives from earl ier s tudies of cortical

synap togenes i s in the fel ine b ra in (49 ,55 ,62 ) . Severa l l ines of ev idence have

revealed an early e labora t ion o f axodendr i t i c synapt ic pa thways and a s o m e -

wha t later, t hough tempora l ly over lapp ing , p h a s e of axosomat ic synapt ic

deve lopment in i m m a t u r e cerebra l cor tex. Desp i t e l imi ta t ions p laced on the

prepara t ion of h u m a n fetal b r a in t i s sue for e lect ron mic ro scopy th is m e t h o d

has already revealed the p re sence of axodendr i t ic synapses in h u m a n fetal

cor tex pr ior to the e n d of the first t r imes te r (30) . T h e ques t i on ar i ses , howeve r ,

as to w h a t me thodo log ica l approaches involv ing l ight mic roscop ic t e c h n i q u e s

could provide in format ion on the p r o b l e m of the relat ive different ia t ion of ax-

osomat ic and axodendr i t i c inpu t s to cort ical n e u r o n s par t icular ly in the per i -

natal per iod . Here aga in the p rob l em of the "cr i t ica l p e r i o d " m a y b e engaged .

For , if there are ind ica t ions that later deve lop ing axosomat ic synapt ic path-

ways are more suscep t ib le than axodendr i t i c p ro jec t ion sys t ems to var ious

insul ts to the immatu re h u m a n bra in , th is could b e of s ignif icance in a t tempts

to unders t and p rocesses of funct ional d i sorgan iza t ion in a var ie ty of synapt ic

t ransac t ions .

T h e th i rd ques t ion posed in th is s tudy relates to the character is t ics of

dendr i t ic sp ines of cort ical neu rons in two cl inical s i tua t ions , infant i le se i -

zures and p roven cases o f D o w n ' s s y n d r o m e w i t h assoc ia ted cardiac a n o m -

al ies . W h a t h a p p e n s to dendr i t ic sp ine deve lopmen t in these cond i t ions in

the y o u n g infant ' s b ra in and h o w specific are the c h a n g e s , i f any?

T h e foregoing ques t ions have b e e n e x a m i n e d in re la t ion to morpholog ica l

obse rva t ions on the i m m a t u r e h u m a n h i p p o c a m p u s . O u r cho i ce of the h ip -

p o c a m p u s as a " w i n d o w " th rough w h i c h to v i e w the p a n o r a m a of develop-

menta l p rocesses in the immatu re cerebra l cor tex has a ba s i s in fact and fancy.

T h e fact is that the h i p p o c a m p u s , at least in labora tory an imals , has b e e n

s tudied in great detai l from the s tandpoin t of its morpholog ica l o rgan iza t ion

( 1 - 7 , 9 , 1 9 , 2 0 , 2 2 , 2 7 , 5 3 , 6 6 ) . T h u s compara t ive neuroana tomica l data should

greatly facili tate analys is of h i p p o c a m p a l o rgan iza t ion in the immatu re h u m a n

bra in . T h e fancy is that the h i p p o c a m p u s (and i ts re lated pro jec t ion sys tems)

plays an impor tan t , a lbei t i l l -defined, role in m e c h a n i s m s of m e m o r y and

learning. T o the extent that th is is true the h i p p o c a m p u s migh t b e a " h i g h pri-

o r i t y " s i te for de tec t ing morpho log ica l a l tera t ions in the immatu re h u m a n

bra in that are assoc ia ted w i th deve lopmenta l re tardat ion.

T h e final ques t i on explored here ex tends the i nqu i ry of the morpho log ica l

subs t ra te of p r imary evoked cort ical potent ia ls in i m m a t u r e labora tory an imals

6. Neuronal Development in the Cerebral Cortex 143

(cf. 2 1 , 3 6 , for ex tens ive references to th is l i terature) to the h u m a n neona te .

O u r p rev ious a t tempts to accoun t for the e lec t rographic features of p r imary

evoked potent ia l s in i m m a t u r e cor tex have e m p h a s i z e d the early d e v e l o p m e n t

of synapt ic inpu t s to dendr i t ic e l emen t s s u b t e n d i n g the m o s t superficial layers

of cor tex as l ike ly genera tors of the p r edominan t ly sur face-negat ive c o m p o -

nen t of these r e sponses ( 3 8 , 4 0 , 4 4 , 4 9 ) . In contras t the appearance of p r o m i n e n t

ini t ia l sur face-pos i t iv i ty of the p r imary r e sponse in older an ima l s has b e e n at-

t r ibu ted to the matura t ion of synapt ic ac t iv i t ies in deeper layers of cortex. P re -

l iminary obse rva t ions address the ques t ion of a pos s ib l e approach to the func-

t ional ma tura t ion of p r imary pro jec t ion cor tex from e x a m i n a t i o n of v isua l

evoked potent ia l s and neurona l m o r p h o l o g y in the h u m a n infant .

MATERIAL AND METHODS

Smal l b locks of unf ixed or formal in-f ixed t i s sue cut n o r m a l to the long i -

tudinal axis of the h i p p o c a m p a l format ion w e r e r e m o v e d from 70 b r a in s s tud-

ied at au topsy in the D e p a r t m e n t o f Pa tho logy , Alber t E i n s t e i n Col lege of

M e d i c i n e . S a m p l e s o f o the r b r a in areas w e r e also r e m o v e d for s tud ies s imi la r

to those desc r ibed in th i s report . S p e c i m e n s w e r e o b t a i n e d from spon t aneous

and i nduced abor t ions , p re te rm infants , full- term n e o n a t e s , and y o u n g infants

and ch i ld ren u p to the age of 10 years . Cl in ica l , genera l pa thologica l , and

neuropa tho log ica l data we re avai lable on all cases w i th the excep t ion of early

s t i l lborn fe tuses .

Cajal and/or B o d i a n s i lver s ta ins , the latter modi f ied accord ing to W e i n z i m e r

(64) were p repared in all cases for examina t i on of axons and axonal p lexus in

the h i p p o c a m p u s (see b e l o w ) . For ty o f the 70 cases were also s tud ied w i t h the

rap id Go lg i and/or G o l g i - C o x m e t h o d s w h i c h y ie lded sat isfactory resul ts in

approx imate ly 30 cases .

DENDRITIC DIFFERENTIATION IN HIPPOCAMPAL NEURONS OF HUMAN FETUS

T h e major morpho log ica l charac ter is t ics o f dendr i t ic g rowth p rocesses in the subpr ima te m a m m a l i a n b ra in have b e e n deta i led in Golg i p repara t ions m o s t recent ly b y Mores t ( 3 1 - 3 3 ) . For p resen t pu rposes it is sufficient to ind ica te that m a n y of the charac ter i s t ics of dendr i t ic di f ferent ia t ion e m p h a s i z e d in Golg i s tudies of labora tory an imals can b e ident i f ied in the h u m a n fetal h ip -p o c a m p u s b e t w e e n 18 and 26 w e e k s ges ta t iona l age (g .a . ) . Sat is factory rapid Golg i p repara t ions of several fetal b r a in s 2 0 - 2 2 w e e k s g.a. i l lustrate the mos t p r o m i n e n t morpho log ica l aspec ts o f dendr i t i c d i f ferent ia t ion o b s e r v e d in the h u m a n h i p p o c a m p u s .

144 Dominick P . Purpura

FIGURE 1. Golgi rapid preparations of pyramidal neurons of the hippocampus from a 22-week-old fetus (spontaneous abortion). A, B , D, G, and H. Cells of the regio superior (CA!-CA 2 ) ; C, E, and F , cells from regio inferior (CA 3 ) . Apical dendrites are well developed in comparison to basilar dendrites. Varicosities, nodulations, and irregular segments of lumpy enlargements are seen in apical dendrites and to a lesser extent in basilar dendrites. A branch of a basilar dendrite of cell H exhibits a terminal enlargement which gives rise to two fine filopodia (at arrow). Further descrip-tion in text. Magnification marker, 50 ^tm unless noted otherwise.


E x a m p l e s of the overt charac ter is t ics of pyramida l n e u r o n s of the h i p -

p o c a m p u s from a 2 2 - w e e k - o l d fetus are s h o w n in F igure 1. Cel ls of the regio

super ior ( C A j - C A g ) are s h o w n in F igures 1 A , B , G , and H. Ap ica l dendr i t es of

these e l emen t s are wel l deve loped w h e r e a s bas i la r dendr i t es are th in , few in

n u m b e r , and shor t in l ength . Irregular e x p a n s i o n s of the apical dendr i t es and

the i r b r a n c h e s are o b s e r v e d in F igures 1 A and G . Regular ly spaced var icos i t i es

in a b r anch of the apical dendr i te in F igure I B are also f requent ly encoun te red .

It is no t u n c o m m o n for fine th in b r a n c h e s to ar ise from ra ther large expan-

s ions of m o r e p rox ima l dendr i t ic s e g m e n t s (Figures 1 C , D , and G ) . S o m e

n e u r o n s of the reg io super io r have apical dendr i tes w h i c h are of un i form

d iamete r and lack the i r regular i t ies and var icos i t i es of n e i g h b o r i n g neu rons .

S u c h a cell e x h i b i t i n g m o r e mature apical dendr i t ic charac ter is t ics is s een in

F igure 1H. Ap ica l dendr i tes o f n e u r o n s in regio infer ior ( C A 3 ) are shor ter ,

more i r regular and genera l ly exh ib i t var icos i t i es (Figures I E and F ) .

Bas i la r dendr i t es of h i p p o c a m p a l pyramida l n e u r o n s in the 2 2 - w e e k - o l d

fetus typical ly exh ib i t p r o m i n e n t i rregularly spaced en la rgemen t s or va r icos -

i t ies w h i c h m a y g ive r ise to o n e or two f i lopodia (Figure 1H , ar row) . Deta i l s

of these and o ther g rowth p rocesses are s u m m a r i z e d in F igure 2 .

A laterally g rowing dendr i t ic b r a n c h e m e r g i n g from the cell b o d y of a C A 3

n e u r o n is s een in F igure 2 A . A large e x p a n s i o n of the dendr i te occurs distal to

its o r ig in . F r o m this expans ion ar ise several th ick and irregularly small

b r a n c h e s w i t h va r i cose and l u m p y en l a rgemen t s . A n apical b r a n c h of a den-

drite in F igure 2 B t e rmina te s in an expanded process (s ingle ar row) w h i c h at

h ighe r magni f ica t ion (Figure 2C) is seen to cons i s t of a g rowth c o n e w i t h sev-

eral finer f i lopodia- l ike p rocesses . T o the r ight , ano ther t e rmina l dendr i t ic

p rocess has the form of a b u l b o u s en la rgemen t (double a r rows) . F igure 2 D

s h o w s an unusua l format ion of a fine p rocess w i th a t e rmina l b u l b a r i s ing

direct ly from a b i furca t ion po in t of a dendr i t ic t ree. Irregular , th ick dendr i t ic

s e g m e n t s charac ter ize the o ther l i m b of the apical p rocess . F igu res 2 E and F

i l lustrate the morpho log ica l differences b e t w e e n the var icose and i r regular

en l a rgemen t s of g r o w i n g bas i l a r dendr i tes and the smoo th , regular con tour of

the axon ar i s ing from a pyramida l neu ron . Deta i l s of a bas i la r dendr i t e of th is

e l emen t are s h o w n in F igure 2 F . T h e p r imary b r a n c h po in t p resen t s an ex-

p a n d e d s e g m e n t from w h i c h a fine sp icule e m e r g e s . S imi l a r sp icules and

f i lopodia are s een on var icos i t i es distal to the b r anch po in t (Figure 2 F ) . Severa l

addi t ional examples o f sp icu les and f i lopodia e m e r g i n g from var icose and

l u m p y en la rgemen t s o f apical dendr i t e s are n o t e d in F igures 2 G - N . T h e s e fig-

ures i l lustrate the w i d e var ia t ion i n the charac ter i s t ics of t hese dendr i t ic

g rowth p roces ses found in different h i p p o c a m p a l neu rons . W h i l e no difficulty

is encoun te r ed in iden t i fy ing f i lopodia in F igure 2 K , it is no t cer ta in w h e t h e r

the fine dendr i t ic p rocesses in F igures 2 M and N represen t f i lopodia, dendr i t ic

sp icules , or i nc ip i en t dendr i t ic sp ines . F inal ly , F igure 2 0 is an en la rgemen t of

a fine dendr i t ic p rocess w h i c h t e rmina tes in a c lawl ike expans ion .


FIGURE 2. Details of dendritic growth processes seen in rapid Golgi preparations of hippocampal

neurons from a 22-week-old fetus. A. A cell has a lateral dendrite with a large distal expansion

that gives rise to several branches and branchlets. B. Two forms of dendritic growth cones (at

single and double arrows) observed in a single field. Inset (C) at higher magnification shows that

multiple fine processes arise from the terminal process. D. Asymmetrical formation of dendritic

branches from main apical stem. E . Basilar dendrite of well-developed pyramidal neuron exhibits

multiple varicosities. F . Higher magnification of E . Note spicules and filopodia arise from varicos-


Dendr i t i c g rowth p rocesses s imi lar to those i l lustrated in F igu res 1 and 2

from a 2 2 - w e e k - o l d fetus we re obse rved in s o m e w h a t y o u n g e r ( 1 8 - 2 0 weeks )

as wel l as older fe tuses (up to 2 5 - 2 6 w e e k s ) . In five cases s p a n n i n g the per iod

of 2 8 - 3 3 w e e k s of ges ta t ion , p r o m i n e n t dendr i t ic va r icos i t i e s , l u m p y enlarge-

m e n t s , and t e rmina l and pre te rmina l g rowth cones or f i lopodia a r i s ing from

var icose swel l ings were not obse rved in pyramida l neu rons of the h ip -

p o c a m p u s . Indeed in the 33 -week -o ld infant dendr i t ic g rowth appears to occur

in a m o r e un i fo rm fash ion (Figures 3 D - G ) , s ince m o s t major dendr i t ic sys tems

appear to have a t ta ined thei r genera l b r a n c h i n g charac ter i s t ics . At th is s tage

s o m e fine exc rescences are de tec tab le on dendr i tes of C A 3 n e u r o n s (Figures

3 D , E , and F) par t icular ly on p rox imal s e g m e n t s that are in re la t ion to m o s s y

fiber p ro jec t ions . S o m a and dendr i t ic sp icules and sp ines in apparen t synapt ic

re lat ion wi th a m o s s y fiber are ev iden t on a neu ron in C A 4 (Figure 3 C ) . In the

33 -week -o ld infant bas i la r dendr i t ic g rowth of h i p p o c a m p a l pyramida l

neurons has p rogressed at a m u c h s lower rate than apical dendr i t ic e labora t ion

and in s o m e cells nodu la t ion is ev iden t on th in bas i la r dendr i t es (Figure 3 G ) .

N o a t tempt wi l l b e m a d e to desc r ibe the character is t ics of dendr i t es of cells

of the fascia dentata . It is of in teres t , h o w e v e r , that the var ie ty of g rowth

p rocesses obse rved in h ippocampa l pyramida l neu rons in the 2 0 - 2 9 - w e e k - o l d

fetus are no t p rominen t ly d i sp layed in denta te granule cells . E i the r such

growth p rocesses occur at an ear l ier ges ta t ional age than that e x a m i n e d he re or

dendr i tes of m o s t g ranule cells g r o w and differentiate b y s imple l inear ex ten-

s ion of t e rmina l p rocesses and diffuse i nc r emen t in dendr i t ic vo lume . It is s ig-

nif icant that in the 3 3 - w e e k - o l d infant some granule cells m a y exh ib i t den-

dri tes that are th ick and mature in over t features (wi th the excep t ion of sp ines

(Figure 3 B ) , w h e r e a s o thers in the same area m a y b e th in and exh ib i t va r icos -

i t ies (Figure 3 A ) . M o r e deta i led s tudy o f the g rowth and different ia t ion of

fascia denta ta neu rons in the h u m a n fetal b r a in is an o b v i o u s neces s i t y in

order to evaluate structural features obse rved in Golg i s tud ies of later deve lop-

ing pa thologica l p rocesses (54) .

T h e obse rva t i ons s u m m a r i z e d in F igures 1-3 sugges t that the pe r iod span-

n i n g 2 0 - 2 8 w e e k s of ges ta t ion is a p h a s e of m a x i m u m dendr i t i c g rowth and

different ia t ion of pyramida l neu rons of the h i p p o c a m p u s . T h e data also in-

dicate that m a n y of the features w h i c h character ize dendr i t ic g rowth as de-

sc r ibed in i m m a t u r e labora tory an imals ( 3 1 - 3 3 ) are de tec tab le in the cerebra l

cor tex of the h u m a n fetus at midges t a t ion . A l t h o u g h cells in different sec t ions

of the h i p p o c a m p u s exh ib i t different t empora l pa t terns of dendr i t ic g rowth

and different ia t ion dur ing the per iod defined in th i s s tudy no ev idence has

ities and nodules. The initial axonal segment is smooth and of relatively uniform diameter. G. Segments of apical dendrites show varicosities and nodulation. H. Higher magnification of branching segment of cell in G. I-N. Examples of primitive spines, spicules, and filopodia-like processes, some of which arise from lumpy enlargements (K and L). O. Fine dendritic branch exhibits a clawlike terminal expansion. Magnification markers, 5 fjum unless noted otherwise.


FIGURE 3. Characteristics of neurons in fascia dentata (A and B) and hippocampus (C-G) of a 33-week-old premature infant (respiratory distress syndrome) Golgi-Cox preparation. Fascia dentata granule cells are well developed at this stage. C. Neuron in CA 4 (hilus of the fascia dentata) has pronounced somadendritic spines which appear to be contacted by a large mossy fiber. D, E, and F. Large pyramids of CA 3 . Note remarkable expansion of apical dendritic and lateral dendritic branches in D. Proximal segments of cells in E and F have thornlike excrescences suggestive of mossy fiber termination sites. Basilar dendrites of all cells shown are poorly developed at this stage and some show fine modulation. Spines are detectable in some apical dendrites. Magnifica-tion markers, 50 /xm.

b e e n ob t a ined for a s econd phase of dendr i t ic dif ferent ia t ion of pyramida l

neu rons after 30 w e e k s . T h i s sugges ts that the matura t iona l cycle of h i p -

pocampa l pyramida l neu rons is relat ively synch ronous th roughout the h i p -

p o c a m p u s and argues agains t s ignif icant addi t ion of n e w l y formed pyramida l

neu rons to the h i p p o c a m p u s at later s tages of h u m a n fetal b ra in deve lopment .


AN APPROACH TO THE COMPARATIVE DEVELOPMENT OF AXOSOMATIC AND AXODENDRITIC SYNAPTIC PATHWAYS

IN IMMATURE HUMAN HIPPOCAMPUS

There is no substitute for quantitative electron microscopic studies of the develop-

ment and distribution of synaptic contacts on different parts of the somadendritic

membrane of a neuron in any meaningful investigation of synaptogenesis. The re

are, h o w e v e r , approaches to th i s p r o b l e m w h i c h pe rmi t r ea sonab le ex tens ion

of avai lable morpho log ica l data from labora tory an imals to l ight m ic roscope

s tudies of the h u m a n i m m a t u r e h i p p o c a m p u s . Fo r example , the appearance

and e labora t ion of dendr i t ic sp icules and sp ines on h i p p o c a m p a l n e u r o n s

w h i c h occur at abou t 2 2 - 2 6 w e e k s g.a. (Figures 4 A and B) and p roceed rapid ly

thereafter to comple t ion b y the e n d of the fifth to s ix th pos tna ta l m o n t h (Fig-

ure 4 C ) p rov ide ind i rec t (l ight mic roscopy) ev idence for the relat ively preco-

c ious d e v e l o p m e n t o f axosp inodendr i t i c synapses . But it m u s t b e b o r n e in

m i n d that e lec t ron mic roscop ic data are avai lable w h i c h ind ica te that cort ical

axodendr i t i c synapses related to dendr i t ic t runks m a k e the i r appea rance b e -

fore dendr i t ic s p i n e s are de tec tab le in the k i t ten (62) . S imi l a r types of cort ical

axodendr i t i c synapses in the h u m a n fetus are de tec tab le as early as 8.5 w e e k s

(pos tovula t ion) (30) . T h e ques t i on ar ises , w h a t can b e inferred c o n c e r n i n g the

FIGURE 4. General appearance of fine dendritic processes of apical dendrites of hippocampal pyramidal neurons at different developmental stages. Golgi rapid preparations. A and B. Twenty-six-week-old fetus. A. A thick dendrite shows a fine branchlet and several spines clustered dis-tally. B . A thin dendrite shows a variety of branchlets, spicules, and filopodia-like processes. C. Thin dendrite from a 6-month-old infant, normal brain. Some heterogeneity in spine morphology is evident. D. Example of spine distribution on a fine dendritic process from a 7-year-old child, accident case, normal brain. Magnification markers, 5 /mm.

A i Bl CjrDl


deve lopmen t of p r o m i n e n t axosomat ic synapt ic pa thways? In th is regard the

h i p p o c a m p u s is a par t icular ly favorable structure for ana lys is .

It has long b e e n e s t ab l i shed (9 ,27) that the axons and axon collaterals that

form a dense fibrillar p lexus i n the s t ra tum pyramida le of the h i p p o c a m p u s

der ive from in t r ins ic n e u r o n s ( in te rneurons) located a b o v e , in the s t ra tum

rad ia tum- lacunosum, and b e l o w the pyramida l layer in the s t ra tum or iens . A

FIGURE 5. Summary diagram from Cajal (9) of the location and axonal distribution of intrinsic

neurons (interneurons) of the hippocampus of the 1-month-old rabbit. Cells A, B , C, D, and E are

located in the stratum oriens. Axons (C) enter the stratum pyramidale (F) and either collateralize

profusely in the stratum pyramidale or pass through (F) and send recurrent collaterals back into

the stratum pyramidale. The basketlike network of axon terminals of these cells is readily appreci-

ated. It is estimated that the axon of a single neuron (e.g., B or E) may be in synaptic relation with

200-500 pyramidal cell bodies of the stratum pyramidale [cf. Andersen (1)]. The morphological na-

ture of the plexus in relation to the cell body of a single pyramidal neuron is well illustrated in

Figures 6H and


reproduc t ion of a s u m m a r y d rawing from the classic s tudies of Cajal (9) amply

i l lustrates the genera l na ture and or ig in of the p lexus of the s t ra tum pyrami -

dale in the 1-month-o ld rabb i t (Figure 5 ) . Typica l ly the axons of i n t e rneu rons

t e rmina te in per icel lular baske t a r r angement s on m a n y pyramida l cell b o d i e s .

Elect ron mic ro scopy has conf i rmed that the t e rmina ls of the per icel lular baske t

p lexus cons t i tu te the major axosomat ic inpu t to h i p p o c a m p a l pyramida l

neu rons (1 ,11) . O f par t icular s igni f icance for the p resen t p r o b l e m are the o b -

serva t ions that axosomat ic synapses on h i p p o c a m p a l pyramida l neu rons in the

k i t ten deve lop postnata l ly w h e n axodendr i t i c pa thways are wel l e s t ab l i shed

(46 ,55) . Addi t iona l ly , u n p u b l i s h e d s tudies from our labora tory ind ica te that

the pe r iod of m a x i m u m axosomat ic synap togenes i s in the fel ine h i p p o c a m p u s

co inc ides w i t h the e labora t ion of the per icel lular ba ske t p lexus . T h e s e dif-

ferent l ines of e v i d e n c e sugges t that the dens i ty o f the axonal p lexus of the

s t ra tum pyramida le o f the h i p p o c a m p u s in the deve lop ing h u m a n b ra in re-

flects in a genera l sense the n u m b e r of p re te rmina l axosomat ic synapt ic proc-

esses fo rmed b y axons of h i p p o c a m p a l in te rneurons . O n e a s s u m p t i o n is nec -

essa ry i f there is to b e a se r ious a t t empt to ut i l ize axon -p l exus dens i ty as a

pa ramete r of ma tu ra t ion of synapt ic s u b s y s t e m s . T h i s is that the failure to de-

tect the per icel lular p lexus in Cajal or B o d i a n si lver s ta ins is no t due to failure

of the h is to logica l m e t h o d s per se . For tunate ly , in all the mater ia l u t i l ized in

the p resen t ser ies no difficulty w a s encoun te red in s ta in ing axons bo rde r ing

the upper and lower l imi t s o f the s t ra tum pyramida le , as wel l as the fiber

sys tems of the s t ra tum or iens and s t r a tum- lacunosum, even in the mos t i m m a -

ture b ra ins (18 w e e k s g .a . ) .

A s u m m a r y of the obse rva t ions on the deve lopmen t of the axonal p lexus of

the s t ra tum pyramida le is s h o w n in F igure 6 in samples o f Cajal s i lver prepa-

ra t ions of the h i p p o c a m p u s . In all the cases in F igure 6 genera l neu ropa tho -

logical e x a m i n a t i o n of the b ra in s ind ica ted no rma l b ra in for age (Figures

6 A - G ) or no neuropa tho log ica l f indings in the case o f the adult b r a in of F igure

6H.

T h e s t ra tum pyramida le o f the h i p p o c a m p u s from a 2 2 - w e e k - o l d s t i l lborn

fetus is i l lustrated in F igures 6 A and A x . N o fibers are de tec tab le in m o r e

superficial l aminae of the s t ra tum pyramida le (Figure 6 A ) , bu t a few coarse

fibers pene t ra te the lower borde r s o f the pyramida l layer (F igure 6 A ) . F igures

6 B - D were o b t a i n e d from premature infants w h o surv ived for a few hours to a

few days after the onse t o f respi ra tory dis t ress .

At 27 w e e k s g.a. (F igures 6 B and B x ) axons are m o r e f requent ly encoun te red

in re la t ion to pyramida l n e u r o n s deep in the s t ra tum pyramida le and some are

no ted abou t major dendr i t ic b r a n c h e s (at a r row in F igure 6 B ) . A x o n s pass ing

th rough the m o r e superficial parts of the s t ra tum pyramida le exh ib i t c lose ap-

pos i t ion to pyramida l neu rons (ar row in F igure 6B X ) b u t lack fine collaterals

and b ranches . Essen t ia l ly s imi lar f indings are no t ed at 28 w e e k s g.a. (Figure

6C and C x ) . B y 33 w e e k s g.a. a greater dens i ty of axons appears in re la t ion to

deep ly ing h i p p o c a m p a l pyramida l neu rons (Figure 6 D ) . Essen t ia l ly no change

l O u m

FIGURE 6. Development of the axon plexus of the stratum pyramidale of the hippocampus in the human brain. Cajal preparations. A and Av Twenty-two-week-old fetus; B and B l 7 27-weeks, pre-term infant. E, F, F l r G, and Gv Two weeks postnatally, full-term infant. H and Hv Normal adult. Note that in very immature hippocampus only a few axons are detectable, especially in relation to deeper lying pyramidal neurons (B, Blf C, Clf and D, Dt). There is a dramatic increase in the den-sity of the axon plexus in the 2-week-old full-term infant. In the adult the dense basketlike plexus consists of many very fine axons and axon terminals with fine boutons. The apical dendrite is free of the plexus (H and Ht). Further description in text.

G G. H,4


has occur red in the appearance o f axons related to superficial cells in the 33

w e e k s g.a. infant from that at 28 w e e k s g.a. H o w e v e r , b e t w e e n 36 w e e k s g.a.

and for several w e e k s after b i r th in the full- term infant there is a r emarkab le

inc rease in the ca l ibe r and dens i ty of the axonal p lexus of the s t ra tum pyrami -

dale (Figure 6 E ) . Careful e x a m i n a t i o n at different focal p lanes reveals m a n y

pericel lular a r r angemen t s of axons w h i c h collateral ize a m o n g several pyram-

idal neu rons (Figures 6 F , F l 7 G , and G 2 ) . W h i l e there is cons ide rab le matu-

ra t ion of the p lexus in the i m m e d i a t e pos tnata l pe r iod the pericel lular net -

w o r k still lacks the dens i ty and character is t ics o f the p lexus o b s e r v e d i n the

adult b r a in (Figures 6 H and H ^ .

To the ex tent that the p rogress ive appearance in si lver p repara t ions of the

axonal p lexus in the s t ra tum pyramida le o f the immatu re h u m a n h i p p o c a m p u s

prov ides ind i rec t ev idence for the e labora t ion of p re te rmina l axosomat ic syn-

aptic p rocesses it m a y b e inferred from the s tudies s u m m a r i z e d above that

these synapt ic i npu t s deve lop re la t ively late in h u m a n ges ta t ion in c o m p a r i s o n

to axodendr i t i c and part icular ly axosp inodendr i t i c synapt ic p ro jec t ions to

pyramida l n e u r o n s . T h i s inference is consonan t w i th p rev ious f indings in the

immatu re fel ine b ra in ( 4 6 , 4 9 , 5 5 , 6 2 ) . T h e resul ts o f F igure 6 also p rov ide a

useful and rela t ively s imple l ight mic roscop ica l approach to eva lua t ion of the

"ma tu ra t iona l s t a tu s " of the h i p p o c a m p u s from the s tandpoin t of deve lop-

m e n t and e labora t ion of axon collaterals and pre te rmina l p rocesses in a wel l -

defined cort ical locat ion . In the final ana lys is the n u m b e r o f axosomat i c syn-

apses on pyramida l neu rons m u s t b e a funct ion of the n u m b e r of axonal

pre te rmina ls that cons t i tu te the p resynap t ic e l emen t of t hese synapses . A n d

s ince there is n o difficulty in reso lv ing axons in the range of 1 /nm (and poss ib ly

less) in good Cajal or B o d i a n prepara t ions of the immatu re h i p p o c a m p u s it

m a y b e a rgued that the n u m b e r of such small d i ame te r axons bea r s a direct

re la t ionsh ip to the n u m b e r of fine axonal p re te rmina l p rocesses .

T h e fo regoing in fe rences are w o r t h y o f cons ide ra t ion in a t t empts to ident i fy

factors w h i c h could conce ivab ly delay, modi fy , or in terrupt the p rocess of ax-

onal i nvas ion , g rowth , and axon collateral prol i ferat ion in the s t ra tum pyrami -

dale dur ing the th i rd t r imes te r and in the i m m e d i a t e pos tna ta l pe r iod . O b -

v ious ly such factors as me tabo l i c or card iorespi ra tory d i s tu rbances w i th or

w i thou t under ly ing gene t ic de t e rminan t s are l ikely to exert w i d e s p r e a d alter-

a t ions in deve lopmen ta l p rocesses . T h e p r o b l e m he re , howeve r , is o n e of de-

tec t ion o f deve lopmen ta l even t s part icular ly vu lnerab le to such d i s tu rbances .

Severa l i l lus t ra t ions serve to define the p r o b l e m as it re lates to the deve lop-

m e n t of the axonal p lexus of the s t ra tum pyramida le .

Examples of Cajal s i lver p repara t ions of the s t ra tum pyramida le in four cases

r ep resen t ing two pairs of age-re la ted infants are s h o w n in F igure 7. T h e

no rma l appearance of the axonal p lexus in a 2 -week -o ld full- term infant w h o

s u c c u m b e d fo l lowing acute respi ra tory d is t ress (Figure 7A) i s c o m p a r e d wi th

the appearance of the s t ra tum pyramida le i n a 1 .5-week-old infant wi th

D o w n ' s s y n d r o m e and Te t ra logy of Falot (Figure 7 B ) . Apar t from the reduc t ion


FIGURE 7. Comparison of the density of the axonal plexus of the stratum pyramidale in different clinical conditions. (Cajal preparations.) A. Relatively normal density of the plexus of the hip-pocampus in a 2-week-old full-term infant who succumbed following acute respiratory distress. B . Absence of the axonal plexus in a 2-week-old full-term infant with Down's syndrome and Tetralogy of Falot. C. Absence of the axonal plexus in a 6-week-old full-term infant with cyanotic heart disease. D. Development and persistence of the axonal plexus in a 6-week-old full-term in-fant with cerebro-hepatorenal syndrome. Note twisted appearance of dendrites of some pyramidal neurons in this condition. Magnification markers, 10 fim.


in n e u r o n cell s ize there is n o s ignif icant difference in the dens i ty of n e u r o n s

in the sec t ion from the D o w n ' s case (Figure 7B) and in the age-re la ted " c o n -

trol 7 ' (F igure 7 A ) . In cont ras t there is a s t r ik ing a b s e n c e of the axonal p lexus in

the infant w i t h D o w n ' s s y n d r o m e and assoc ia ted mul t ip le congen i t a l a n o m -

alies . It cannot b e conc luded from avai lable data that the decreased dens i ty

or loss of the axonal p lexus of the s t ra tum pyramida le seen in F igure 7B is a

direct express ion of the gene t ic abno rma l i t y o f D o w n ' s synd rome . It shou ld b e

no ted that o n e of a n u m b e r of pos s ib l e factors con t r ibu t ing in the D o w n ' s case

was the p re sence of congen i ta l cyanot ic hear t d i sease w h i c h could conce ivab ly

inf luence the per inata l deve lopmen t of the axonal p lexus . Tha t p ro longed and

unremi t t ing cyanot ic hear t d i sease a lone could retard or p revent the growth

and prol i ferat ion of axons in th is s u b s y s t e m is clear from the f indings in

ano ther case s h o w n in F igure 7C from a 6 -week-o ld infant (full- term). Desp i t e

the p resence of we l l -deve loped pyramida l neu rons in th i s infant the axonal

p lexus i s vir tual ly absen t . H o w e v e r , such i s no t the s i tua t ion in an age-re la ted

case of ce rebro -hepa to rena l s y n d r o m e (Figure 6D) that w a s s tud ied exten-

s ively in regard to the me tabo l i c defect r e spons ib l e for th i s s y n d r o m e (18) .

W h i l e the dens i ty of the axonal p lexus in the s t ra tum pyramida le i n the 6-

week -o ld infant w i t h ce rebro -hepa to rena l s y n d r o m e is less than expec ted for

this age , it is c lear that the pe rox i somal and mi tochondr i a l defects under ly ing

th is d isorder have no t had a major inf luence on axon p lexus deve lopment .

Ra the r , it is sugges ted from examina t i on of all avai lable cases to date that the

factor of p ro longed cerebra l h y p o x e m i a (and p robab ly assoc ia ted b lood and

t i ssue p H changes ) is a s ignif icant deterrent to the relat ively late deve lopmen t

of synapt ic pa thways , such as those subse rved b y the axonal p lexus of the h i p -

pocampa l s t ra tum pyramida le . T h i s ra ises the ques t ion as to w h a t re la t ion

exis ts b e t w e e n changes in the character is t ics of th i s axonal p lexus and the

matura t ion o f dendr i t ic sp ines of pyramida l neu rons in the h i p p o c a m p u s in

cases o f deve lopmenta l re tardat ion in y o u n g infants?

OBSERVATIONS ON DENDRITIC SPINES AND FINE DENDRITIC PROCESSES IN DEVELOPMENTAL RETARDATION

There is n o w a g rowing b o d y of data that dendr i t ic b r a n c h i n g pat terns as

seen in Golg i p repara t ions are s ignif icant ly different in the cerebra l cor tex of

s o m e , bu t not all, cases of p rofound men ta l re tardat ion (cf. Hu t t en locher ,

Chap te r 5 , th is vo lume) . Mar in -Pad i l l a (28 ,29) has appl ied the rapid Golg i

m e t h o d in a deta i led s tudy of dendr i t ic sp ines o f cort ical neu rons in two cases

of c h r o m o s o m a l abnorma l i t i e s k n o w n to b e assoc ia ted w i th men ta l retarda-

t ion. In o n e case , that o f an 18 -mon th -o ld re tarded female w i t h conf i rmed

t r i somy 2 1 , mo to r cor tex dendr i t es exh ib i t ed var iab le reduc t ion in sp ines w i t h

m a n y s e g m e n t s comple te ly free of sp ines . O t h e r s e g m e n t s m i g h t b e covered


w i t h small sp ines p o s s e s s i n g fine pedic les and bare ly v i s ib l e t e rmina l heads

(28) . In the second case , that of a n e w b o r n female wi th conf i rmed D 2 ( 1 3 - 1 5 )

t r i somy, reduc t ion , nonun i fo rm d i s t r ibu t ion and b izar re abnorma l i t i e s were

obse rved in dendr i t ic s p i n e s o f m o t o r cor tex n e u r o n s (29) . E m p h a s i s w a s

p laced b y Mar in -Pad i l l a on the ve ry long (hai r l ike) , th in , i r regular , tor tuous ,

and d i so r ien ted appearance of dendr i t ic sp ines in the D1 ( 1 3 - 1 5 ) t r i somy case .

W e have e x a m i n e d the h i p p o c a m p a l format ion for c h a n g e s in dendr i t ic

sp ines in three infants of s imi lar age each o f w h o m had cl inical ev idence of

deve lopmenta l re tardat ion. T w o infants , ages 8 and 9 m o n t h s , we re conf i rmed

cases o f t r i somy 21 wi th classic s t igmata of D o w n ' s s y n d r o m e and associa ted

mul t ip le cardiac anomal i e s . A thi rd infant , (8 m o n t h s ) w i th a c l inical h i s tory of

in t rac table genera l ized se izures , spast ic quadrap leg ia , and failure to a t ta in any

mo to r or behav io ra l m i l e s tones is p resen ted as a case of severe deve lopmenta l

re tardat ion of u n k n o w n e t io logy.

Rou t ine neuropa tho log ica l examina t ion of the cerebra l cor tex in the 8-

mon th -o ld infant w i th se izures and failure to deve lop revealed nonspec i f ic

react ive m e n i n g i t i s , focal , mi ld . O t h e r w i s e n o al terat ions were found. S tud ies

of the h ippocampa l format ion d isc losed marked loss o f neu rons in the S o m m e r

sector w h i c h was sharply demarca ted from the regio infer ior (Figure 8 A ) . (It is

poss ib ly s ignif icant that a l though an Apgar score of 9 w a s recorded at b i r th

the infant r equ i red pos i t ive pressure oxygen and resusc i ta t ion for 5 minu t e s in

the del ivery room. ) E x a m i n a t i o n of the s t ra tum pyramida le o f the preserved

C A 3 sector failed to d isc lose the axonal p lexus character is t ic of th is cell layer

(Figure 8 B ) . In genera l , neu rons in C A 3 h ad dendr i tes wi th we l l -deve loped

and n u m e r o u s sp ines (Figure 8 C ) . Rare ly s o m e segmen t s o f these dendr i tes

s h o w e d u n e v e n d i s t r ibu t ions of sp ines or sp ine loss . Su rv iv ing n e u r o n s in the

C A j sector , ou ts ide the reg ion of m a x i m u m cell loss , pos se s sed dendr i t es w i t h

a pauc i ty of b r a n c h e s . S u c h dendr i tes exh ib i t ed var icos i t i es and s e g m e n t s

wi th cons ide rab le sp ine loss (Figure 8 D ) . In some segmen t s clusters o f den-

dritic sp ines , sp icules , and b izar re f i lopodia-l ike p rocesses we re obse rved .

T h e f i lopodia-l ike p rocesses we re ex t remely fine and could s o m e t i m e s b e

ident if ied only b y vir tue o f the te rmina l heads (Figure 8 D , ar row) . Dendr i t e s

of neu rons in the s u b i c u l u m and p re sub icu lum exh ib i t ed b izarre and p leo-

morph ic sp ines , sp icules , and f i lopodia-l ike p rocesses (Figures 8 E - G ) . T h e a b -

normal character is t ics o f these dendr i t ic p rocesses are readi ly apprec ia ted b y

compar i son wi th dendr i t ic sp ines of h i p p o c a m p a l neu rons in a no rmal 6-

mon th -o ld infant w i th a nega t ive neuro logica l h i s to ry and normal b ra in for

age (Figures 8 0 - Q ) . It is of in teres t that the dendr i t ic sp ine character is t ics in

the 8 -month-o ld infant w i t h se izures and deve lopmenta l re tardat ion r e semble

in m a n y respects the descr ip t ions of abnorma l sp ines repor ted b y M a r i n -

Padi l la (29) in a case o f t r i somy D x ( 1 3 - 1 5 ) .

T w o cases of conf i rmed t r i somy 21 wi th s t igmata of D o w n ' s synd rome

e m p h a s i z e the p r o b l e m of further def ining the deve lopmenta l a l terat ions in

dendr i t ic sp ines and axonal p lexus of the s t ra tum pyramida le in a c h r o m o -

FIGURE 8. A-G. Examples of neuronal elements of the hippocampal formation of an 8-month-old infant with intractable seizures and developmental retardation. A. Nissl preparation, at arrow, beginning of region of maximal neuronal loss in the Sommer sector. B . Bodian preparation for neurofibrils. Cell in the surviving C A 3 region. The axonal plexus is not observed in the entire stratum pyramidale. C. Large dendrite of a C A 3 neuron. D. Dendrite of a neuron in CA1 with a paucity of abnormal spines and spicules. A fine filopodia-like process is barely detectable (at arrow) but the terminal head is visible. E -G. Abnormal appearing fine dendritic processes of pyramidal neurons in the subiculum and presubiculum. H-K. Elements of the hippocampus of an 8-month-old infant with confirmed trisomy 21 (Down's syndrome). H. Bodian preparation shows absence of axonal plexus of the stratum pyramidale. I -K. Abnormal appearance of dendrites and dendritic fine processes of hippocampal pyramidal neurons. L-N. Hippocampal elements of a 9-month-old infant with confirmed trisomy 21. L. Cajal preparation. The axonal plexus is well developed. M. Apical dendrite with some reduction in spine density. N. Basilar dendrite with paucity of spines and spine abnormalities. O-Q. Dendrites of hippocampal pyramidal neurons of a 6-month-old infant with negative neurological history, normal brain. Magnification markers, 2.5 jiim, unless noted otherwise.


somal d isorder k n o w n to b e assoc ia ted w i th men ta l re tardat ion. In b o t h cases

congeni ta l cardiac anoma l i e s were present . O n e infant , 8 m o n t h s old at the

t ime of death , was b o r n at 33 w e e k s g.a. w i t h A - V c o m m u n i s and patent

ductus an te r iosus . T h e pa t i en t n e v e r s h o w e d cl inical s igns o f ca rd iopu lmonary

insuff ic iency a l though a small r ight to left shunt w a s no ted at the t ime of car-

diac ca theter iza t ion at 5 m o n t h s of age . Elec t ive l iga t ion of the pa tent ductus

and b a n d i n g of the pu lmona ry artery were carr ied out uneventful ly . Pos t -

opera t ively the pa t ien t deve loped low cardiac output , suffered genera l ized

myoc lon ic se izures , and three cardiac arrests before a fatal fourth. Mul t ip le

coronal sec t ions and mic roscop ic examina t ion of the cerebra l cor tex revealed

no abnormal i t i e s in hema toxy l in and eos in prepara t ions .

Examina t ion of the s t ra tum pyramida le o f the h i p p o c a m p u s in th is case

revealed we l l -deve loped pyramida l neu rons bu t a vir tual a b s e n c e of the axonal

plexus (Figure 8 H ) . M a n y dendr i tes s h o w e d r eg ions o f poor impregna t ion

sugges t ive of cyst ic degenera t ion . S p i n e loss w a s p r o m i n e n t and sp ine dis-

t r ibu t ion w a s irregular . Addi t iona l ly , sp ines we re fine and hai r l ike and re-

s e m b l e d f i lopodia-l ike p rocesses (Figures 8 I - K ) . Dendr i t i c b r a n c h i n g pat terns

were not s ignif icant ly different from those of the 6 -month -o ld normal infant.

T h e s econd case of t r i somy 21 w a s a full-term ma le w i th external s t igmata of

D o w n ' s syndrome and Tet ra logy of Falot . T h e pa t ien t w a s admi t t ed at the age

of 9 m o n t h s for e lect ive cardiac ca the ter iza t ion w h i c h w a s compl ica ted b y

c o m m o n iliac t h rombos i s . D e a t h occurred 10 days thereafter .

T h e s t ra tum pyramida le of the h i p p o c a m p u s in th i s infant revea led a wel l-

deve loped axonal p lexus (Figure 8 L ) . R e m a r k a b l y few abnormal i t i e s were

no ted in the dendr i t ic b r a n c h i n g pat tern or dendr i t ic sp ines . T h e i m p r e s s i o n

was ga ined that mos t apical dendr i tes of pyramida l neu rons exh ib i t ed a m i n o r

reduct ion in sp ines (Figure 8 M ) . In s t r ik ing contras t bas i lar dendr i t e s had

m u c h fewer sp ines and s h o w e d m a n y o f the sp ine abnormal i t i e s , inc lud ing

fine f i lopodia-l ike p rocesses obse rved in the two o ther cases in th is ser ies

(Figure 8 N ) .

T h e foregoing two examples of D o w n ' s s y n d r o m e in y o u n g infants of s imi -

lar age (8 and 9 m o n t h s ) , b o t h w i t h assoc ia ted cardiac anomal i e s , s h o w e d s ig-

nificant a l terat ions in dendr i t ic " s p i n e " structure o f h ippocampa l neu rons .

T h e s e obse rva t ions are cons i s t en t wi th the f indings of Mar in -Pad i l l a (28 ,29) in

cases o f c h r o m o s o m a l abnorma l i t i e s k n o w n to b e assoc ia ted w i th men ta l retar-

dat ion. O n e of the two infants w i t h D o w n ' s s y n d r o m e also e x h i b i t e d a m a r k e d

a t tenuat ion of the axonal p lexus of the s t ra tum pyramida le . T h e o ther did not ,

even though cardiac abnormal i t i e s (Tetralogy of Falot) we re m o r e severe .

Pe rhaps it is s ignif icant that the infant w i t h the a t tenuated axonal p lexus w a s

bo rn at 33 w e e k s g .a . , thus c o m p o u n d i n g the r i sk factor of D o w n ' s s y n d r o m e

and congeni ta l hear t d isease on deve lopmenta l p rocesses regula t ing axon

plexus matura t ion . N o n e of the s t igmata of D o w n ' s syndrome was obse rved in the 8 -month-o ld


infant wi th in t rac table se izures and deve lopmenta l re tardat ion. Ye t dendr i t ic

sp ine abnorma l i t i e s on h i p p o c a m p a l neu rons we re p r o m i n e n t and m o r e i m -

press ive than in the two cases of D o w n ' s s y n d r o m e of s imi la r age . In v i e w of

these obse rva t ions it s e e m s unl ike ly that specific abnorma l i t i e s in dendr i t ic

sp ine structure charac ter ize the dendr i t ic sp ines in cases of specific c h r o m o -

somal abnorma l i t i e s k n o w n to b e assoc ia ted wi th men ta l re tardat ion. T h e

same m a y b e sa id in regard to the deve lopmen t of the axon p lexus of the h ip -

p o c a m p u s . P rematur i ty and assoc ia ted cardiac anoma l i e s and/or p ro longed

neona ta l h y p o x e m i a m a y b e more impor tan t in in f luenc ing the deve lopmen t

of the axon plexus than the p re sence of D o w n ' s s y n d r o m e per se .

It m u s t b e e m p h a s i z e d that the three infants s tud ied in th is se r ies we re

approx imate ly the same age at the t ime of dea th ( 8 - 9 m o n t h s ) . Conce ivab ly ,

had they l ived to at tain early ch i ldhood or ado lescence , the early changes in

dendr i t ic p rocesses no ted he re m i g h t have p rogressed to the stage desc r ibed

b y Mar in -Pad i l l a in an 18 -mon th -o ld infant w i th t r i somy 21 (28) . T h e r e is an

impor tan t impl i ca t ion in th i s , name ly , that in such cond i t i ons as D o w n ' s

s y n d r o m e cort ical neu rona l g rowth and different ia t ion inc lud ing ear ly synap-

togenes i s m a y p roceed wi th s o m e tempora l delays th rough the last phases of

antenata l d e v e l o p m e n t and pe rhaps th rough m o s t of the first yea r of infancy.

Thereaf ter degenera t ive c h a n g e s in e l emen t s o f the neurop i l (axonal t e rmina ls ,

axodendr i t i c and axosp inodendr i t i c synapses ) m a y occur at a var iab le t empo

wi th c o n s e q u e n t loss of or b izar re a l terat ions in dendr i t ic sp ine s , the appear-

ance of dendr i t ic b r a n c h i n g pat tern abnorma l i t i e s , and a t tenua t ion and degen-

era t ion of axonal te rminals . M u c h w o r k wil l b e r equ i red to further define spe-

cific s tages in th i s c o n t i n u u m of morpho log ica l a l tera t ions and the i r var ious

in ter re la t ions .

MORPHOLOGICAL SUBSTRATE OF VISUAL EVOKED POTENTIALS IN THE PRETERM INFANT

There appears to b e s o m e ag reemen t that the v isua l evoked r e sponse (VER)

to pho t ic s t imula t ion in p re te rm infants r ang ing in age from 24 w e e k s g.a. to

abou t 30 w e e k s g.a. is charac ter ized b y an ini t ia l sur face-negat ive def lect ion of

var iable dura t ion ( 1 5 , 1 6 , 2 3 , 2 4 , 6 1 , 6 3 ) . Acco rd ing to p u b l i s h e d data at approxi -

mate ly 32 w e e k s g.a. ( ± 2 weeks ) the p r o m i n e n t nega t iv i ty i s p receded b y a

smaller pos i t ive def lect ion w h o s e deve lopmen t hera lds the p rogress ive devel-

o p m e n t of the early pos i t i ve -nega t i ve c o m p o n e n t of the V E R that is character-

ist ically obse rved in the full- term neona t e ( 1 5 , 1 6 , 2 3 , 2 4 ) . F e w inves t iga tors con-

ce rned w i t h the ma tu ra t ion of the V E R in the h u m a n infant have res is ted

specula t ion as to the pos s ib l e deve lopmenta l c h a n g e s in neurona l and synapt ic

o rgan iza t ions r e spons ib l e for these dramat ic changes in the early c o m p o n e n t s

of the V E R . S o m e have sought clues to the matura t iona l s tatus of the visual

160 Dominick P . Purpura

cortex in the pre te rm infant in the s tudies o f C o n e l (14) and R a b i n o w i c z

(51,52) despi te the fact that these authors have not repor ted neurohis to log ica l

obse rva t ions in p re te rm infants in the s ixth and seven th m o n t h s of ges ta t ion .

O t h e r inves t iga tors have re l ied heav i ly u p o n compara t ive m o r p h o p h y s i o -

logical data o b t a i n e d in different spec ies of labora tory an imals (21 ,36 ,49 ) . N o

s tudies have b e e n repor ted in w h i c h the morpho logy of the v isua l cor tex w a s

e x a m i n e d wi th the rapid Golg i m e t h o d in p re te rm infants in w h i c h elec-

t rophys io log ica l data on the character is t ics of the V E R were o b t a i n e d pr ior to

death of the infant. Such correlat ive morphophys io log ica l s tud ies are current ly

b e i n g pursued in the an t ic ipa t ion that the m o r p h o g e n e t i c even t s under ly ing

the t rans i t ion in early c o m p o n e n t s of the V E P in the p re te rm infant m a y b e

be t te r def ined.

At th is t ime only the bas i c plan of approach and s o m e pre l iminary obse rva-

t ions can b e repor ted to ind ica te the potent ia l va lue of the m e t h o d . T h e case

selected for d i scuss ion is that a p re te rm infant b o r n at 29 w e e k s g.a. (bi r th

we igh t 1330 g m ) , w i t h respi ra tory dis t ress r equ i r ing ass i s ted resp i ra t ion and

oxygena t ion . T h r e e w e e k s after b i r th (32 w e e k s g.a .) the phot ic evoked po ten-

tials (VEP) s h o w n in F igure 9 we re recorded . Dea th occurred several days later

C3 A , y v

3 sec 32 weeks

FIGURE 9 . Averaged visual evoked potentials recorded from central, parietal, temporal, and oc-

cipital sites, referred to the ipsilateral ear. Stroboscopic stimulation, ISI 8 seconds. Infant 3 2 weeks

estimated gestational age ( 3 weeks postpartum). Prominent early negativity at occipital lead ( 0 1 ) is

preceded by a small positivity. Late components are especially noteworthy and indicative of con-

siderable complexity in the VEP at this age. Golgi preparations of the visual cortex from this in-

fant are illustrated in Figure 1 0 . (From M. Eisengart, M. W. Kremenitzer, J . A. Kreuzer, and H. G.

Vaughan, Jr . , unpublished data.)

P3

15

Ol


after b r i e f r epea ted ep i sodes of cardiac arrest. V E P ' s recorded at 32 w e e k s from

the occ ipu t e x h i b i t e d a p r o m i n e n t early nega t iv i ty p receded b y a small surface

pos i t iv i ty . R e m a r k a b l y complex long la tency wavefo rms we re also obse rved

in th is infant fo l lowing the p o s i t i v e - n e g a t i v e ini t ia l c o m p o n e n t s . T h e V E P ' s

recorded at th i s age m a y represen t a t rans i t iona l s tage from the m o r e i m m a -

ture conf igura t ion ( i . e . , in i t ia l large surface nega t iv i ty ) to the early ma tu re

form ( i . e . , d iphas ic pos i t i ve -nega t i ve in i t ia l c o m p o n e n t s ) .

T h e genera l morpho log ica l features of several of the m o s t p r o m i n e n t types

of n e u r o n s encoun te r ed in the v isua l cor tex are i l lustrated in F igure 10 in ex-

amples from rap id Golg i p repara t ions o b t a i n e d from the 3 2 - w e e k p re te rm in-

fant w h o s e V E P ' s are s h o w n in F igure 9. Smal l and m e d i u m pyramida l and

pyramidal - l ike cells of the superficial layers of the v isua l cor tex are i l lustrated

in F igures 1 0 A - C . Apica l dendr i tes o f these e l emen t s are in a state of con-

t inu ing g rowth as ind ica ted b y the p re sence of m a n y fine p rocesses , va r icos -

i t i es , and l u m p y en la rgemen t s on shafts and b r a n c h e s of the apica l dendr i t ic

sys tem. Bas i la r dendr i t es , in contras t to apical dendr i t es , are poor ly deve loped

and in m a n y ins t ances are r ep resen ted b y a few pro top lasmic ex t ens ions from

cell b o d i e s (F igures 10B and C) . Bas i la r dendr i tes of deepe r ly ing m e d i u m pyr-

amids appear s o m e w h a t m o r e deve loped (Figure 10D) bu t t hey do not exh ib i t

the ex tens ive d e v e l o p m e n t of the bas i la r dendr i tes of layer V py ramids (Figure

10M) or M e y n e r t cells (Figure 10P) .

A m o s t s t r ik ing f inding is the p resence of we l l -deve loped n e u r o n s t e rmed

b y Cajal (9) cellules a double bouquet dendritique (F igures 1 0 E - H ) . Charac ter -

is t ical ly these are fusiform cells w i t h g roups of dendr i tes o r ig ina t ing from

b o t h poles and s u b t e n d i n g layers superficial and deep to the cell b o d y (11 ,59) .

T h e dendr i tes exh ib i t a ver t ica l o r ien ta t ion and no t in f requent ly the superfi-

cially d i rec ted dendr i tes penet ra te th rough the molecu la r layer (F igures 10G

and H ) . T h e fine axons of these cells are difficult to v i sua l ize in the i r en t i re ty

bu t in genera l they conform to the " h o r s e - t a i l " descr ip t ion appl ied recent ly b y

Szen tago tha i (59) w h o has sugges ted the i r impor tan t i nvo lvemen t in car t r idge-

l ike c l i m b i n g fiber axodendr i t i c synapses on apical dendr i tes of deep ly ing

pyramids . It is of in teres t that the cell b o d y of the cellule a double bouquet

dendritique w a s m o s t f requent ly encoun te red in the superficial ha l f o f the

v isua l cor tex. D e s c e n d i n g dendr i tes of these e l e m e n t s t e rmina t ed as deep as

the layer of M e y n e r t cells ( layer V ) . Cel ls of layer V I w i t h arciform ascend ing

axons were also ident i f ied in the p resen t case (F igures 101 and M ) . Stel late cells

of the s u b g e n n a r i a n layer were found to have ex tens ive , we l l -deve loped den-

dri tes (Figures ION and O ) that were often longer than the bas i la r dendr i t es of

layer V and VI py ramids .

Dendr i t i c sp ines we re no t o b s e r v e d on the vast major i ty of apical dendr i t ic

shafts or s ide b r a n c h e s of pyramida l n e u r o n s w h o s e cell b o d i e s we re located

in layers III—VI. H o w e v e r , occas iona l ly small local ized s e g m e n t s of apical

dendr i t ic shafts pas s ing th rough layers IV and V pos se s sed p r imi t ive dendr i t ic

sp ines , sp icu les , or f i lopodia- l ike p rocesses (F igures 1 0 J - L ) . Surpr i s ing ly , the

FIGURE 10. Morphological characteristics of some prominent neuron types in the visual cortex of a 33-week-old preterm infant (born at 29 weeks g.a.) Golgi rapid preparations. A-C . Small and medium pyramids of layers II and III. Dendritic growth processes are detectable on apical den-drites of most of these elements. Basilar dendrites are poorly developed. D. Pyramidal neuron of layer III. E-H. Examples of Cajal's cellules a double bouquet dendritique. Note, in G and H superfi-cially directed dendrites of these cells penetrate the molecular layer. I. Neuron of layer VI with ar-ciform axon ascending in close relation to its apical dendrite (arrows). J . Higher magnification of midapical region (axon on the right). The dendrite is free of typical spines. K and L. Low and higher magnification, respectively, of an apical dendrite of a layer V cell. A localized cluster of primitive dendritic fine processes (spines?) is evident in the higher magnification photo (L). M. Pyramidal neuron of layer VI with axon collateral well-visualized. Basilar dendrites well devel-oped. N-O. Large stellates and pyramidal neurons of layers V and VI. P. Meynert cell with thorny excrescences on proximal segments of basilar dendrites. Magnification markers, 10 fim, unless noted otherwise.

162


bas i la r dendr i tes of M e y n e r t cells exh ib i t ed c o n s p i c u o u s thorny p rocesses .

T h e foregoing s u m m a r y descr ip t ion of several types of v isua l cor tex neu rons

encoun te red in a 32 -week -o ld p re te rm infant should b e cons ide red on ly a first

app rox ima t ion to the m a i n p r o b l e m w h i c h l ies ahead in ca tegor iz ing re la t ions

b e t w e e n deve lopmen ta l changes in cort ical n e u r o n s and matura t iona l changes

in the V E P . M a n y types of cells such as the cells w i t h doub le dendr i t ic

bouque t , w h i c h are though t to play an impor tan t role in t rans laminar co -

lumnar ope ra t ions in v isua l cor tex (11 ,59 ) , as wel l as pyramida l neu rons and

stellate cells of the subgenna r i an layer appear r emarkab ly wel l -deve loped in

the p resen t case . O n the o ther h a n d superficial py ramids are in a state of ac-

t ive dendr i t ic different ia t ion. T h e a b s e n c e of sp ines on dendr i t ic shafts of

mos t var ie t ies of pyramida l neu rons is s t r ik ing. T h e f inding that small seg-

m e n t s of dendr i t ic shafts in layers IV and V e x h i b i t e d small clusters of sp ines

and spicules is of in teres t from the s t andpo in t of the re la t ion of these dendr i t ic

p rocesses to the pos s ib l e prol i ferat ion of axon collaterals o f stel late cells (59) . A

more in t r igu ing ques t i on , howeve r , is h o w do the genicu locor t ica l afferents

engage cort ical n e u r o n s at th is deve lopmenta l s tage? T h e r e is good ev idence

that in adult an imals lateral geniculocor t ica l afferents t e rmina te p r e d o m i n a n t l y

in re la t ion to dendr i t ic sp ines of e l emen t s in layers IV and III (10 ,13 ,17 ,59 )

p r o b a b l y avo id ing the apical dendr i tes of pyramida l n e u r o n s (59) . If this is the

case in the mature cor tex , w h a t can b e inferred c o n c e r n i n g the m o d e of te rmi-

na t ion of geniculocor t ica l afferents in the i m m a t u r e b ra in w h e n dendr i t ic

sp ines are not p rominen t , as in the mater ia l i l lustrated in F igure 10 . S ince

wel l - local ized V E P s are de tec tab le at th i s t i m e [and m u c h ear l ier ( 1 5 , 2 3 , 6 1 , 6 3 ) ]

it m a y b e sugges ted that the immatu r i ty of the V E P ( i . e . , p r o m i n e n c e of early

nega t ive c o m p o n e n t ) is related in s o m e obscure fash ion to the type of synapt ic

e n g a g e m e n t of geniculocor t ica l afferents as wel l as the m o d e of d i s t r ibu t ion of

these afferents. T h i s p r o b l e m has b e e n dealt w i t h p rev ious ly in connec t i on

wi th the i m m a t u r e fel ine v isua l cor tex (26) and awai t s further examina t i on .

COMMENT: TOWARD A DEVELOPMENTAL NEUROBIOLOGY OF MENTAL RETARDATION?

T h e presen t s tudies have explored several approaches to deve lopmenta l

even ts in the i m m a t u r e h u m a n b ra in that have b e e n useful in on togene t i c in-

ves t iga t ions in labora tory an imals . W h i l e the obse rva t ions have p rov ided ad-

di t ional in format ion on the morpho log ica l features o f i m m a t u r e neu rons in the

h i p p o c a m p u s and v i sua l cor tex in p re te rm infants they have con t r ibu ted little

to the p r o b l e m of the re la t ionsh ip of aber rant deve lopmen t to men ta l sub -

normal i ty . N o r shou ld th is b e o the rwi se ; for th is p r o b l e m is no t l ikely to y ie ld

to the m i n i m a l effort exempl i f ied here b y a t ten t ion to a few aspects of

neurona l d e v e l o p m e n t in two selected areas of the h u m a n bra in . Never the less


the p r o b l e m has b e e n e n g a g e d wi th s t ra tegies that have s o m e mer i t in the i r

appeal to ques t i ons o f genera l neu rob io log ica l in teres t . Severa l f indings sum-

mar ized above i l lustrate th is v i ewpo in t .

Def in i t ion of the pe r iod in h u m a n fetal deve lopmen t dur ing w h i c h dendr i t ic

different ia t ion occurs is clearly of in teres t in speci fy ing even ts of cons ide rab le

impor t ance in cort ical deve lopmen t (9) . D e n d r i t e s of cort ical n e u r o n s p rov ide

more than 9 0 % of the surface area for synapt ic inpu t s to cort ical n e u r o n s (58)

even t h o u g h the total area occup ied b y pos t synap t ic m e m b r a n e is a very small

fraction of the avai lable somadendr i t i c surface area (12 ,13) . No tw i th s t and ing

th i s , dendr i t ic g rowth and dendr i t i c b r a n c h i n g pat terns are genera l ly cons id -

ered to b e essen t ia l pa ramete rs o f the p robab i l i ty o f neurona l in te rac t ion in

normal and pa thologica l s i tua t ions (cf. Hut ten locher , Chap te r 5 , th is vo lume) .

Unfor tuna te ly it r ema ins to b e e s t ab l i shed w h e t h e r s ignif icant devia t ions

from " n o r m a l " b r a n c h i n g pat terns necessa r i ly imply abno rma l neurona l in-

teract ion. O the r cr i t ical factors are l ikely to b e the n u m b e r , d i s t r ibu t ion , and

geomet ry of dendr i t ic sp ines , w h i c h are morpho log ica l ref lect ions of the major

m o d e of synapt ic e n g a g e m e n t o f cort ical n e u r o n s b y ext r ins ic afferents

( 1 3 , 1 7 , 2 5 , 5 9 ) . S igni f ican t c h a n g e s in the n u m b e r , d i s t r ibu t ion , and geomet ry

of dendr i t ic sp ines shou ld have impor tan t c o n s e q u e n c e s for neurona l opera-

t ions (54 ,65) p rov id ing of course that all dendr i t ic sp ines are s i tes of synapt ic

contacts . T h i s is p r o b a b l y t rue in normal ma tu re b ra in bu t does not necessa r -

ily ho ld for cond i t ions in w h i c h dendr i tes exh ib i t abnormal ly appear ing

sp ines , sp icules , or f i lopodia- l ike p rocesses , as in the p resen t s tud ies and

those of Mar in -Pad i l l a (28 ,29) . T h u s , phys io log ica l impl ica t ions of dendr i t ic

sp ine abnormal i t i e s obse rved in Go lg i mater ia l can on ly b e fantas ized unt i l it

is e s t ab l i shed in e lec t ron m i c r o s c o p e s tudies that a b n o r m a l dendr i t ic " s p i n e s "

in immatu re cor tex are in synapt ic re la t ion w i t h axonal p rocesses . R e c e n t e lec-

t ron mic ro sco pe s tud ies of dendr i tes and dendr i t ic sp ines of n e u r o n s in pa tho-

logical s p e c i m e n s of h i p p o c a m p a l t i s sue r e m o v e d at the t ime of surgery for

tempora l lobe ep i l epsy are re levant to th is p r o b l e m (8) .

T h e present s tud ies have e m p h a s i z e d ano the r s t ra tegy for ana lys is of

neurona l deve lopmen t in the h u m a n bra in . T h i s approach r ecogn izes the use-

fulness of fiber s ta ins for d e t e r m i n i n g the matura t iona l features of a remark-

able axonal p lexus wel l -charac te r ized in compara t ive neuroana tomica l s tudies

of the h i p p o c a m p u s (9 ,27) . T h e fact that the vas t major i ty of axons in th i s

p lexus t e rmina te in baske t l ike ne tworks that enve lop pyramida l cell b o d i e s

has a l ready b e e n no t ed (1 ,4 ) . M o r e impor tan t ly , phys io log ica l obse rva t ions

suggest that axosomat ic synapses formed b y th is ba ske t p lexus are i nh ib i t o ry

in funct ion (1 ,3 ) . I ndeed it has b e e n p r o p o s e d that the baske t l ike p lexus of ax-

osomat i c te rminals on to h i p p o c a m p a l pyramida l neu rons cons t i tu tes one of

the mos t powerful i n h i b i t o r y sys t ems in the cerebra l cor tex (1) . H e n c e the s ig-

nif icance of a t t empts to def ine its t empora l pat tern of deve lopmen t and the


poss ib l e factors in f luenc ing axon te rmina l prol i ferat ion. Da ta on th is po in t are

far from comple te .

Ava i l ab le in format ion sugges t s that the axon p lexus of the h i p p o c a m p u s

m a y b e exqu i s i t e ly sens i t ive to a var ie ty of factors w h i c h place the p re te rm

and full-term infant at r isk. W h a t effect a t t enua t ion of i n h i b i t o r y synapt ic

even ts m i g h t exer t on the normal funct ional opera t ions of the h i p p o c a m p u s is

not k n o w n , s ince so little is unders tood abou t the na ture of these opera t ions

to b e g i n wi th . It is p robab ly no c o i n c i d e n c e that the axonal p lexus of the h ip -

p o c a m p u s w a s found to b e severe ly a t t enua ted in the case desc r ibed above

w i t h infant i le se izures and deve lopmen ta l re tardat ion . C o n c e i v a b l y the loss of

axosomat ic i nh ib i t o ry inpu t s u b s e q u e n t to a t tenua t ion of the axonal p lexus

m i g h t p rov ide o n e m e c h a n i s m for h i p p o c a m p a l i nvo lvemen t in se izures of in-

fancy (43) . Clear ly th i s p r o b l e m is a m e n a b l e to expe r imen ta l i n q u i r y in i m m a -

ture labora tory an ima l s and is wor thy of c lose pursu i t for its r e levancy to the

p r o b l e m of the pa thogenes i s of t empora l l obe se izures in ear ly ch i ldhood .

A n o t h e r approach to the s tudy of deve lopmen ta l even t s in the immatu re

h u m a n b ra in has b e e n i l lustrated b y p re l imina ry obse rva t ions on the m o r p h o -

logical features of v i sua l cor tex n e u r o n s in a p re te rm infant in w h i c h v isua l

evoked potent ia l s (VEPs) we re recorded several days pr ior to death . T h e spe-

cific a im of th is approach is to correlate morpho log ica l c h a n g e s in cort ical

neu rons w i th deve lopmenta l changes in early c o m p o n e n t s of V E P s . A for-

m i d a b l e task cons ide r ing that expe r imen ta l s tudies have y ie lded few u n e q u i v -

ocal data c o n c e r n i n g the morpho log ica l subs t ra te of any var ie ty of evoked cor-

t ical po ten t ia l in labora tory an ima l s . Never the less exper imenta l ly de r ived data

mus t con t inue to p rov ide the b a s i s for ana lys is o f s t ruc ture- func t ion re la t ions

in the h u m a n bra in .

Reg i s t ra t ion o f V E P s and e x a m i n a t i o n o f the matura t iona l s ta tus of v i sua l

cort ical n e u r o n s in infants w h o fail to surv ive can p rov ide a wea l th of data on

several impor tan t ques t i ons . T h e i ssue of the m o d e of synapt ic e n g a g e m e n t of

cort ical e l emen t s b y p r imary geniculocor t ica l afferents in p re t e rm infants p r io r

to the e labora t ion of dendr i t ic sp ines is of no little impor t ance as no ted

previous ly . T h e re la t ionsh ip b e t w e e n tempora l pat terns of apical and bas i la r

dendr i t ic g rowth in n e u r o n s at different cort ical dep ths to character is t ics of

V E P s in ear ly and late p re te rm infants is ano the r ques t i on that is a m e n a b l e to

inves t iga t ion . F ina l ly , there r ema ins the p r o b l e m of p rov id ing a sat isfactory

descr ip t ion of the matura t ion of cer ta in spectacular e l emen t s such as the

cellules a double bouquet dendritique, w h i c h m a y play a centra l role in the

morpho log ica l subs t ra te for funct ional co lumnar o rgan iza t ions (11 ,59 ) .

T h e several approaches de sc r ibed in th is repor t obv ious ly reflect the specia l

in teres t of the au thor and therefore ho ld n o c la im to pr ior i ty o f impor t ance .

Hopeful ly they m a y b e v i e w e d as the m o s t tenta t ive g rop ings toward a devel-

opmen ta l n e u r o b i o l o g y of the h u m a n b ra in w h i c h wil l have as one of i ts


pr imary ob jec t ives an adequa te under s t and ing of the morphophys io log ica l

bas i s of menta l re tardat ion.

SUMMARY

Severa l approaches ut i l ized in on togene t i c inves t iga t ions in labora tory an-

imals have b e e n explored in p re l iminary s tudies of morphogene t i c events in

the h u m a n cerebra l cortex.

1. Golg i s tudies of dendr i t ic g rowth c o n e s , f i lopodia, and o ther develop-

menta l p rocesses have pe rmi t t ed specif icat ion of the max ima l phase of den-

dritic g rowth and different ia t ion o f pyramida l neu rons in the h i p p o c a m p u s .

Th i s pe r iod spans the twen t ie th to twen ty -e igh th w e e k of fetal deve lopment .

2 . S tud ies of the t empora l pat tern of appearance of the axonal p lexus of the

s t ra tum pyramida le sugges t that axosomat ic synapt ic pa thways in the h i p -

p o c a m p u s develop relat ively late in respec t to the appearance of axo-

sp inodendr i t i c inputs .

3. Dendr i t i c sp ine deve lopmen t is ev iden t at 26 w e e k s g.a. in the h i p -

p o c a m p u s bu t no t in the v isua l cortex. M o s t h i p p o c a m p a l pyramida l neu rons

have acqu i red a full c o m p l e m e n t of sp ines b y 6 m o n t h s postnatal ly . T h e pres-

ence of severe me tabo l i c and card iorespi ra tory d i s tu rbances and/or c h r o m o -

somal abnormal i t i e s s ignif icant ly inf luences dendr i t ic sp ine morpho logy and

deve lopment .

4 . T h e genera l morpho log ica l features of several var ie t ies of n e u r o n s in the

v isual cortex of a 32 -week -o ld p re te rm infant are cons ide red in respec t to the

e lec t rographic charac ter is t ics of th is infant ' s v isua l evoked r e sponses . T h e o b -

servat ions in th is and o the r cases i l lustrate the m a n n e r in w h i c h on togene t i c

p rob lems suscep t ib le to i nqu i ry in labora tory an imals can serve to gu ide s imi -

lar morphophys io log ica l s tud ies of normal and aber rant deve lopmenta l events

in the h u m a n bra in .

ACKNOWLEDGMENTS

The expert technical assistance of Mrs. Marie Buschke in the preparation of the histological material is gratefully acknowledged. Mr. Stanley Brown contributed greatly to the photographic work illustrated in this report. This study could not have been carried out without the dedicated services of these two fine persons.

This work was supported in part by grants from the NINDS NS-07512, NICHD HD-01799, and the Alfred P. Sloan Foundation.

REFERENCES

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3. Andersen, P., Eccles, J . C , and Loyning, Y. Location of postsynaptic inhibitory synapses on hippocampal pyramids. / . Neurophysiol., 1964, 27: 592-607.

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5. Blackstad, T., and Kjaerheim, A. Special axo-dendritic synapses in the hippocampal cortex: Electron and light microscopic studies on the layer of mossy fibers. / . Comp. Neurol., 1961, 117: 133-159.

6. Blackstad, T. W. On the termination of some afferents to the hippocampus and fascia dentata. An experimental study in the rat. Acta Anat., 1958, 35: 202-214.

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18. Goldfischer, S., Moore, C. L. , Johnson, A. B . , Spiro, A. J . , Valsamis, M. P., Wisniewski, H. K. , Ritch, R. H., Norton, W. T. , Rapin, I., and Gartner, L. M. Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome. Science, 1973, 182: 62-64.

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premature newborns. Electroenceph. clin. Neurophysiol, 1964, 16: 575-581. 25. Jones, E. G., and Powell, T. P. S. An electron microscopic study of the laminar pattern and

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30. Molliver, M. E . , Kostovic, L , and Van Der Loos, H. The development of synapses in cerebral cortex of the human fetus. Brain Res., 1973, 50: 403^107.

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34. Nafstad, P. H. J . An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata. Z. Zellforsch. Mikrosk. Anat., 1967', 76: 532-542.

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37. Poliakov, G. I. Some results of research into the development of the neuronal structure of the cortical ends of the analyzers in man. / . Comp. Neurol., 1961, 117: 197-212.

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40. Purpura, D. P. Analysis of axodendritic synaptic organizations in immature cerebral cortex. Ann. N.Y. Acad. Sci., 1961, 94 : 604-654.

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46. Purpura, D. P., and Pappas, G. D. Structural characteristics of neurons in feline hippocampus during postnatal ontogenesis. Exp. Neurol, 1968, 22 : 379-393.

47. Purpura, D. P., Prelevic, S., and Santini, M. Postsynaptic potentials and spike variations in the feline hippocampus during postnatal ontogenesis. Exp. Neurol, 1968, 22: 4 0 8 ^ 2 2 .


48. Purpura, D. P., and Shofer, R. J . Excitatory action of dibutyryl cyclic adenosine monophos-phate on immature cerebral cortex. Brain Res., 1972, 38: 179-181.

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6A Discussion: CNS Maturation and

Behavorial Development

G U E N T E R R O S E

Mental Retardation Research Center,



As a psycho log i s t , I have chosen to focus on p r o b l e m s w h i c h ar ise in

relat ing behavioral even t s to i nd ices of matura t ion _ o b t a i n e d b y o ther d is -

c ip l ines . T h e ou t s t and ing con t r ibu t ions of Drs . "Purpura and Hut t en loche r

cover ing ana tomica l and phys io log ica l c h a n g e s dur ing neu rona l deve lopmen t

p rov ide a b a s i s for such compar i sons . Fur the rmore , the de l inea t ion of d i s t inc-

t ive pat terns of e m e r g e n t b e h a v i o r s in n e o n a t e s , w h i c h parallel the c o n c o m i -

tant ana tomica l -phys io log ica l di f ferent ia t ion o f neura l subs t ra tes , as repor ted

in these s tud ies , wil l p rov ide an impor tan t inves t iga to ry tool in under -

s tand ing the matura t ion of no rmal as wel l as a b n o r m a l s t ructural-funct ional

re la t ionsh ips .

A crucial a s s u m p t i o n under ly ing th is type of research is that there are rela-

t ively abrupt c h a n g e s in the ne rvous sys t em w h i c h are reflected in the sudden

e m e r g e n c e of the po ten t ia l for n e w behav io r . T h e difficulties in demons t r a t ing

these re la t ionsh ips exper imenta l ly have t ended to e rode the concep t of cri t ical

s tages in deve lopmen t . H o w e v e r , a few successes in such corre la t ions lend

some suppor t to the concep t of th resho ld age , and the e lus iveness o f m a n y

s t ructura l - funct ional corre la t ions m a y b e exp la ined in te rms of i nadequa t e

fundamenta l concep t s and t e c h n i q u e s . I w o u l d l ike to focus br ief ly on such

p rob l ems b y ra i s ing two major po in t s .

First of all, wh i l e mos t s tudies of behav io ra l or central ne rvous sys t em

(CNS) matura t ion e m p h a s i z e gradual , usual ly l inear , inc reases in a m o u n t or

prof ic iency of the p h e n o m e n o n u n d e r inves t iga t ion , there are , in contrast ,

1 7 1

172 Guenter Rose

other s tudies w h i c h ind ica te non l inea r inc reases , inc reases then decreases , or

events u n i q u e to a par t icular g rowth pe r iod , such as p s e u d o s p i n e s on the cell

b o d i e s of neu rons . Typ ica l a g e - u n i q u e al terat ions are especia l ly seen in b i o -

chemica l s tudies o f matura t ion . W i t h regard to b e h a v i o r , gross act ivi ty levels

in rat pups , for example (4) , do no t con t inue to inc rease w i t h matura t ion , bu t

peak at approx imate ly 1 5 - 2 0 days of age , then decrease at later ages . Cor t ica l

sp indles dur ing s low w a v e s leep are p resen t b e t w e e n 1 5 - 2 5 days of age in

k i t tens . After the twenty-fif th day, the sp ind les d i sappea r on ly to reappear at

approx imate ly 6 w e e k s of age (3) . It i s useful , I th ink , to r ecogn ize that there

are even ts u n i q u e to a par t icular deve lopmenta l pe r iod w h i c h m a y or m a y not

b e eas i ly ident i f ied as the precursors of later behav io ra l or phys io log ica l

r e sponses bu t w h i c h have s igni f icance w i t h i n a c i r cumsc r ibed age-per iod .

O f pe rhaps greater impor t ance , is the fact that differential matura t iona l rates

w i th in funct ional sys t ems m u s t b e cons ide red , especia l ly w h e n cons t ruc t ing

neura l mode l s o f i m m a t u r e behav io r s . Dr . D o b b i n g has e m p h a s i z e d the dif-

ferent rate of deve lopmen t o f the cerebel lar as o p p o s e d to the cerebra l cortex.

Paral le l ing Dr . D o b b i n g ' s data on h u m a n s , F igure 1 i l lustrates the compara t ive

g rowth rates of se lec ted subcor t ica l nuc le i in the k i t ten b ra in , expressed as the

percent of total v o l u m e seen in the adult. N o t e , for example , the h i g h growth

rate of the lateral genicula te b o d y in contras t to the red nuc leus dur ing the first

30 days of life.

HIGH GROWTH RATE'-

Day 1 5 10 15 2 0 2 5 3 5 X 1 5 10 15 20 25 35 X 1 5 10 15 20 25 35 X 1 5 10 15 20 25 35 X

MEDIUM GROWTH RATE:

Dayl 5 10 15 2025 35 X 1 5 10 15 20 25 35 X h 5 10 15 20 25 35 X 1 5 10 15 20 2535 X

400

80

60

40

20

LOW GROWTH RATE'

Red Nucleus

J I 100

80

60'

40'

?0-

Day 1 5 10 15 2025 35

Septal Region

J 1 5 10 15 20 25 35 X

Ventral Anterior N.

J I 1 5 10 15 2025 35 X

Lateral Dorsal N.

/

1 5 10 15 2 0 2 5 3 5 X

FIGURE 1. Comparative growth rates of selected brain nuclei in kittens, expressed as percentages

of total volume in the adult.

Globus Pallidus Lateral Geniculate Body

. Inferior Colliculus L Lateral Lemniscus

Putamen [ Substantia Nigra J- Superior Colliculus • Dorsomedial N.

100

80

6 0 '

40

20 •

X

6A. CNS Maturation and Behavioral Development 1 7 3

If, as w e expect , the e lec t rophys io logica l data to b e o b t a i n e d suppor t the

ana tomica l data, it w o u l d ind ica te that adult neuro logica l mode l s of behav io ra l

p h e n o m e n a canno t b e eas i ly appl ied to deve lop ing behav io r s s ince the func-

t ional capab i l i ty of parts o f the m o d e l var ies w i th age . T h i s fact shou ld con-

t r ibute to a focus on neuro log ica l mode l s w h i c h c o m p l e m e n t b e h a v i o r s u n i q u e

to par t icular s tages of deve lopmen t , d e e m p h a s i z i n g the noncr i t ica l re l iance on

es t ab l i shed adult mode l s .

T h e s econd major po in t that I w i s h to m a k e relates to the difficulties in o b -

ta in ing mean ing fu l behav io ra l data pe r se from i m m a t u r e an ima l s , i . e . , data to

b e u t i l ized for s u b s e q u e n t corre la t ions w i th o the r i nd ices of matura t ion . O u r

o w n research , for example , is p r imar i ly conce rned w i t h the re la t ionsh ip

b e t w e e n e m e r g i n g v isua l behav io ra l capab i l i t i e s and e lec t rophys io log ica l indi -

ces of the deve lop ing v isua l sys t em, as ref lected b y a l tera t ions in the cort ical

potent ia l evoked b y l ight inpu t (6 ,9 ) . In unanes the t i zed k i t t ens , th i s cons i s t s

of a charac ter is t ic s e q u e n c e of wavefo rm changes b e g i n n i n g w i t h a s ingle

long- la tency nega t ive c o m p o n e n t at 0 - 4 days of age and e n d i n g w i t h an adult-

l ike complex W-shaped w a v e at 2 5 - 3 5 days of age . W i t h the excep t ion of a

recent b r i e f repor t (5 ) , s imi lar subcor t ica l a l tera t ions have yet to b e de l inea ted .

In a s se s s ing v i sua l ma tura t ion at the behav io ra l level , two approaches pre-

domina te : t hose w h i c h look at un lea rned reflexive k inds of b e h a v i o r , and

those dea l ing w i t h l ea rned behav io r . T h e former inc ludes tes ts of eye o p e n i n g ,

acui ty u t i l iz ing op tok ine t i c r e sponses , dep th pe rcep t ion v ia v i sua l cliff t ech-

n i q u e s , ex tens ive and gu ided p lac ing , v i sua l fo l lowing, etc. To date , w e can

make only a rough approx ima t ion conce rn ing neura l re la t ionsh ips b y s ta t ing

that m o s t of the more complex v isua l b e h a v i o r s s e e m to e m e r g e w h e n the

v isual cort ical r e sponse has a t ta ined a mature l ike wave fo rm at approx imate ly 4

w e e k s of age . T h i s ignores the m o s t cer ta in inf luence of subcor t ica l even ts

w h i c h to date r ema in unexp lored .

In unde r t ak ing deve lopmenta l s tudies such as the a b o v e , as wel l as those at-

t empt ing an a s s e s s m e n t of ear ly l ea rn ing-d i sc r imina t ive ab i l i t i es , there are a

n u m b e r of u n i q u e conceptua l and methodo log ica l p r o b l e m s one m u s t cons ide r

(1): (a) b o t h the sensory r e sponse to var ious s t imul i as wel l a,s the mo to r

express ion of b e h a v i o r var ies w i th age ; (b) mot iva t iona l levels vary w i t h age ,

as do the value of va r ious re inforcers , e .g . , y o u n g e r an imals w i t h h i g h e r me ta -

bol ic ra tes and genera l act ivi ty are l ikely to b e hungr i e r after a g iven pe r iod of

food depr iva t ion ; (c) un lea rned preferences to cer ta in s t imul i u sed in l ea rn ing

s tudies , such as l ight versus dark, m a y vary w i t h age ; and (d) finally, early ex-

per ien t ia l factors can play a role in e s t ab l i sh ing lea rn ing abi l i ty , e .g . , the

p rev ious expe r i ence of the an imal m a y m a k e that an ima l m o r e or less e m o -

t ional in a nove l test s i tua t ion w h i c h m a y i m p e d e or e n h a n c e l ea rn ing de-

p e n d i n g on the task.

B e c a u s e of all or s o m e of these factors , few, if any , s tudies have es t ab l i shed

v isua l d i s c r imina t ion capabi l i t i es i n k i t t ens b e l o w 6 w e e k s of age . To over-

c o m e s o m e of the r e sponse , mot iva t ion , and re in fo rcement p r o b l e m s , w e

174 Guenter Rose

dev i sed a s y s t e m of t ra in ing the an ima l s on a b l ack -wh i t e d i sc r imina t ion

u t i l iz ing a Y-water m a z e (7) . T h e k i t tens are capab le of s w i m m i n g b y 1 w e e k of

age , detes t the water , and h e n c e are mot iva ted to escape . T w o age groups were

s tudied: o n e , b e g i n n i n g at 35 days of age w h e n e v o k e d potent ia l s are mature -

l ike , and the s econd b e g i n n i n g at abou t 3 w e e k s of age w h e n evoked potent ia l s

are still immatu re . Bo th g roups had 1 w e e k of p re t ra in ing before actual tes t ing.

F igure 2 schemat ica l ly s u m m a r i z e s the data w h i c h ind ica ted that the age at

w h i c h the k i t t ens per formed a b o v e chance level , regardless of w h e n they started

the in i t ia l t ra in ing , s e e m e d to b e related to a t ime z o n e , b e t w e e n 25 and 35 days

of age , w h e n a mature- l ike v i sua l e v o k e d potent ia l is ach ieved . Aga in , th is is

a very crude correla t ion; it m a y b e ve ry task-specif ic . Fur the rmore , it is very

hard to s h o w a one - to -one co r r e spondence in these s i tua t ions unless one i s

actually record ing the e lec t rophys io logica l r e sponses dur ing the acqu i s i t i on of

the task.

W e have recent ly carr ied ou t a s tudy of th i s latter type (8) . B e g i n n i n g at 10 days

of age , k i t tens were in a Pavlovian cond i t i on ing pa rad igm wi th l ight as the

cond i t i oned s t imulus and leg flexion as the cond i t i oned r e sponse . Cort ical

evoked potent ia l and G S R measu re s were taken dur ing the t ra in ing . A l though

the an imals still d id no t s h o w behav io ra l cond i t i on ing unt i l abou t 6 w e e k s of

age , a difference b e t w e e n control and exper imenta l an imals wi th regard to

phys io log ica l measu re s w a s s e e n as early as 2 w e e k s of age . T h i s s tudy sugges ts

that wh i l e the assoc ia t ive phase of l ea rn ing m a y occur early in deve lopment , the

overt mo to r express ion of l ea rn ing occurs later w h e n neu ra l -muscu la r sys tems

are sufficiently mature .

mature-like units?

-A'A A

5 0 A

A G E IN W E E K S

FIGURE 2 . Schematic representation of black-white discrimination in Y-water maze (kittens). Learning curves beginning at 3 and 5 weeks of age based on actual data; hypothetical curve begin-ning at 1 week of age. Electrophysiological ( 9 ) and behavioral ( 2 ) data taken from previous studies.

co IOC UJ CO

O 0-00 LU K 5C

o UJ cc cr. o o

c

6A. CNS Maturation and Behavioral Development 175

H e n c e , w e are n o w sea rch ing for u n i q u e w a y s of e s t ab l i sh ing the onse t of

l ea rn ing ab i l i t i es w h i c h take the inab i l i ty o f m o t o r express ion in to account . I

shall conc lude th i s d i scuss ion wi th two examples .

In pi lot s tud ies on classic cond i t i on ing in our labora tory , Dr . R o b e r t

N o r m a n se lec ted the cond i t i oned e y e b l i n k as a mode l r e sponse sy s t em, b e -

cause it is a re lat ively s imple and h igh ly s te reo typed reflex, u n e n c u m b e r e d b y

postural or g a m m a m o t o r f eedback m e c h a n i s m s . Fur the rmore , ex tens ive

s tudies in adult an imals have p rov ided no rma t ive data on s t imulus and

r e sponse paramete rs for th i s pa rad igm. W e have a t t empted to t rain k i t t ens b y

pa i r ing an audi tory c o n d i t i o n e d s t imulus (CS) (1 k H z , 4 0 0 m s e c , 85 db) w i th a

shock to the eye (40 V , 2 m s e c ) , as the u n c o n d i t i o n e d s t imulus (US) . O u r in i -

tial s tudies ind ica ted that , i n d e p e n d e n t of w h e n t ra in ing was in i t i a ted , and

i n d e p e n d e n t of the n u m b e r of t r a in ing tr ials , cond i t i oned r e sponses could not

b e e s t ab l i shed unt i l 30 days of age . H o w e v e r , it w a s no t i ced that often w h e n

cond i t i on ing did appear , r e sponses w e r e seen on the first trials of a ses s ion ,

imply ing that acqu i s i t i on had b e g u n ini t ia l ly in earl ier s e s s ions , b u t w i th an

a b s e n c e of m o t o r express ion . T h i s idea w a s tes ted in a s u b s e q u e n t s tudy b y

t ra in ing exper imen ta l k i t t ens for 7 days b e g i n n i n g at age 21 days wh i l e control

l i t termates r ece ived r a n d o m presen ta t ions of b o t h C S and U S . After 2 days '

rest , b o t h groups rece ived cond i t i on ing tr ials. T h e an imals w h i c h had pre-

v ious pa i r ings s h o w e d i m m e d i a t e cond i t i on ing , often on the first t r ials , wh i l e

the control a n i m a l took 2 0 0 - 3 0 0 trials to cond i t ion (Figure 3 ) . W e tenta t ive ly

conc luded that the pr ior expe r i ence of pa i red p resen ta t ion of the C S and U S

was faci l i tated at s o m e earl ier age ( 2 1 - 2 7 days) bu t could not b e expressed

because of s o m e matura t iona l b lock b e y o n d the assoc ia t ive s tage in neura l

p rocess ing .

A C N S feedback des ign p rov ides ano ther al ternate t e c h n i q u e to th i s di-

l e m m a . In pi lot s tud ies , Dr . Jer ry Col l ins , Dr . R o b e r t N o r m a n , and I a t t empted

to answer the fo l lowing: Cou ld w e demons t ra t e l ea rn ing in very immatu re

sys t ems b y b y p a s s i n g m o t o r r e sponses , t rea t ing the po ten t ia l e v o k e d b y l ight

flash as an i n d e p e n d e n t va r iab le , and re inforc ing it d i rect ly to p roduce spec i -

fied c h a n g e s ? T h e des i red change in th is case wou ld b e an inc rease or de-

crease of the w h o l e v i sua l e lectrocort ical r e sponse (VEP) to e s t ab l i sh learn ing

abi l i ty pe r se . Add i t iona l payoff w o u l d occur i f w e could se lec t ively cond i t i on

part icular c o m p o n e n t s of the V E P .

To date , w e h a v e b e e n ab le to t rain e ight 1 V 2 - 3 - w e e k - o l d k i t tens to inc rease

or decrease , b y as m u c h as 5 0 0 % , an i m m a t u r e long- la tency nega t ive V E P

c o m p o n e n t (N 2 ) r ecorded from the v isua l cor tex , u s ing tone as a d i sc r imina t ive

s t imulus (SD), l ight flash as the C S , and mi ld electr ical shock as a nega t ive

reinforcer in a shock -avo idance pa rad igm (10) . A m p l i t u d e a l tera t ions in

yoked controls (e .g . , l ight flash only; n o n c o n t i n g e n t shock) r eached a max-

i m u m increase or decrease of 3 5 % from base l i ne , d e p e n d i n g on age . Th i s

occurs in b o t h awake k i t tens w i th imp lan t ed e lec t rodes and in Flaxedi l ized

prepara t ions . T h e resul ts are not due to trivial factors such as or ienta t ion

176 Guenter Rose

7 5 1

TRAINING T E S T DAYS

AGE IN DAYS

FIGURE 3 . Conditioned eyeblink magnitude (integrated E M G from orbicularis oculi) in arbitrary units averaged over 1 0 0 trials per daily session. Conditioned (solid line) group received condi-tioning trials on all days, while the random (dashed line) group received random presentation of CS and US during days 2 1 - 2 7 and were switched to paired presentation on days 3 0 - 3 2 . The imme-diate acquisition of the conditioned group on test days indicates that the response was "learned" during initial training in the absence of motor expression.

toward the l ight and pupi l lary changes . T h e evoked potent ia l inc reases and

decreases can , howeve r , b e in terpre ted as ind ica t ing that tonic state alter-

a t ions , as opposed to d iscre te neura l r e sponses , have b e e n cond i t i oned .

A n example of the data o b t a i n e d in such s tudies is s een in F igure 4 . A cr i te-

r ion specif ied b y a t ime-ampl i tude " w i n d o w " was set so as to r equ i re a 14-

day-old ki t ten (awake , chron ic implant ) to lower the ampl i tude of a N 2 c o m -

ponen t recorded from the v isua l cortex. T h i s an imal s h o w e d good control

th rough three cycles of acqu i s i t ion and ex t inc t ion . (The first ex t inc t ion pe r iod

was only part ial ly comple ted . ) T h e evoked potent ia ls sampled dur ing the s a m e

trials bu t from the midec tosy lv ian cor tex r e m a i n e d relat ively s tab le , pro-

v id ing an addi t ional control , sugges t ing that the changes seen in the v isua l

cor tex were not s imply due to per iphera l p h e n o m e n a , such as pupi l lary dila-

t ion . It sugges t s , fur thermore , that the m e c h a n i s m s w h i c h modu la t ed the

changes are e i the r con ta ined w i t h i n a specific cort ical locus or that va r ious path-

w a y s to the cor tex can have separate ga t ing m e c h a n i s m s w h i c h can funct ion

i n d e p e n d e n t l y of one ano ther dur ing the e s t ab l i shmen t of b r a i n - b e h a v i o r

r e la t ionsh ips .

AV

ER

AG

E

CR

M

AG

NIT

UD

E

CONDITIONED BLINK IN KITTENS I O O _

6A. CNS Maturation and Behavioral Development 177

T h e y o u n g e s t an imal successful ly cond i t i oned w a s 12 days of age , w h i c h , to

our k n o w l e d g e , is the earl iest age at w h i c h l ea rn ing invo lv ing the v isua l

moda l i ty h a s b e e n demons t ra t ed . It re inforces our o p i n i o n that o ther s tud ies

u s ing m o t o r r e sponse s have fai led to ident i fy the ear l ies t age o f v isua l

l ea rn ing , due to the immatu r i ty o f r e sponse sys t ems . A s m e n t i o n e d above , it

wou ld have b e e n sufficient in these s tud ies to have demons t r a t ed any signifi-

cant ampl i tude c h a n g e s over cont ro ls . T h e fact that it can b e conf ined to

specif ied w a v e s is an added advan tage in k i t tens in v i e w of hypo these s

c o n c e r n i n g the ana tomica l and funct ional ba s i s o f wavefo rm changes dur ing

matura t ion . T h e t e c h n i q u e , i f sufficiently va l ida ted , b e c o m e s a potent ia l ex-

pe r imen ta l tool for further research in a t t empt ing to i m p e d e or accelerate e lec-

t rophys io logica l ma tu ra t ion , a c c o m p a n i e d b y tes t ing for behav io ra l effects or

a l terat ion or C N S modif ica t ion . In addi t ion , t ransfer s tud ies invo lv ing evoked

potent ia l s or o the r e lec t rophys io log ica l act ivi ty can b e under t aken . S t e r m a n ,

for example , has operan t ly cond i t i oned a subs tan t ia l inc rease in the senso ry

moto r r h y t h m recorded from the cor tex of adult cats and then demons t r a t ed

specific t ransfer effects. S u c h cond i t i on ing p roduces a s ignif icant inc rease in

CONDITIONING OF N 2 COMPONENT - - CHRONIC K I T T E N ( I4day)

~* 8 E •€ 1 21 4 5 RE 16 1 4 8 12 16 1 4 5 12 16 20

ACQUISITION EXT. REACOUISITION EXTINCTION REACOUISITION

BLOCKS OF 5 0 T R I A L S

FIGURE 4. An example of feedback conditioning of an N 2 component evoked by light flash in the visual cortex (solid line). The criterion was set so as to require the kitten to lower the evoked po-tential amplitude at a prespecified latency. This animal showed good control through three cycles of acquisition and extinction. The evoked potentials sampled during the same trials but from the midectosylvian cortex (dotted line) remained relatively stable, suggesting that the changes in visual cortex were not simple peripheral phenomenon. Each data point represents the algebraic summation of 50 responses.

AM

PL

ITU

DE

(u

v.)

178 Guenter Rose

s leep sp indles (12) and a s ignif icant increase in the th reshold of se izures in-

duced b y hydraz ine c o m p o u n d s (13) . F inal ly , S t e r m a n (11) demons t ra ted the

effect iveness of S M R cond i t i on ing in r educ ing the i nc idence of grand mal

se izures i n o n e epi lept ic pa t ient .

It is our o p i n i o n that these al ternate t e c h n i q u e s are part icularly useful in

deal ing w i t h deve lop ing an ima l s and h u m a n s w h e r e one is faced w i t h u n i q u e

a s ses smen t p rob l ems , bu t also finds cons ide rab le potent ia l for C N S plast ici ty.

W i t h regard to the p resen t s y m p o s i u m , w e wou ld s t ress that any mode l s of

menta l re tardat ion, espec ia l ly in early deve lopment , m u s t take in to account

not only the poss ib i l i ty of C N S re tardat ion in the h i g h e r centers , bu t per iph-

eral s enso r imoto r def ic iencies as wel l .

REFERENCES

1. Campbell, B . A. Developmental studies of learning and motivation in infra-primate mammals. In: Early Behavior: Comparative and Developmental Approaches. (H. W. Stevenson, E. W. Hess, and H. L. Reingold, Eds.). Wiley, New York, 1967: 43 -71 .

2. Fagen, L. Unpublished observations.

3. Jouvet-Mounier, D., Actic, L . , and Lacote, D. Ontogenesis of the states of sleep in rat, cat, and guinea pig during the first postnatal month. Dev. Psychobiol, 1969, 2: 216-239.

4. Moorcroft, W. A. , Lytle, L . , and Campbell, B. A. Ontogeny of spontaneous locomotor activity in the rat. / . Comp. Physiol. Psychol, 1971, 75: 59-67.

5. Norman, J . L., and Wilson, P. P. Development of subcortical evoked potentials in kittens. Brain Res., 1973, 55: 4 4 6 ^ 5 0 .

6. Rose, G. H. The relationship of electrophysiological and behavioral indices of visual develop-ment in mammals. In: Neural Ontogeny and Behavior. (M. B . Sterman, D. J . McGinty, and A. M. Adinolfi, Eds.). Academic Press, New York, 1971: 145-183.

7. Rose, G. H., and Collins, J. P. Light-dark discrimination and reversal learning in early post-natal kittens. Develop. Psychobiol, in press.

8. Rose, G. H., and Dalhouse, A. Neurophysiological correlates of learning in young kittens. Electroenceph. clin. Neurophysiol, 1974, 37: 428.

9. Rose, G. H., Gruenau, S. P., and Spencer, J . W. Maturation of visual electrocortical responses in unanesthetized kittens: Effects of barbiturate anesthesia. Electroenceph. clin. Neurophysiol, 1972, 33: 141-158.

10. Rose, G. H., Norman, R. J . , Naifeh, K., and Collins, J . P. Plasticity of visual evoked potentials in kittens demonstrated by operant conditioning. 9. Physiol Behav. in press.

11. Sterman, M. B . , and Friar, L. Suppression of seizures in an epileptic following sensorimotor EEG feedback training. Electroenceph. clin. Neurophysiol, 1972, 33: 89-95.

12. Sterman, M. B . , Howe, R. C , and MacDonald, L. R. Facilitation of spindle-burst sleep by con-ditioning of the electroencephalographic activity while awake. Science, 1970, 167: 1146-1148.

13. Sterman, M. B . , LoPresti, R. W. , and Fairchild, M. D. Electroencephalographic and Behavioral Studies of Monomethyl Hydrazine Toxicity in the Cat. Tech. Rep. AMRL-TR-69-3. Wright-Pat-terson Air Force Base, Air Systems Command, 1969.

6B Discussion: Development of Postsynaptic

Potentials Recorded from Immature Neurons in Kitten Visual Cortex

C H E S T E R D . H U L L and D . R. G. F U L L E R




T h i s in format ion on the c h a n g e d m o r p h o l o g y of n e u r o n s in the h u m a n

ce r eb rum, g a i n e d from s o m e of na ture ' s unfor tunate expe r imen t s , is in teres t -

ing . It cer ta in ly s t r eng thens the sugges t ion that , to a cons ide rab le degree ,

severe men ta l re tardat ion resul ts f rom the func t ion ing of ma ldeve loped

n e u r o n s w h i c h have fewer no rma l (and poss ib ly m o r e abnormal ) synapses

w i th each other . T h e case for the abnormal ly func t ion ing n e u r o n in men ta l re-

tardat ion , ra ther than loss o f n e u r o n s pe r s e , i s s t r eng thened b y W e i n s t e i n

and T e u b e r ' s s tud ies (7) of Wor ld W a r II v i c t ims of cranial pene t ra t ing w o u n d s

in w h o m little or no loss of m e a s u r e d in te l l igence occurred in spi te o f ex ten-

s ive cort ical loss (7) . H o w e v e r , the effects of i m p o v e r i s h m e n t in neurona l

g rowth and synapt ic contac ts on neurona l funct ions are not yet very clear. A

f requent ly sugges ted m o d e l is the i m m a t u r e neu ron in w h i c h the dendr i t ic

g rowth is still cur ta i led , as is the n u m b e r of synapt ic contac t s . In th i s regard,

w e are repor t ing s o m e resul ts c o n c e r n i n g the e lec t rophys io log ica l deve lop-

m e n t of the i m m a t u r e neu ron w h i c h w e feel are general ly suppor t ive of Dr .

Purpura ' s v i e w s (4) on synap togenes i s in the cerebra l cor tex.

Recen t ly , w e have b e e n e n g a g e d in the a s se s smen t of age-re la ted c h a n g e s in

pos t synap t ic po ten t ia l s r ecorded from n e u r o n s in the v isua l cor tex of the

ki t ten. Reco rd ings have b e e n m a d e from ki t tens r ang ing from 4 - 1 7 days of

pos tnata l age . For k i t ten v i sua l cor tex , th i s age span b racke t s the onse t of in-

1 7 9

180 Chester D . Hull and D . R. G. Fuller

t ense synapt ic deve lopmen t and funct ional change as represen ted b y alter-

a t ions in l ight evoked potent ia ls recorded wi th macroe lec t rodes from the cor-

tical surface. Es t ima t ions b y Cragg of the n u m b e r of synapses per neu ron in

k i t ten cor tex (2) ind ica te a low rate o f synapt ic deve lopmen t up to the post-

pa r tum age of 8 days (112 synapses per n e u r o n ) , w h e n a rap id g rowth sets in ,

w h i c h b y age 18 days , has r eached 7000 synapses /neuron .

W h i l e the rapid rate of synapt ic g rowth con t inues unt i l abou t 37 days of

age , it is abou t 20 days of age w h e n mye l in first appears in th is cortex. T h u s ,

the effects of mye l ina t i on o f th is part o f the v isua l sys t em are p r o b a b l y no t

r e spons ib le for the changes w e are repor t ing here .

As Dr . R o s e has ind ica ted , and as has b e e n repor ted e l sewhere (3 ,6 ) , the

l ight evoked potent ia ls recorded from the cort ical surface undergo a ser ies of

progress ive changes in wavefo rm and la tency of r e sponse . For the per iod of

about 500 m s e c fo l lowing the onse t of a l ight flash, the p r e d o m i n a n t r e sponse

at the ear l ies t age is a re lat ively late nega t ive w a v e fol lowed, in turn, b y a

longer pos i t ive r e sponse . W i t h inc reas ing age , shor ter l a tency c o m p o n e n t s ap-

pear and the later c o m p o n e n t s of the r e sponse t end to b e a t tenuated unt i l a

relat ively adult form is r eached b y abou t 35 days of age .

It should b e m a d e clear that w e have conf ined our a t ten t ion to the earl ier

more tempora l ly re l iable c o m p o n e n t s of a complex r e sponse w h i c h , par t icu-

larly in y o u n g e r k i t t ens , can have a qu i te long dura t ion. T h i s is i l lustrated in

F igure 1. T h e b o t t o m traces of F igure 1 s h o w a s low record ing from the v isual

cortex of a 4 -day-o ld k i t ten , i l lustrat ing several cort ical r e sponse s e v o k e d b y a

100 -msec flash w h o s e in tens i ty is approx imate ly 17 m L . Note the long ser ies of

LIGHT EVOKED SURFACE POTENTIALS

FIGURE 1. Example of a light evoked surface potential recorded from a 4-day kitten with a macroelectrode. Bottom of figure shows a slow recording of 4 consecutive responses evoked by a 100-msec flash (1 per 20 seconds). Note long afterdischarge. Upper right: expanded time scale. Ver-tical distance between these two traces represents one standard deviation. Calibration: vertical base at left of figures are 100 fiV, positive upward. Horizontal line for average represents onset and duration of 100-msec flash. Vertical calibration is 50 JJLV.

6B. Postsynaptic Potentials Recorded from Immature Neurons 181

LIGHT EVOKED P.S.P.s

FIGURE 2. Example of intracellular record of light evoked PSP. Bottom of figure is a slow

recording of 4 consecutive responses evoked by a 50-msec flash (1 per 10 seconds). Upper left: 1

response shown on expanded time scale. Onset and duration of 50-msec flash shown immediately

below. Upper right: average of 10 consecutive light evoked PSPs. Spikes were removed before

averaging. Vertical bars at left are 10 mV. Vertical bar for average is 5 mV. Duration and onset of

50-msec light flash is shown by horizontal line.

h igh ampl i tude w a v e s evoked b y a s ingle flash. T h e trace at the upper left

s h o w s o n e of these r e sponses on an e x p a n d e d t ime scale . At the uppe r r ight is

an average of 10 consecu t ive r e sponses on a still more expanded t ime scale and

represen ts the part of the l ight evoked potent ia l w i t h w h i c h the r e m a i n d e r of

th is report wil l b e conce rned . T h e v isua l evoked potent ia l in the y o u n g ki t ten

is h igh ly d e p e n d e n t on rate of s t imula t ion . Fo r th is reason , low rates o f s t imu-

lat ion (one per 1 0 , or o n e per 20 seconds were employed ) .

As a further w o r d on me thodo logy , all k i t t ens we re p repared unde r e i ther

gas or shor t -ac t ing barb i tu ra te anes thes i a , and were s u b s e q u e n t l y admin i s -

tered local anes thes i a , para lyzed , and artificially vent i la ted . Eye l ids we re sepa-

rated and re t racted b y m e a n s of a po lye thy lene cy l inder w i t h a flared b a s e ,

w h i c h w a s inse r ted unde r the eye l ids . L igh t w a s in t roduced into the re t ina b y

m e a n s of a Maxwe l l i an v i e w sys tem and the ent i re re t ina w a s i l lumina ted .

As w e p rev ious ly ind ica ted , our goal is to e x a m i n e the deve lopmen t of

P S P s dur ing the onse t of rap id synapt ic deve lopment . Techn ica l ly , w e have

found the intracel lular r ecord ing from these i m m a t u r e cort ical n e u r o n s to b e

difficult and have no t yet b e e n able to vary the s t imula t ion paramete rs to any

s ignif icant degree . As o ther inves t iga tors have repor ted (5) , w e have found

these n e u r o n s to b e ex t remely fragile and difficult to impa le for long per iods of

t ime . A n example of one of our be t t e r record ings taken from an 8-day-old

k i t ten is s h o w n in F igure 2 . As in F igure 1, the r e sponse is s h o w n on different

t ime b a s e s . T h e b o t t o m traces s h o w re sponses of a s lowly depola r iz ing cell to

a ser ies of s t imula t ions . At th is t ime scale , the on ly clearly ident i f iable

r e sponse is an i nh ib i t o ry one . A s ingle r e sponse at an e x p a n d e d t ime scale is

182 Chester D. Hull and D. R. G. Puller

s h o w n in the uppe r left of the figure in w h i c h an E P S P appears to b e evoked

pr ior to the i nh ib i t o ry r e sponse . T h e sp ikes we re " s l i c ed off" b y m e a n s of a

compu te r p rogram, and then ten consecu t ive r e sponses were averaged to gen-

erate the t rac ing s h o w n on the upper r ight , i nd ica t ing the pos t synap t ic

r e sponse to b e a conven t iona l exc i t a to ry - inh ib i to ry s e q u e n c e . E x a m p l e s of

these averaged P S P s we re then g rouped accord ing to ages as s h o w n in F igure

3 , co r r e spond ing to age before i n t ense synapt ic deve lopmen t (first co lumn) ,

age at the b e g i n n i n g of rapid synapt ic deve lopmen t ( second co lumn) , and the

age jus t before the onse t o f mye l in i za t ion ( third co lumn) . Trac ings b e l o w the

dashed l ine are averages of the average P S P s for the co r respond ing age

groups . Cons ide r ing these averages at the b o t t o m as an ind ica t ion of the con-

t r ibu t ion of the addi t ional synapses per neu ron , at least for th is type of s t imu-

la t ion, the mos t p r o m i n e n t feature w o u l d appear to b e an exci ta tory process

g rowing out of a p rev ious ly laid d o w n inh ib i t o ry p rocess , and p r o b a b l y corre-

sponds to the sur face-negat ive potent ia l repor ted b y Dr . R o s e . A s further

synapt ic deve lopmen t takes p lace , th is exci ta tory r e sponse b e c o m e s sharper

and decreases in la tency.

P.S.P.s with Averages

4-7 Days 8-12 Days 13-17 Days

FIGURE 3. Averaged PSPs arranged according to age group. Each average is based on 4 to 10 sep-

arate responses. Traces below dashed line are average of the average PSPs and are based on different

numbers of averaged PSPs. 4-7 days (N = 6), 8-12 days (N = 5), and 13-17 days (N = 15). Calibra-

tion: vertical line is 5 mV; horizontal line is 100 msec. Onset of light flash occurred at the begin-

ning of the trace.

6B. Postsynaptic Potentials Recorded from Immature Neurons 183

SYNTHESIS of a S.E.P.

Syn.

Rec.

P.S.P.

P.S.P.

FIGURE 4. Synthesis of surface evoked potential. Top trace is weighted combination of the two PSPs shown at bottom. The latency of these PSPs were changed in the synthesis to achieve a better description of the actual surface evoked potential—second trace from top. In this case, negative is up for the surface evoked potential and down for the PSPs. Calibration for the PSPs is 5 mV (vertical bar) and 50 /JLV for the surface evoked potential. Horizontal is 100 msec.

O n e of the long- te rm purposes of m a k i n g these record ings is to es tab l i sh a

compu te r i zed " b a n k " of averaged P S P s of ind iv idua l n e u r o n s . A t t emp t s wil l

t hen b e m a d e to syn thes i ze a g iven surface evoked poten t ia l , at a g iven age ,

from a w e i g h t e d c o m b i n a t i o n of P S P s from th is b a n k as a po ten t ia l i ndex of

ind iv idua l differences in the rate of ma tura t ion of the v isua l sy s t em in ind i -

v idua l an imals . A n example of th is syn thes i s for the surface e v o k e d po ten t ia l

of F igure 1 is s h o w n in F igure 4 . No te that the surface evoked po ten t ia l has

b e e n inver ted such that a nega t ive def lect ion is n o w upward .

T h e r e i s , of course , no neces s i t y that any of the changes in these evoked po-

tent ia l c h a n g e s are reflect ing the concomi t an t addi t ion of n e w synapses .

D e v e l o p m e n t a l a l tera t ions in ind iv idua l synapt ic contac ts from " i m m a t u r e " to

mature form is also p o s s i b l e (1) . C o n t r i b u t i o n s from ma tu ra t ion of the v i sua l

pa thway from the re t ina onward also can and p robab ly do con t r ibu te . T h e pat-

tern of synap togenes i s s h o w n b y these record ings is cons i s t en t w i t h Dr . Pur-

pura ' s conc lus ions (4) that the earl iest deve lopmen t in the cor tex is a long

inh ib i to ry p rocess on w h i c h s u b s e q u e n t synapt ic deve lopmen t bu i lds .

REFERENCES

1. Aghajanian, G. K. , and Bloom, F. E. The formation of synaptic junctions in developing rat brain: A quantitative electron microscopic study. Brain Res., 1967, 6: 716-727.

184 Chester D. Hull and D. R. G. Fuller

2. Cragg, B. G. The development of synapses in cat visual cortex. Invest. Ophthalmol., 1972, 1 1 : 377-385.

3. Ellingson, R. J . , and Wilcott, R. C. Development of evoked responses in visual and auditory cortices of kittens. / . Neurophysiol, 1960, 23: 363-375.

4. Purpura, D. P. Intracellular studies of synaptic organizations in the mammalian brain. In: Structure and Function of Synapses. (G. D. Pappas and D. P. Purpura, Eds.). Raven Press, New York, 1972: 257-302.

5. Purpura, D. P., Shofer, R. J . , and Scarff, T. Properties of synaptic activities and spike potentials of neurons in immature cortex. / . Neurophysiol, 1965, 28: 925-942.

6. Rose, G. H., and Lindsley, D. B. Development of visually evoked potentials in kittens: Specific and non-specific responses. / . Neurophysiol, 1968, 3 1 : 607-623.

7. Weinstein, S., and Teuber, H. Effects of penetrating brain injury on intelligence test scores. Science, 1957, 125: 1036-1037.

7 Neurophysiological and Anatomical

Interrelationships of the Basal Ganglia

N. A. BUCHWALD, C. D. HULL, and M. S. LEVINE

Mental Retardation Research Center, Brain Research Institute, and

Departments of Anatomy and Psychiatry,



T h e te rm " m e n t a l r e t a rda t ion" is u s e d to desc r ibe m a n y different cogn i t ive

dysfunct ions . At o n e ex t reme are the ca tas t rophic c h a n g e s found in the as-

saults on the ne rvous sys t em w h i c h resul t f rom i n b o r n me tabo l i c errors such

as those occur r ing in T a y - S a c h s d i sease and me tach roma t i c l eukodys t rophy .

At the o ther ex t reme are the u n d e t e r m i n e d subt le changes in the ne rvous

sys t em w h i c h are " p r o g r a m m e d i n " b y deve lopmenta l ly early exper ien t ia l

factors and w h i c h impa i r an ind iv idua l ' s abi l i ty to acqu i re knowledge . Inter-

med ia t e b e t w e e n these ex t remes is a b road range of dysfunct ions that involve

complex in te rac t ions a m o n g local ized b r a in pa tho logy , the t ime of onse t of the

pa tho logy , and exper ien t ia l factors.

M a n y research s t ra tegies have b e e n u s e d to inves t iga te these cogn i t ive dys-funct ions . O n e of these s t ra tegies is to abst ract from the total ne rvous sys t em, an ana tomica l ly wel l -def ined set of in terre la ted s t ructures , man ipu la t e t h e m exper imenta l ly dur ing prenata l or early pos tnata l per iods of deve lopmen t , and t hen to assess the o u t c o m e s o f t hese man ipu l a t i ons in t e rms of a l tera t ions of b ra in funct ion and behav io r . A fruitful example of th is s t ra tegy is exempl i f ied b y the research o n the role o f the ce r ebe l lum repor ted b y A l t m a n i n th i s vol -u m e (2).

T h e sy s t em w e have focused on is the basa l gangl ia , a g roup of te lencephal ic and d iencepha l i c nuc le i w h i c h inc lude the caudate nuc leus , the pu t amen , the

1 8 7

188 N. A. Buchwald, C. D. Hull, and M. S. Levine

globus pal l idus , the subs tan t i a n igra , and the in t e rconnec t ions of these struc-

tures . To date , m o s t of the research in our o w n labora tory has conce rned

s tudies in adult an ima l s , bu t w e have n o w b e g u n to ex tend our work to the

deve lopmenta l sphere us ing the research s trategy ind ica ted above .

There are good reasons for cons ide r ing deve lopmenta l s tudies o f the basa l

gangl ia to b e of impor tance in p rov id ing a mode l o f the k ind of cogn i t ive b ra in

deficits w h i c h m i g h t result i n menta l re tardat ion. T h e s e r easons relate, on the

one h a n d , to cons ide ra t ion of the impor tan t funct ions w h i c h the basa l gangl ia

have dur ing deve lopmen t , and o n the other , to the a t t rac t iveness o f the basa l

gangl ia for exper imenta l inves t iga t ions because of the relat ive ease w i th w h i c h

they can b e s tudied and man ipu la t ed .

W i t h regard to the i r funct ions dur ing deve lopmen t , current ev idence

sugges ts that the basa l gangl ia , and part icular ly the s t r ia tum, play a s ignif icant

role w i th respect to spar ing of behav io ra l deficits s u b s e q u e n t to per ina ta l cor-

tical damage . For example , in p r imates , exper imen ta l l es ions i n the h e a d of

the caudate nuc leus m a d e in in fancy lead to pers i s t ing " c o g n i t i v e " deficits in

adul thood , as demons t ra ted b y the an ima l s ' failure to per form delayed-

response or s imi lar tasks invo lv ing m n e m o n i c abi l i t ies (33) . S imi la r deficits in

de layed- response per formance occur in adult m o n k e y s in w h i c h prefrontal

cort ical l es ions have b e e n made . H o w e v e r , these deficits do no t appear if the

cort ical l e s ions are m a d e in in fancy (13) . P r e sumab ly , the s t r ia tum in the

deve lop ing an imal is ab le to a s s u m e funct ions w h i c h the cor tex controls at

matur i ty . O n e migh t suppose , therefore , that the s t r ia tum in infancy is an

impor tan t c o m p o n e n t o f the b ra in m e c h a n i s m s subse rv ing the per formance of

complex b e h a v i o r s , gradual ly r e l i nqu i sh ing th i s role w i th deve lopmen t of the

cortex. G o l d m a n , in he r chapte r in th is v o l u m e , d i scusses the data and the

impl ica t ions of the complex re la t ionsh ip b e t w e e n s t r ia tum and cortex dur ing

deve lopmen t (12) . In addi t ion to the exper imenta l data, c l inical ev idence is

also avai lable sugges t ing that prenata l damage invo lv ing the basa l gangl ia

f requent ly results i n men ta l re tardat ion. M u c h of th is ev idence c o m e s from

cases of severe men ta l d i sabi l i ty in w h i c h abnormal i t i e s of the ventr icular

e p e n d y m a l l i n ing are a c c o m p a n i e d b y drast ic d is tor t ions of the caudate nu-

cleus (51) . In th i s s i tua t ion , and in m o s t cases in w h i c h morpho log ica l changes

in the b ra in have b e e n correlated w i t h ex i s tence of " m e n t a l re ta rda t ion ," the

behav io ra l deficit is on ly part of a synd rome of abnormal i t i e s , m a n y of w h i c h

are apparent ly unre la ted to the r educed level o f cogn i t ion . T h e sufferer is the

v i c t im of w h a t a m o u n t s to a ca tas t rophic mul t io rgan d i sease process resu l t ing

in death , h e n c e , the avai labi l i ty of the b ra in for s tudy. W h e t h e r the cellular

pa tho logy d iscovered in these d isease s tates can provide leads to under-

s tanding mi lde r forms of re tardat ion is u n k n o w n . H o w e v e r , b ra ins from such

ca tas t rophies (with the excep t ion of an occas iona l s p e c i m e n m a d e avai lable b y

accidenta l death or b y unre la ted d isease) are the on ly h u m a n mater ia l gener -

ally avai lable for s tudy [cf. chapters b y Purpura (46) and Hut ten locher (24) in

th i s v o l u m e ] .

7. Neurophysiological and Anatomical Interrelationships 189

A l though our par t icular in teres t is focused on n o n m o t o r i c aspec ts of basa l

gang l ion ic funct ion , dysfunct ions of the basa l gangl ia are cer ta in ly related to

" ex t r apy ramida l " m o t o r d isorders , inc lud ing P a r k i n s o n ' s and H u n t i n g t o n ' s

d i seases . T h e search for any s ingle concep t ( i . e . , m o t o r funct ion , sensory

funct ion, a t tent ion) to express the role of the basa l gangl ia in regula t ing b e h a v -

ior , h o w e v e r , m a y not b e meaningfu l . W h i l e the in terna l s t ructure of each

of the nuc le i c o m p o s i n g the basa l gangl ia is relat ively h o m o g e n e o u s , the topo-

graphica l d i s t r ibu t ion of cor t icostr ia ta l inpu t s (27) sugges t s that different

r eg ions of the s t r ia tum m a y serve different func t ions . Expe r imen ta l damage or

ab la t ion of the caudate nuc leus has n o w b e e n s h o w n to result i n m i n i m a l

motor ic or me tabo l i c def ic iencies , even though more subt le cogn i t ive and

motor ic abnorma l i t i e s can b e demons t ra t ed . Vi l l ab lanca and M a r c u s , in the i r

chapter in th i s v o l u m e , d i scuss th i s sub jec t and also detai l expe r imen ta l

m e t h o d s for caudate nuc lear ab la t ion , w h i c h do not result in gross cort ical

damage (52) .

F rom a pract ical po in t of v i e w , the basa l gangl ia , and part icular ly the

s t r ia tum, are at t ract ive for inves t iga t ion b e c a u s e of the i r b i o c h e m i c a l , ana tom-

ical , and e lec t rophys io logica l proper t ies . B iochemica l l y they are r ich in a

n u m b e r of pu ta t ive neuro t ransmi t te r s . T h e s e inc lude d o p a m i n e , ace ty lchol ine ,

and y - aminobu ty r i c acid ( G A B A ) , as wel l as the i r assoc ia ted e n z y m e s

( 7 , 8 , 1 4 , 1 9 , 3 8 , 4 4 ) . T h e s igni f icance of the h igh striatal d o p a m i n e con ten t has

b e e n the subjec t of cons ide rab le inves t iga t ion . Its dep le t ion w i t h phar-

macologica l agents and w i t h l e s ions in te r rup t ing the nigrost r ia ta l p a t h w a y are

wel l k n o w n p h e n o m e n a (18 ,40) . Hel ler and Hoffman d iscuss these mat ters in

the i r chapter (17) . Str iatal d o p a m i n e appears relat ively late in the deve lopmen t

of the ne rvous sys tem. T e n n y s o n et al. desc r ibe the course and na ture of the

deve lopmen t of the d o p a m i n e - c o n t a i n i n g fibers e m a n a t i n g from the sub -

s tant ia n ig ra in the i r chap te r (50) . T h e role p layed b y d o p a m i n e in regula t ion

of str iatal neura l act ivi ty , h o w e v e r , r ema ins e lus ive , and in the presen t

chapter , w e wil l p resen t s o m e data on th i s sub jec t . T h e r e i s i nc r ea s ing evi -

dence that G A B A is p resen t in a large fraction of striatal ou tput f ibers , and

that the synapt ic effects of g a b a m i n e r g i c f ibers on the i r target cells is i n h i b i -

tory ( 2 6 , 3 2 , 4 3 ^ 5 ) . T h e r e is little doub t that the concen t ra t ion of ace ty lchol ine

in the s t r ia tum is the h ighes t in the b ra in , a l though exact in vivo quan t i t a t ive

es t imates o f the concen t ra t ion of th is subs t ance have b e e n difficult to m a k e

because of the rap id des t ruc t ion of ace ty lchol ine b y cho l ines te rase . L e s i o n s of

b ra in s i tes w h i c h supply afferents to the s t r ia tum ( in t ra laminar tha lamus ,

cor tex, or subs t an t i a n igra) p roduce little or no change in the striatal con ten t of

e n z y m e s w h i c h are invo lved in m e t a b o l i s m of ace ty lchol ine (37) . T h e impl ica -

t ion of these s tudies is that the s tr iatopetal axons do no t ut i l ize ace ty lchol ine

as the i r synapt ic t ransmi t te r . Recen t work (25) in w h i c h striatal ace ty lchol ine

con ten t w a s m e a s u r e d direct ly b y b io log ica l assay , s eems to ind ica te , h o w -

ever , that a chol inerg ic inpu t does i n d e e d e m a n a t e from cer ta in nigral cells.

T h i s fact, of course , does no t cont radic t the p robab i l i t y that the bu lk of ace-


ty lchol ine is located in axon te rmina ls o f in t e rneurons . In contras t to ace-

ty lchol ine , the striatal d o p a m i n e con ten t is r e m o v e d a lmost comple te ly b y the

des t ruct ion of n igrost r ia ta l afferents.

T h e r ema inde r o f th i s chapte r wil l b e addressed to r ev i ewing our current

knowlege of the re la t ionsh ips o f the neu rophys io logy and ana tomy of the

basa l gangl ia in adult an imals w i th the impl ica t ions of th is knowledge for

s tudies in no rma l and a b n o r m a l aspects of the deve lopmenta l p rocess . W e will

b e g i n w i th a b r i e f desc r ip t ion of the a n a t o m y of the basa l gangl ia .

ANATOMY OF THE BASAL GANGLIA

For th i s d i scuss ion , the basa l gangl ia are defined to inc lude the caudate

nuc leus and p u t a m e n (s t r ia tum), the two shel ls o f the g lobus pal l idus ( the

inne r shell is called nuc leus en topenduncu la r i s in carnivores) and b e c a u s e of

its close ana tomica l and funct ional re la t ionsh ip wi th the o ther basa l gang l ion ic

c o m p o n e n t s , the subs tan t i a n igra . S o m e authors use the te rm neos t r i a tum or

corpus s t r ia tum as ident ica l w i th s t r ia tum. F igure 1 is a schemat ic of these

s tructures and inc ludes s o m e of the pr inc ipal connec t i ons of the basa l gangl ia .

T h e s t r ia tum rece ives major inputs from cerebra l cor tex, in t ra laminar thal-

a m u s , and the subs tan t i a n igra .

All or near ly all parts o f the cerebral cor tex project u p o n the s t r ia tum in a

topographica l m a n n e r (27 ,53) . R e c e n t work b y H e i m e r (15) even cons iders that

the cor tex of the rh inencepha lon pro jec t ing on the nuc leus a c c u m b e n s repre-

sents a l imb ic ex tens ion of the basa l gangl ion ic sys tem. Pro jec t ions from the

contralateral cortex to the s t r ia tum have also b e e n desc r ibed (27) .

Cerebral Cortex

Area 4 ; Cerebral Cortex j Contralateral

Thalamus ^ Caudate Substantia

VL !CM Putamen Nigra

i 1 Globus Pallidus

FIGURE 1. Schematic of the principal connections of the basal ganglia.


Tha l amic afferents reach the s t r ia tum from the in t ra laminar tha lamic nuc le i ,

pr incipal ly the central m e d i a n and the parafascicular nuc le i (39) . The re is no

secure ana tomica l ev idence conce rn ing the topography of th i s d i s t r ibu t ion .

T h e r e m a i n i n g direct afferents to the s t r ia tum c o m e from the m i d b r a i n , the

bu lk of w h i c h are nigrofugal . At least part of th i s p ro jec t ion i s m a d e up of

d o p a m i n e - c o n t a i n i n g fibers (3 ,4 ,40 ) . Str iatal afferents no t o r ig ina t ing in the

n igra inc lude se ro ton in -con ta in ing fibers (16) .

Ex tens ive detai ls of the neurona l m a k e - u p and synapt ic contacts of striatal

neu rons have b e e n p rov ided recent ly b y K e m p and Powel l ( 2 8 - 3 1 ) . M o r e than

9 0 % of the neu rons of the caudate nuc leus and p u t a m e n appear to b e small to

m e d i u m - s i z e d in t e rneurons w i t h ex tens ive dendr i t ic fields. T h e s e n e u r o n s are

d i s t r ibu ted h o m o g e n e o u s l y th roughou t the s t r ia tum. L e s s t han 5 % of striatal

neu rons have long axons w h i c h project ou t s ide the nuc leus .

K e m p and Powel l ' s work ( 2 8 - 3 1 ) detai ls the pat tern of synap t ic contac ts

on the shor t -axoned striatal i n t e rneurons . Dendr i t e s o f these n e u r o n s are

dense ly covered w i t h sp ines , except at the i r b a s e s . T h e major i ty of synapses

formed b y inpu t f ibers are axodendr i t ic . I n c o m i n g axons typical ly c ross the

dendr i tes of a n u m b e r of these neu rons at r ight angles , m a k i n g synapt ic con-

tacts en passant. A n ind iv idua l striatal in t e rneuron m a y rece ive inpu t s from

cort ical , tha lamic , and nigra l f ibers. Cor t ica l and tha lamic i npu t s are equa l ly

d i s t r ibu ted over the sp iny reg ions of the dendr i tes . H o w e v e r , cort ical afferents

are m o r e n u m e r o u s at the dendr i t ic ba se . Nigra l inputs are sparser . T h e i r

exact t e rmina t ions have no t b e e n ascer ta ined .

T h e two pr inc ipa l s t ructures r ece iv ing striatal efferents are the g lobus pal-

l idus and the subs tan t i a n igra . In addi t ion to the striatal p ro jec t ion , a major

input to the pa l l idum (and n igra) c o m e s from the sub tha l amic nuc leus (9) . A

nigropal l ida l p a t h w a y has also b e e n de sc r ibed (9) .

In contras t to the s t r ia tum, m o s t n e u r o n s i n the g lobus pal l idus are large

and fusiform ( 1 , 1 0 , 3 0 ) . Synap t i c contacts on these neu rons are usual ly m a d e

b y axons runn ing parallel to the dendr i tes . T h e major i ty of the neu rons are

l ong -axoned and te rmina te extranuclear ly . Sho r t - axoned in t e rneurons are

scarce . Acco rd ing to b o t h K e m p and Powel l (30) and F o x (10) , the m o s t

c o m m o n synapt ic e n d i n g s are te rmina ls of striatal ou tput cells w h i c h appear

large and con ta in large ves ic les . Smal l t e rmina ls m a y represen t e n d i n g s of

sub tha lamic or n igra l inpu ts to the pa l l idum, wh i l e rare large end ings w i t h

few ves ic les m a y b e the t e rmina t ion of intrapal l idal axonal b r a n c h e s .

T h e subs tan t i a n ig ra pos se s se s a cellular appearance ve ry s imi la r to that of

the g lobus pal l idus . T h e majo r i npu t to the n igra c o m e s from the s t r ia tum.

Apparen t ly , str iatofugal fibers m a k e contac t w i t h dendr i tes o f n igra l cells in

the ret icular por t ion of the subs tan t i a n igra .

T h e major p ro jec t ions of the g lobus pal l idus are to the vent ra l -an ter ior and

ventral- lateral nuc le i o f the tha lamus . Pall idal p ro jec t ions also occur to the

sub tha lamic nuc leus and to the m i d b r a i n t e g m e n t u m (42) . T h e tha lamic path-


w a y (Figure 1) serves to close the loop b e t w e e n the basa l gangl ia and the

cerebra l cor tex, a l though pall idal efferents also synapse on in t ra laminar tha-

lamic neu rons and are thus part of a m o r e local basa l gang l ion ic - tha lamic-

basa l gang l ion ic circui t . Recen t ly , Nau ta (41) has p rov ided ana tomica l evi-

dence for the ex i s t ence of an impor tan t pa thway from the pa l l idum to the

habenu la r nuc leus .

A l though the n igra , as wel l as the pa l l idum, posses ses a tha lamic afferent

(9) , m o r e a t ten t ion has b e e n pa id to the fine nigrost r ia ta l f ibers. T h e ex is tence

of th i s pa thway , e s t ab l i shed b y f luorescent mic roscop ic t e c h n i q u e s , has re-

cent ly b e e n conf i rmed b y the F i n k - H e i m e r m e t h o d (40) . Chapte rs b y Hel ler

and Hoffman (17) and T e n n y s o n et al. (50) in th is v o l u m e desc r ibe facets of the

nigrost r ia ta l pa thway in detai l .

ELECTROPHYSIOLOGY OF THE BASAL GANGLIA

W e be l i eve that it is n o w poss ib l e to under s t and the neu rophys io logy of the

basa l gangl ia . De ta i led in format ion on the synapt ic re la t ionsh ips o f basa l

gangl ion ic nuc le i has b e e n p rov ided b y recent defini t ive s tudies us ing mor -

phologica l ( 2 8 - 3 1 ) and e lec t rophys io logica l ( 5 , 6 , 2 0 - 2 2 , 3 4 ) t e c h n i q u e s . T h e ac-

count w h i c h follows is essent ia l ly a s u m m a r y of our o w n work on basa l

gang l ion ic neu rophys io logy . T h o s e detai ls w h i c h w e be l i eve can b e useful

e i ther n o w or in the near future in under s t and ing basa l gangl ion ic funct ion in

the deve lop ing o rgan i sm are s t ressed. Firs t , w e wil l cons ide r the in t r ins ic

act ivi ty of caudate and pall idal neu rons , and second , the r e sponses w h i c h can

b e evoked in these n e u r o n s b y s t imula t ion of the i r afferents.

Intrinsic Activity

N e u r o n s in the caudate nuc leus and p u t a m e n fire more s lowly than do

neu rons in m a n y o ther b ra in s i tes ( 6 , 2 0 , 3 5 , 4 7 , 4 8 ) . W h e n sys temat ic measu re -

m e n t s are m a d e unde r cond i t ions in w h i c h the states of consc iousness of the

exper imenta l sub jec t s are taken in to account , the m e a n firing rate of caudate

neu rons is found to b e s lower than that of n e u r o n s in the tha lamus or of

neu rons in the neocor tex (35) . Tab le I expresses th is result in t e rms of the

m e a n interval b e t w e e n recorded neurona l sp ikes . Trans la ted in to spikes /sec-

ond , caudate n e u r o n s fired 0 .67/second, cort ical neu rons 1.36/second, and

tha lamic n e u r o n s (ventral an te r io r and ventra l lateral nuc le i ) 1 .96/second. In a

separate s tudy (34) of n e u r o n s in the g lobus pal l idus and en topeduncu la r

nuc leus , it was found that cells in these s tructures general ly fired at m u c h

h ighe r rates (5 .1 /second) . T h e s lower overall m e a n firing rate of caudate

neu rons is due to a larger n u m b e r of caudate cells w i th very long m e a n inter-

sp ike intervals (greater than 2000 m s e c ) , and a relat ively smal ler n u m b e r of fast

7. Neurophysiological and Anatomical Interrelationships

Table I Mean Interspike Intervals in Normal Catsa

1 9 3

No. of Cats Number of Units Mean Interspike Interval (msec)

Caudate Cortex VA-VL thalamus

234 110 151

1656 738 510

" These values are derived from experiments performed on paralyzed, locally anesthetized cats.

firing caudate cells ( m e a n in te rsp ike intervals less than 400 m s e c ) . In contrast ,

mos t cells in the cor tex and tha lamus have shor t m e a n in te r sp ike intervals

(less than 4 0 0 msec ) w i th relat ively few cells w i th long m e a n in te r sp ike in -

tervals (Figure 2 , left pane l ) .

T h e low firing rate of striatal neu rons m a y resul t from the i r in t r ins ic ana tom-

NORMAL

Control Side Experimental Side

MONOCAUDATE

Experimental Sid* THALAMUS

Jl 200 600 1000 1400 1800 >2000

200 600 1000 1400 1800 > 2 0 0 0 2 0 0 600 1000 MOO 1S00 >2000 200 600 WOO 1400 l«00 >2000

MEAN ISI (msec) FIGURE 2. Distributions of mean interspike intervals (ISIs) for caudate, cortical, and thalamic units in intact cats (left panel) and in cats with one caudate nucleus removed (monocaudate) (right panel). Unilateral caudate ablation produced a slowing of unit firing in the contralateral caudate seen by the increase in the proportion of slowly firing caudate units in monocaudate cats. Bin widths = 200 msec. Frequency x 100 indicates percent of total number of units in each bin. Fig-ure adapted from Levine et al. (35).

Control Side

THALAMUS 60- p

50-

40-

30-

20-

10-

FREQ

UENC

Y x

(100)

O 3

O

O

OO

O

OO

O

I i

i 1

1

1

r~r

-i

1

1

1—

I

CORTEX I

I

CAUDATE I

CAUDATE

f

7 4 4


7000 H 0 # N o r m a l Cats

O O Prelesion Monkeys

# # MFB Cats

EXPERIMENTAL C O N T R O L

SIDE

FIGURE 3 . Overall group mean interspike intervals for intact cats and monkeys with unilateral lesions. Unilateral MFB and SN lesions in cats interrupted the nigrostriatal pathway and depleted dopamine and its biosynthetic enzymes from the ipsilateral caudate nucleus. SPN lesions in cats and unilateral lesions in monkeys did not produce depletion of dopamine in the ipsilateral caudate. (See text for further description.) Figure adapted from Hull et al. ( 2 3 ) .

ical , phys ica l , or chemica l character is t ics and/or from the na ture of synapt ic

inpu ts to t h e m from str iatopetal axons . T h e latter poss ib i l i ty w a s inves -

t igated in an expe r imen t d e s i g n e d to e x a m i n e the c o n s e q u e n c e s of in ter rup-

t ion of the nigrost r ia ta l pa thway on spon taneous firing rates o f caudate nu-

cleus neu rons (23) . Uni la tera l l e s ions in the subs tan t i a n ig ra ( S N ) , jus t dorsal

to the n igra (SPN) or m o r e rostral ly, in the lateral hypo tha l amus a long the

course of the nigrost r ia ta l p a t h w a y ( M F B le s ions ) , all p roduced an a symmet ry

in caudate nuc leus firing rep resen ted ma in ly b y a marked s lowing of s ingle

uni t firing rates in the caudate nuc leus contralateral to the l es ion (Figure 3 ) .

In ter rupt ion of the dopamine rg i c nigrostr ia ta l pa thway did not appear to b e

the p r ime cause of this pos t l es ion a symmet ry . T h e contralateral s lowing oc -

curred w h e t h e r or no t pos t les ion dep le t ions in concen t ra t ions of ipsi la teral

striatal d o p a m i n e or a l terat ions in the act ivi ty of its b iosyn the t i c e n z y m e s ,

ty ros ine hydroxylase and D O P A decarboxylase occurred .

P o s t m o r t e m examina t ion of the b ra ins of an imals in th is s tudy ind ica ted

that the l es ions p robab ly in te r rupted striatal outputs as wel l as striatal inputs .

W e thought that the contralateral s lowing m i g h t have b e e n a c o n s e q u e n c e of

in ter rupt ing striatal ou tputs w h i c h b y mul t i synap t ic pa thways p roduced a

tonic i n h i b i t i o n in the s t r ia tum on the oppos i t e s ide of the b ra in . T o test th i s

hypo thes i s , w e r e m o v e d the caudate nuc leus on one s ide us ing the surgical

ME

AN

IS

I (m

sec)


t e c h n i q u e repor ted b y Vi l l ab lanca (52) . Fo l lowing th is opera t ion , a marked

s lowing of un i t firing in the r e m a i n i n g caudate nuc leus occur red (Figure 2 ,

r ight panel ) (35) . T h i s effect w a s qu i t e s imi la r to that p roduced b y the uni la t -

eral l e s ions in the p rev ious expe r imen t (23) and prov ides pos i t ive ev idence to

sugges t that in te r rupt ion of str iatofugal fibers had i n d e e d b e e n of impor tance .

T h e ques t i on as to h o w ipsi lateral l e s ions p roduce contralateral n e u r o p h y s -

iological effects still r ema ins to b e answered , a l though recent expe r imen t s

have p rov ided p o s s i b l e answers to th is ques t ion . W h e n the firing rates of cor-

tical and tha lamic un i t s we re e x a m i n e d in an imals p rev ious ly sub jec ted to

uni la tera l caudate ab la t ion , a s l ight s lowing was found in the tha lamus ipsi lat-

eral to the l e s ion and in the cor tex on b o t h s ides of the b r a in (35) . T h i s

s lowing w a s s tat is t ical ly ins igni f icant for cort ical neu rons . Fo r tha lamic un i t s ,

h o w e v e r , a l though overall m e a n firing rates d id no t appear to b e s ignif icant ly

al tered b y the l e s ions , a s ignif icant decrease appeared in the occur rence of

uni t s wi th the shor tes t m e a n in te r sp ike intervals (fastest firing rates) (Figure 2 ,

r ight panel ) . T h e fact that d i s t r ibu t iona l differences in tha lamic firing rates oc-

curred pos t l e s ion , wh i l e overall m e a n rates were u n c h a n g e d is p r o b a b l y re-

lated to the neurona l he t e rogene i ty of the tha lamus compared to the s t r ia tum.

T h e poss ib i l i ty that the tha lamus is involved in the al terat ion of neu rona l

firing rates of the contralateral s t r ia tum, w h i c h occurs after r emova l of stria-

tofugal in f luences , s e e m s reasonab le in v i e w of the fact that the target cells o f

the s t r ia tum in g lobus pal l idus and subs tan t i a n ig ra project to b o t h in t ra lam-

inar and ventra l an te r ior and ventrolateral tha lamic nuclear g roups (42) . To

prov ide a m o r e direct test o f tha lamic i nvo lvemen t , w e p repared an imals w i t h

c o m b i n e d l e s ions in the area of the med ia l forebra in bund l e (MFB) and in the

ipsi la teral in t ra laminar reg ion (the parafascicular-central m e d i a n nuc le i

( C M - P F ) of the tha lamus) . Tab le II shows that in an imals w i th c o m b i n e d

l e s ions the s ide- to -s ide a symmet ry in caudate firing rates p roduced b y damag-

ing striatal outputs a lone no longer ex is ted . T a k e n toge ther , t hese results

p rov ide ev idence that the l ow spon taneous firing rates of striatal neu rons are

at least part ly due to the na ture of the synapt ic inpu ts to th is nuc leus from the

tha lamus . T h e data also provide a phys io log ica l clue to the even t s w h i c h

m i g h t under l i e the part ial success of tha lamic les ions in a l levia t ing s y m p t o m s

in cer ta in ex t rapyramida l d i seases .

Table II Mean Interspike Intervals (ISIs) for Caudate Neurons Recorded from Intact Cats, Cats with Unilateral MFB Lesions and Combined MFB and CM-PF Lesions

Combined Intact Cats Unilateral MFB Lesion MFB and CM-PF Lesions

Left Right Ipsilateral Contralateral Ipsilateral Contralateral

Mean ISI (msec) 1768 1544 955 4786 1238 1095


Synaptic Responses of Neurons in the Caudate Nucleus and the Globus Pallidus

CAUDATE NUCLEUS

In a s sess ing the e lec t rophys io logica l character is t ics of caudate nuclear

neu rons , it is impor tan t to m a k e intracel lular record ings because even in

r e sponse to s t imula t ion o f exci ta tory inpu t s , caudate neu rons f requent ly re-

m a i n polar ized b e l o w the i r firing levels . In a ser ies of expe r imen t s ( 5 , 6 , 2 0 - 2 2 ) ,

w e s tud ied the r e sponses of caudate neu rons to exc i ta t ion of k n o w n ana tomi -

cal i npu t s (cor tex, in t ra laminar tha lamus , and subs tan t i a n igra) and to a

n u m b e r of per iphera l inputs . S u c h s t imula t ion a lmost a lways e l ic i ted ini t ia l

exci ta t ion in caudate n e u r o n s , w h i c h w a s fol lowed b y a longer per iod of

i n h i b i t i o n (an E P S P - I P S P s e q u e n c e ) . T h e only major excep t ion to the E P S P -

IPSP s e q u e n c e evoked in caudate cells occurred w h e n the in t ra laminar thal-

amus (the central median-parafasc icu la r reg ion) w a s s t imula ted . T h i s s t imula-

t ion evoked relat ively pure E P S P s in 3 0 - 4 0 % of caudate n e u r o n s impa led (6) .

W e be l i eve that the in i t ia l exc i ta t ion evoked in caudate n e u r o n s b y s t imula-

t ion of s t r iopetal axons is a c o n s e q u e n c e of act ivat ion of exci ta tory inpu ts on

the dendr i tes o f the u b i q u i t o u s shor t -axoned in te rneurons in the caudate

nuc leus . T h e succeed ing I P S P s are genera ted internal ly and are med ia t ed b y

these in t e rneurons w h i c h project on to each other . In m o s t in s t ances , input

axons to the s t r ia tum form contac ts w i th a n u m b e r of caudate in te rneurons .

H o w e v e r , in those cases in w h i c h i n c o m i n g exci ta tory axons t e rmina te on a

smal l n u m b e r of caudate n e u r o n s , a " p u r e " E P S P wou ld b e genera ted .

Tab le III s u m m a r i z e s r e sponses of caudate neu rons to s t imula t ion of cor tex,

Table III Postsynaptic Response of Caudate Neurons

Brain Site Stimulated No. Tested EPSP-IPSP EPSP Only IPSP Only No Response

Substantia nigra 18 14 3 1 Fields of Forel and subthalamus 8 3 1 1 3 Supranigral midbrain 9 7 2 Caudate n. 7 7 Supragenieulate n. 3 1 2 Lat. posterior n. 13 5 4 4 Ventral lat. and ventral ant. nn 23 19 2 1 1 Ant. thalamic radiations 29 25 3 1 Medial dorsal n. 13 8 2 3 Central median and 32 18 10 4

parafascicularis Precruciate cortex 41 30 4 7 Peripheral stimulation 18 14 4

214 151 24 11 28


tha lamus , and subs tan t i a n igra and to a n u m b e r of pe r iphera l s t imul i .

R e s p o n s e s are classif ied accord ing to the pos t synap t ic s e q u e n c e recorded in-

tracellularly.

D u r i n g the course of the inves t iga t ion of the synapt ic r e sponses of striatal

neu rons , w e also s tud ied the result of c o m b i n a t i o n s of s t imul i on the evoked

pos t synapt ic r e sponses . A n impor tan t f inding of these expe r imen t s (21) w a s

the demons t r a t ion of the p repo tency o f cort ical over tha lamic or nigral act iva-

t ion. W h e n s t imul i to two different b ra in s i tes we re pa i red , a p reced ing s t im-

ulus to the cor tex p reven ted or a t tenuated the exc i ta t ion p roduced b y a tha-

lamic or n igra l s t imulus (Figures 4 and 5 ) . Conver se ly , a p reced ing tha lamic or

n igral s t imulus fai led to suppress cort ical ly evoked exc i ta t ion (Figures 4 and 5 ) .

T h e cor t icostr ia ta l funct ional re la t ionships e m p h a s i z e d in these s tud ies m a y

b e of impor t ance w i th regard to the c h a n g i n g roles of cor tex and s t r ia tum

dur ing deve lopmen t sugges ted b y the les ion expe r imen t s m e n t i o n e d earl ier in

th is chapter . T h e results of those expe r imen t s imp ly that the s t r ia tum in the

infant opera tes i ndependen t l y of the less ma tu re cerebra l cortex. Inc reas ing / / c o r t i c a l i z a t i o n , , as the o r g a n i s m develops m a y inc lude synapt ic " c a p t u r e " of

the s t r ia tum b y the cor tex , as ind ica ted b y our e lec t rophys io logica l f indings in

mature an imals . E lec t rophys io log ica l expe r imen t s to inves t iga te the t ime of

onse t of th i s synapt ic " c a p t u r e " are current ly b e i n g carr ied out in our labora-

tory.

FIGURE 4. Suppression of a central median (TH) evoked EPSP in a caudate neuron by a cortically

evoked IPSP. Shocks delivered to the cortex at 10 V, 0.5 msec duration, and 0.3 Hz. Pulse duration

for thalamic shock was the same as for the cortex. Responses to 10 and 15 V intensity shocks to the

TH alone are shown in middle and right, upper traces; responses to cortex alone (upper left trace).

Paired cortex (CX) and 10-V TH response, lower left trace; paired cortex and 15-V TH response,

lower right trace. All traces are averages of ten consecutive responses. Figure adapted from Hull

ET AL (21).

198 N. A. Buchwald, C. D. Hull and M. S. Levine

FIGURE 5 . Asymmetry of cortical and nigral paired stimuli on caudate neuronal PSPs. Top traces

are responses to cortical (CX) and nigral (BS) stimuli alone. In the lower left, the nigral stimulus

precedes the cortical stimulus by 5 0 msec. In the lower right, the cortical stimulus precedes the

nigral by 7 0 msec. All traces are averages of ten consecutive responses. All are recorded from the

same neuron. Figure adapted from Hull et al. ( 2 1 ) .

STRIATAL OUTPUTS

Morpho log ica l s tud ies s h o w that striatal outputs are few in n u m b e r and

rest r ic ted in s i tes of t e rmina t ion to the g lobus pal l idus and subs tan t ia n igra

( 2 8 - 3 0 ) . Less than 5 % of caudate neu rons are l ong-axoned and thus , can-

didates for output cells. In an expe r imen t u s ing intracellular record ing tech-

n i q u e s (11) , w e impa led caudate neu rons and ident if ied those w h i c h re-

sponded ant idromical ly to pall idal or nigral s t imula t ion as p re sumpt ive output

neu rons , u s ing the cr i ter ia o f lack of prepotent ia l s , abi l i ty to fol low h igh

f requenc ies of s t imula t ion , and " c o l l i s i o n " wi th o r thodromic act ion potent ia ls .

A total of 5 . 4 % (8 of 148 cells tes ted) of the neu rons impa led proved to b e an-

t idromic . In contrast w i th caudate in te rneurons , output cells appeared to be

more polar ized wi th respect to the i r firing levels and in r e sponse to ac t ivat ion

of s tr iopetal f ibers , fewer I P S P s occurred after the ini t ia l exci ta t ion .

GLOBUS PALLIDUS AND ENTOPEDUNCULAR NUCLEUS

Exci ta t ion of striatal outputs evokes a mix ture of ini t ia l ly exci ta tory and in i -

tially i nh ib i t o ry pos t synap t ic r e sponses in pall idal and en topeduncu la r

neu rons , in contras t to the 9 3 % of ini t ia l ly exci ta tory r e sponses of caudate

neu rons to ac t iva t ion of s t r ia topetal s i tes (see Tab le IV) . In a recent expe r imen t

(34) in w h i c h intracellular r e sponses were recorded from more than 200 pallidal

and en topeduncu la r neu rons , ini t ia l E P S P s or E P S P - I P S P s e q u e n c e s occurred

7 2 % of the t ime , in i t ia l IPSPs occurred 2 8 % of the t ime . T h u s , the exci ta tory

caudate inputs were conver ted in to a spec t rum of exci ta t ion and i n h i b i t i o n

at the pall idal level . Tab le V summar i ze s the r e sponses o f pall idal and en-

C x - B S INTERACTION


Table IV Comparison of Percentages of Different Postsynaptic Potential Types Evoked by Stimulation of Caudate or Pallidal Afferents in

Caudate and Pallidal Neurons

EPSP-IPSP (%) EPSP Alone (%) IPSP Alone (%)

Caudate 80 13 7 Globus pallidus 64 8 28

topeduncu la r neu rons to s t imula t ion of caudate , cor tex, tha lamus and a

n u m b e r o f per iphera l inpu t s (audi tory and somes the t i c ) . A s s h o w n in Tab le

V , pall idal cells r e sponded (wi th m u c h longer la tenc ies) to a var ie ty of sensory

inputs . S u c h in te rac t ions b e t w e e n striatal and sensory inf luences sugges t a

p r ime in tegra t ive role of the str iatopall idal sys tem. W h e t h e r ac t iva t ion of

caudate ou tput n e u r o n s evokes I P S P s and E P S P s in the pa l l idum v ia separate

striatal efferents or w h e t h e r I P S P s evoked in pall idal neu rons b y caudate s t im-

ulat ion are i nduced b y o ther m e c h a n i s m s (e .g . , i n h i b i t i o n of the impa led

n e u r o n b y a pall idal in te rneuron) canno t b e answered direct ly from these

expe r imen t s . H o w e v e r , the relat ive scarci ty of in t e rneurons and the lack of

collaterals f rom the m a n y large neu rons in the pall idal and en topeduncu la r

nuc leus (10) sugges t that secondary i n h i b i t i o n does no t occur . Therefore , the

synapt ic r e sponses of pall idal neu rons p r o b a b l y reflect the na ture of striatal

i npu t s ra ther than the p re sence of in te rneurona l collateral synapses .

It is of in teres t to no te that pall idal and en topeduncu la r neu rons are poly-

Table V Postsynaptic Responses of Pallidal and Entopeduncular Neurons

Brain Site Stimulated No. Tested EPSP-IPSP EPSP Only IPSP Only No Response

Recordings in globus pallidus

Caudate n. 58 18 2 9 29

Precruciate cortex 10 7 2 1 Central median and 8 6 2

parafascicularis Peripheral 22 5 2 4 11

Recordings in entopeduncular nucleus

Caudate n. 77 37 4 18 18

Precruciate cortex 24 6 1 6 11 Central median and 19 9 1 6 3

parafascicularis Peripheral 77 33 _ 5 _9 30

295 121 15 54 105


sensory , a s ingle cell r e spond ing to a var ie ty of sensory inpu ts (34 ,36 ,49 ) .

A l though the pa thways invo lved in the p roduc t ion of sensory -evoked

r e sponses in pall idal neu rons are u n k n o w n , there is a s t rong poss ib i l i ty that

these pro jec t ions occur v ia the s t r ia tum. B o t h the cons ide ra t ion of the c o n n e c -

t ions of the s t r ia tum to the pa l l idum and the fact that pos t synap t ic potent ia l

shapes and s e q u e n c e s evoked in pall idal neu rons b y ac t iva t ion of caudate and

sensory inpu t s are s imi lar (34) , p rov ide indi rec t ev idence that sensory inputs

to the pa l l idum are med ia t ed b y the s t r ia tum.

FUNCTIONAL INTERRELATIONSHIPS OF THE BASAL GANGLIA

From our knowledge of the ana tomy and neu rophys io logy of the basa l

gangl ia , w e can m a k e guesses as to the i r funct ional in te r re la t ionsh ips . T h e

" h e a d e n d " of the sys t em is the s t r ia tum, w h i c h processes neura l in format ion

sent b y o ther b ra in s t ructures . T h e results of th is p rocess ing is a modula ted

output to the g lobus pal l idus and the subs tan t ia n igra . T h e guessed at func-

t ion of these latter s tructures is to increase the " g a i n " in the sys tem. Smal l

changes in s lowly firing striatal neu rons are t ranslated in to m u c h larger

changes in faster firing pall idal cells. T h e outputs of the basa l gangl ia are

re layed to the cortex from the pa l l idum and n ig ra v ia the tha lamus , and on the

bas i s of recent ev idence , to the l imb ic sys t em via the habenu l a r nuc le i . Feed-

back control o f the s t r ia tum p r o b a b l y occurs b y w a y of s t r ia tonigrostr ia ta l

pa ths and b y indi rec t loops from the pa l l idum and n ig ra v ia the tha lamus

and/or cor tex b a c k to the s t r ia tum.

W e be l i eve that there is n o w e n o u g h knowledge of these in te r re la t ionsh ips

w h i c h , together w i t h in format ion avai lable w i th regard to the process o f matu-

rat ion of the chemica l cons t i tuency of the s t r ia tum, wil l a l low the use of the

basa l gangl ia as a mode l sys t em to s tudy deve lopment . Surgica l or phar-

macologica l insul ts can b e inf l ic ted u p o n the basa l gangl ia dur ing infancy and

the resul ts of these insul ts can b e de t e rmined as the an imal matures .

ACKNOWLEDGMENT

This research was supported by USPHS Grant Nos. MH-07097, HD-05958, and HD-04612.

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23. Hull, C. D. , Levine, M. S., Buchwald, N. A., Heller, A., and Browning, R. A. The spontane-ous firing pattern of forebrain neurons. I. The effects of dopamine and non-dopamine depleting lesions on caudate unit firing patterns. Brain Res., 1974, 73: 241-262.

24. Huttenlocher, P. R. Dendritic and synaptic development and mental defect. Chapter 5, this volume.

25. Jones, B. E . , Guyenet, P., Cheramy, A., Gauchy, C , and Glowinski, J. The in vivo release of


acetylcholine from cat caudate nucleus after pharmacological and surgical manipulation of dopaminergic nigrostriatal neurons. Brain Res., 1973, 64: 355-369.

26. Kataoka, K. , Bak, I. J . , Hassler, R. , Kim, J . S., and Wagner, A. L-Glutamate decarboxylase and choline acetyltransferase activity in substantia nigra and striatum after surgical interruption of the strio-nigral fibres of the baboon. Expl. Brain Res., 1974, 19: 217-227.

27. Kemp, J. M. , and Powell, T. P. S. The cortico-striate projection in the monkey. Brain, 1970, 93: 525-546.

28. Kemp, J . M. , and Powell, T. P. S. The structure of the caudate nucleus of the cat: Light and electron microscopy. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 3 8 3 ^ 0 1 .

29. Kemp, J. M. , and Powell, T. P. S. The synaptic organization of caudate nucleus. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 403-412.

30. Kemp, J . M. , and Powell, T. P. S. Site of termination of fibers in the caudate nucleus. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 413-^27.

31. Kemp, J . M. , and Powell, T. P. S. Termination of fibers from cerebral cortex and thalamus upon dendritic spines in the caudate nucleus: A study with the Golgi method. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 429-439.

32. Kim, J . S., Bak, I. J . , Okada, Y . , and Hassler, R. Role of y-aminobutyric acid in extrapyramidal motor system. II. Some evidence for existence of a type of GABA-rich strionigral neurons. Expl. Brain Res., 1971, 14: 95-104.

33. Kling, A., and Tucker, T. J . Effects of combined lesions of frontal granular cortex and caudate nucleus in neonatal monkey. Brain Res., 1967, 6: 4 2 8 ^ 3 9 .

34. Levine, M. S., Hull, C. D. , and Buchwald, N. A. Pallidal and entopeduncular intracellular responses to striatal, cortical thalamic and sensory inputs. Exp. Neurol, 1974, 44: 4 4 8 ^ 6 0 .

35. Levine, M. S., Hull, C. D. , Buchwald, N. A. , and Villablanca, J . The spontaneous firing pat-terns of forebrain neurons. II. Effects of unilateral caudate nuclear ablation. Brain Res., 1974, 78: 411-424.

36. Lidsky, T. I., Buchwald, N. A., and Hull, C. D. Pallidal and entopeduncular single unit activity in cats during drinking. Soc. Neurosci., 197A, 4: 308.

37. Lynch, G. S., Lucao, P. A., and Deadwyler, S. A. Demonstration of acetylcholinesterase con-taining neurons in caudate nucleus of rat. Brain Res., 1972, 45: 617-621.

38. McCaman, R. E . , and Hunt, J . M. Microdetermination of cholineacetylase in nervous tissue. / . Neurochem., 1965, 12: 253-259.

39. Mehler, W. R. Further notes on the center median nucleus of Luys. In: The Thalamus. (D. Pur-pura and M. Yahr, Eds.). Columbia Univ. Press, New York, 1966: 109-122.

40. Moore, R. Y . , Bhatnagar, R. K., and Heller, A. Anatomical and chemical studies of a nigro-neostriatal projection in the cat. Brain Res., 1971, 30: 119-135.

41. Nauta, H. W. J. Evidence of a pallidohabenular pathway in the cat. / . Comp. Neurol, 1974, 56: 19-28.

42. Nauta, W. J . H., and Mehler, W. R. Fiber connections of the basal ganglia. U. S. Pub. Health Serv., Publ. # 1 9 3 8 , 1969: 6 7 - 7 2 .

43. Obata, K., and Yoshida, M. Caudate evoked inhibition and actions of GABA and other sub-stances on cat pallidal neurons. Brain Res., 1973, 64: 455^459.

44. Okada, Y . , Nitsch-Hassler, C , Kim, J . S., Bak, I. J . , and Hassler, R. Role of y-aminobutyric acid (GABA) in the extrapyramidal motor system. I. Regional distribution of GABA in rabbit, rat, guinea pig and baboon CNS. Expl Brain Res., 1971, 13: 514-518.

45. Precht, W. , and Yoshida, M. Blockage of caudate-evoked inhibition of neurons in the sub-stantia nigra by picrotoxin. Brain Res., 1971, 32: 229-233.

46. Purpura, D. P. Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. Chapter 6, this volume.

47. Purpura, D. P., and Malliani, A. Intracellular studies of the corpus striatum. I. Synaptic poten-tials and discharge characteristics of caudate neurons activated by thalamic stimulation. Brain Res., 1967, 6: 325-340.


48. Rocha-Miranda, C. E. Single unit analysis of cortex-caudate connection. Electroenceph. clin. Neurophysiol, 1965, 19: 237-247.

49. Soltysik, S., Hull, C. D. , and Buchwald, N. A. Caudate and pallidal unit activity during delayed instrumental response performance in monkey. Soc. Neurosci., 197A, 4: 431.

50. Tennyson, V. M. , Mytilineou, C , Heikkila, R., Barrett, R. E. , Cohen, G., Cote, L. , Duffy, P. E. , and Marco, L. Dopamine-containing neurons of the substantia nigra and their terminals in the neostriatum. Chapter 9, this volume.

51. Tobin, A. Mental retardation due to germinal matrix infarction. Science., 1969, 164: 156-158. 52. Villablanca, J . R. , and Marcus, R. Effects of caudate nuclei removal in cats. Comparison with

effects of frontal cortex ablation. Chapter 10, this volume. 53. Webster, K. E. The cortico-striatal projection in the cat. / . Anal, 1965, 99: 329-337.

8 Neuronal Control of Neurochemical Processes

in the Basal Ganglia

ALFRED HELLER and PHILIP C. HOFFMANN Department of Pharmacological and Physiological Sciences,

The University of Chicago,

Chicago, Illinois

T h e n e u r o c h e m i s t r y of the caudate nuc leus and the assoc ia ted basa l gangl ia

is as complex and in t r igu ing a subjec t as is the examina t ion of i ts ana tomica l

o rgan iza t ion and phys io log ica l funct ion. T h i s d iv i s ion of the t e l encepha lon

has a neurona l popula t ion capab le of syn thes i z ing and s tor ing a n u m b e r of

b io logica l ly act ive subs t ances inc lud ing ace ty lcho l ine , y - aminobu ty r i c ac id ,

n o r e p i n e p h r i n e , se ro ton in , and d o p a m i n e . T h e p r e s e n c e of such s u b s t a n c e s ,

all of w h i c h m a y act as chemica l t ransmi t te rs , in a b ra in area w h i c h subse rves

moto r as wel l as behav io ra l func t ions , p rov ides an at tract ive m o d e l for the

s tudy of the role of specific neu rochemica l subs t ances i n phys io log ica l func-

t ions . Ana lyses of th is type , h o w e v e r , r equ i re direct in fo rmat ion as to the

assoc ia t ion of par t icular neu rochemica l subs t ances w i t h specific neu rona l

e l ements . O n e approach to th is p rob l em, w h i c h has p roved par t icular ly useful

in the case of the m o n o a m i n e r g i c sys t ems , is the se lec t ive des t ruc t ion of specific

central pa thways b y l es ions fol lowed b y e x a m i n a t i o n of the n e u r o c h e m i c a l or

h i s tochemica l c h a n g e s p roduced b y such neurona l des t ruc t ion .

T h e l e s ion t e c h n i q u e w a s in i t ia l ly app l ied to the ident i f ica t ion of the m e -

dial forebrain b u n d l e , a tract w h o s e p r e s e n c e is e ssen t ia l for the b i o s y n t h e s i s

and storage of b r a in se ro ton in and n o r e p i n e p h r i n e (for r ev i ew , see

2 4 , 2 5 , 2 7 , 2 8 , 3 0 , 4 7 ) . T h e l e s ion m e t h o d c o m b i n e d wi th the use of the Falck-

Hil larp h i s tochemica l m e t h o d (12 ,13) h a s , in add i t ion , l ed to an ex tens ive

m a p p i n g of central m o n o a m i n e r g i c pa thways (8 ,17 ,63 ) . T h e med ia l forebra in

bund le is a complex tract i n t e r connec t ing the h y p o t h a l a m u s , the basa l te len-

cepha lon , and the m i d b r a i n ( 2 1 , 3 9 , 5 3 ) , and i ts des t ruc t ion leads to a profound

and p e r m a n e n t loss in se ro ton in and n o r e p i n e p h r i n e in all areas of the te len-

205

206 Alfred Heller and Philip C. Hoffmann

cepha lon ( 3 0 , 3 1 , 5 0 , 5 2 ) . T h e p r imary b i o c h e m i c a l deficit w h i c h accounts for

the reduc t ion of b r a in m o n o a m i n e s is a loss of enzymat ic ac t iv i t ies essent ia l for

the i r b i o s y n t h e s i s , inc lud ing tyros ine hydroxylase , 5 -HTP/dopa decar-

boxy lase , and d o p a m i n e /3-hydroxylase ( 2 6 , 3 2 , 3 3 , 4 8 , 5 9 ) . Te lencepha l i c no rep -

i n e p h r i n e or se ro ton in can b e se lect ively reduced b y lateral ve rsus med ia l

p l acemen t of l e s ions in t egmenta l areas pro jec t ing to the med ia l forebra in

bund le (28) (Table I ) .

In the basa l gangl ia , p r imary in teres t has b e e n focused o n the p resence of

d o p a m i n e . In m a m m a l s , the h igh concen t ra t ion o f d o p a m i n e in the basa l

gangl ia is qu i te s t r ik ing as c o m p a r e d to o ther areas of b r a in (6 ,37) and there is

n o w abundan t ev idence that the p resence of th is m o n o a m i n e in the basa l

gangl ia i s a funct ion of the ex i s tence of a direct p ro jec t ion of d o p a m i n e -

produc ing axons to the neos t r i a tum from cell b o d i e s in the subs tan t i a n igra .

Ev idence for such a pa thway is avai lable from a var ie ty o f sources : (a) T h e

d o p a m i n e con ten t of the basa l gangl ia is marked ly reduced in the neos t r i a tum

of pa rk inson ian pa t ien ts in assoc ia t ion wi th cell loss in the subs tan t i a n igra

(10 ,20 ,36 ) ; (b) appl ica t ion of the Falck-Hil larp f luorescence h i s tochemica l

m e t h o d (12 ,13) ha s demons t r a t ed the p resence of d o p a m i n e - c o n t a i n i n g cell

b o d i e s in the pars compac ta of the subs tan t i a n igra and adjacent areas and

d o p a m i n e - c o n t a i n i n g te rmina ls in the neos t r i a tum ( 4 , 7 , 8 , 1 8 ) ; and (c) a var ie ty

of l e s ions invo lv ing des t ruc t ion of the subs tan t i a n igra , the ventra l m i d b r a i n

t e g m e n t u m , or rostrally a long the course of the in ternal capsule all resul t in a

marked reduc t ion in striatal d o p a m i n e ( 1 - 3 , 5 , 1 4 , 2 2 , 4 9 , 5 1 , 5 6 , 5 7 ) .

Desp i t e a good deal of sugges t ive ev idence for a direct n igros t r ia ta l sys t em,

it had no t b e e n pos s ib l e unt i l recent ly to demons t ra t e such a p ro jec t ion b y

conven t iona l ana tomica l t e c h n i q u e s . B y appl ica t ion of the F ink and H e i m e r

modif ica t ion of the Nau ta m e t h o d for s i lver impregna t ion of degenera t ing

axons and te rmina ls (16) it has recent ly b e e n poss ib l e to s tudy the course and

d i s t r ibu t ion of axon degene ra t ion fo l lowing les ions of the subs tan t ia n igra

(46 ,49) . Fo l lowing les ions of the subs tan t i a n igra and ventra l t e g m e n t u m in

Table I Effects of Central Nervous System Lesions Involving Components of the Medial Forebrain Bundle on Telencephalic Serotonin and Norepinephrine in the Rat a

Experimental Lesion % Serotonin1' % Norepinephrine0

Lateral hypothalamus - 8 2 - 8 2

Medial tegmentum - 7 8 ns" Ventrolateral tegmentum ns" - 7 3

a Reprinted with permission from Heller (25). b Control telencephalic serotonin = 0.68 /otg/gm wet weight of tissue. c Control telencephalic norepinephrine = 0.26 Atg/gm wet weight of tissue. ( l ns refers to differences between control and lesion levels not statistically significant, P > 0.05.

8. Neuronal Control of Neurochemical Processes 207

the cat it can b e s h o w n that axons of the nigros t r ia ta l p ro jec t ion pass th rough

the ventral t e g m e n t u m and ascend v ia the in ternal capsule and lateral hypo -

tha lamus to innerva te the p u t a m e n and caudate nuc leus . T h e d i s t r ibu t ion of

degenera t ing te rmina ls is h o m o g e n e o u s in the p u t a m e n w h e r e a s in the cau-

date m o r e t e rmina l s are ev iden t laterally than media l ly . O f par t icular in teres t

is the d i s t r ibu t ion of degenera t ing nigros t r ia ta l axons w i t h i n the hypotha l -

amus . At the rostral tubera l level o f the hypo tha l amus , the fibers run ent i re ly

in the med ia l in ternal capsu le and the adjacent lateral hyp tha lamus . T h u s ,

l e s ions t ransec t ing the lateral hypo tha l amic area and ex tend ing in to the med ia l

part of the in terna l capsule p roduce profound effects on caudate nuc leus

d o p a m i n e , ty ros ine hydroxy lase , and dopa decarboxylase res t r ic ted to the s ide

of the b r a i n con t a in ing the l es ion (49) . T h e m a g n i t u d e of the effect is depen-

dent upon the s ize and exact locat ion of the c o m b i n e d les ion and s o m e wel l -

p laced l es ions cause a near ly total loss of a m i n e s and e n z y m e s . L e s i o n s in-

volv ing only the med ia l in ternal capsule , bu t spar ing the lateral hypo tha l amic

area resul t in decreases in caudate nuc leus d o p a m i n e , ty ros ine hydroxylase ,

and dopa decarboxylase in the range of 4 0 - 5 0 % . L e s i o n s invo lv ing b o t h the

media l in ternal capsu le and the lateral hypo tha l amic area have a m o r e m a r k e d

effect on d o p a m i n e and the e n z y m e s p roduc ing a 7 0 - 9 0 % loss of each .

T h e rate of loss of caudate d o p a m i n e fol lowing les ions of the nigros t r ia ta l

pa thway has b e e n s tud ied in some detai l in the rat (25) . D o p a m i n e is lost

m o r e rapidly fo l lowing t ransec t ion of th i s central m o n o s y n a p t i c ca t echo lamine

pa thway than are se ro ton in and n o r e p i n e p h r i n e fol lowing cer ta in central

les ions (24 ,25) . T h e loss of se ro ton in and n o r e p i n e p h r i n e fo l lowing such

les ions m a y invo lve t ranssynap t ic m e c h a n i s m s (for r ev iew, see 2 5 , 2 9 , 3 0 , 4 7 ) .

Fo l lowing t ransec t ion of the nigrost r ia ta l p ro jec t ion e i the r w i t h i n the d ien-

cepha lon or at the m e s e n c e p h a l i c level b y des t ruc t ion o f the subs tan t i a n igra ,

there is a rapid fall in d o p a m i n e w i t h a 5 0 % reduc t ion b y 2 days and a 7 0 - 8 0 %

reduc t ion in 4 to 6 days (25) . T h i s t ime course is qu i t e s imi la r to that o b s e r v e d

in per iphera l t i s sues fo l lowing direct sympa the t i c denerva t ion and therefore

cons i s ten t w i th a direct central denerva t ion p h e n o m e n o n .

At 1 day fo l lowing les ion p lacemen t , d o p a m i n e is e levated to 3 0 % above

normal (25) . T h i s obse rva t ion is of in teres t in v i e w of the recent f inding that

after l e s ions of the n igros t r ia ta l p a t h w a y at the level of the rostral e n d of the

sup ramammi l l a ry decussa t ion , there is a ve ry rap id inc rease in d o p a m i n e . B y

18 m i n u t e s after the l es ion , d o p a m i n e levels are inc reased b y 6 4 % and this ef-

fect is sus ta ined for at least 20 hours . It has b e e n sugges ted that the inc rease in

d o p a m i n e caused b y axo tomy results from an ini t ia l i n h i b i t i o n of impu l se

flow in the n igroneos t r ia ta l p a t h w a y and a reduc t ion in d o p a m i n e re lease

(64) .

T h e on togen ic deve lopmen t of d o p a m i n e and its b io syn the t i c e n z y m e s in

the rat p rov ides addi t iona l ev idence for a direct n igros t r ia ta l p ro jec t ion . T h e

rat is a useful an ima l for such s tudies s ince the increase of m o n o a m i n e and en-

208 Alfred Heller and Philip C. Hoffmann

z y m e act ivi ty takes place a lmos t ent i re ly postnata l ly and in addi t ion , the b ra in

is of sufficient s ize , e v e n at a y o u n g age , to pe rmi t re l iable d i s sec t ion and

analys is (58) . T h e caudate shows a m o r e or less s teady increase in d o p a m i n e

from abou t 1 5 % of adult va lues at 4 days of age to abou t 5 0 - 6 5 % of the adult

levels at 45 days . T h e inc rease in the b io syn the t i c e n z y m e s , ty ros ine hydroxyl -

ase and dopa decarboxylase , is qu i t e s imi lar to that of dopamine . T h u s , there

is a concurrent deve lopmen t o f a m i n e s and b iosyn the t i c e n z y m e s in this area.

T h i s differs from the sequen t i a l deve lopmen t of the e n z y m e s and norep i -

neph r ine in the m e s e n c e p h a l o n , p o n s - m e d u l l a , and d i encepha lon (58) . T h e

concurrent appearance of e n z y m e s and a m i n e s in the caudate m a y represent

the arrival at ne rve te rminals of e n z y m e s syn thes i zed in the cell b o d i e s and

t ranspor ted b y axoplasmic flow to the caudate in a unicel lular sys t em (15) .

Recen t ly it has b e e n s h o w n that the p l acemen t o f l es ions in the d ien-

cepha lon of the neona ta l rat p revents the postnata l increase of te lencephal ic

ca t echo lamines wi th deve lopmen t (11) . A n i m a l s w i th uni la teral l e s ions placed

at 4 days of age have marked ly lower levels o f n o r e p i n e p h r i n e (—74%) and

d o p a m i n e (—84%) in the ipsi la teral t e lencepha lon at 6 - 1 2 w e e k s , a t ime at

w h i c h ca t echo lamines have r eached adult va lues on the control s ide . N o s ig-

nif icant s ide- to -s ide differences in e i the r n o r e p i n e p h r i n e or d o p a m i n e were

seen in an imals w i t h l e s ions res t r ic ted to the med ia l hypo tha l amus (Table II) .

T h e correlat ion b e t w e e n ana tomica l p l acemen t of the les ion and the m o n o -

a m i n e reduc t ion is ident ica l to that seen in the adult. T h e fibers involved are

p r e sumab ly the s ame as those impl ica ted in m a i n t e n a n c e of adult levels of cat-

e c h o l a m i n e s , i . e . , the med ia l forebra in b u n d l e for n o r e p i n e p h r i n e and the

nigrostr ia ta l p ro jec t ion for d o p a m i n e . Non te rmina l d o p a m i n e - c o n t a i n i n g

axons have b e e n v i sua l ized in n e w b o r n rats [ (42 ,43 ,45 ,55 ) , T e n n y s o n et

ah, Chap te r 9 , th i s v o l u m e ] and it i s l ikely that l e s ions in the neona ta l rat are at

least part ial ly t ransec t ing i m m a t u r e m o n o a m i n e r g i c f ibers . T h e l e s ions m a y

also b e b lock ing the ing rowth of neu rons in to the te lencepha lon . The place-

men t of l es ions in the n e o n a t e w h i c h p reven t ca t echo lamine deve lopmen t in

the caudate nuc leus m a k e s pos s ib l e the s tudy of me tabo l i c and behav io ra l

Table II Effect of Neonatal Unilateral Lesions on Telencephalic Catecholamine Development in the Rat a

Norepinephrine1' Dopamine'' Lesion Group % Change % Change

Lateral hypothalamus and medial internal capsule - 7 4 - 8 4 Medial hypothalamus - 9 + 1

" The lesions were placed at 4 days of age and the animals killed 6 -12 weeks later. Norepinephrine control side value = 0.23 /xg/gm. Dopamine control side value = 0.99 /ug/gm.


p h e n o m e n a in an ima l s w h i c h have matured in the a b s e n c e of n o r m a l levels of

d o p a m i n e in th i s s t ructure.

T h e r e is little doub t that the nigrost r ia ta l p ro jec t ion is a direct dopamine rg i c

input to the neos t r i a tum and that its des t ruc t ion results in the loss o f dop-

a m i n e - c o n t a i n i n g ne rve end ings in th is s t ructure. T h e funct ional con-

s e q u e n c e s of the loss of dopamine rg i c inne rva t ion for the pos t synap t ic cellular

e l emen t s of the caudate have no t yet b e e n e luc ida ted . Neurophys io log ica l

s tudies of the effects of l e s ions r e m o v i n g the dopamine rg i c inpu t in the cat

and m o n k e y have cast doub t on the concep t that there is a s imple re la t ion

b e t w e e n d o p a m i n e and the regula t ion of caudate neurona l ac t iv i ty [(38) ,

B u c h w a l d et ah, Chap te r 7, th is v o l u m e ] . W e have b e e n s tudy ing the pos s ib l e

role of d o p a m i n e in the caudate nuc leus as a regulator of me tabo l i c even t s ,

part icularly w i t h respec t to ca rbohydra te m e t a b o l i s m . In m a n y per iphera l

t i s sues , b o t h e p i n e p h r i n e and n o r e p i n e p h r i n e have b e e n s h o w n to inf luence

bas ic reac t ion s e q u e n c e s invo lved in intracellular m e t a b o l i s m such as gly-

cogenolys i s . T h e classic work of Cor i and Su ther land has e s t ab l i shed the en-

zymat ic ba s i s for e p i n e p h r i n e ' s accelera t ing effect on g lycogenolys i s in l iver

and skeletal musc le . M o r e recent ly , these n e u r o h u m o r s have also b e e n s h o w n

to affect g lycogen syn thes i s (35) . Su the r l and and h i s co-workers have devel-

oped the h y p o t h e s i s o f the " s e c o n d m e s s e n g e r " in w h i c h m a n y d iverse hu-

mora l agents p roduce the i r effects b y ac t iva t ing the e n z y m e , adenyla te cyclase

(60) . T h i s , in turn, resul ts in an inc rease in the intracel lular 3 ' , 5 ' - a d e n o s i n e

m o n o p h o s p h a t e (cyclic A M P ) level w h i c h acts as a regulator for part icular

metabo l ic p rocesses such as g lycogenolys i s and g lycogen syn thes i s .

T h e g lycogenoly t ic r e sponse to ca t echo lamines is of par t icular in teres t s ince

s t imula t ion of pa rasympa the t i c and sympa the t i c ne rves to the l iver can mark-

edly alter the rates o f g lycogenolys i s and g lycogen syn thes i s t h rough a d e m -

ons t ra ted effect on the act iv i t ies o f the e n z y m e s control l ing these p rocesses ,

name ly , g lycogen syn the tase and phosphory la se (61 ,62) . T h i s r e sponse to sym-

pathe t ic s t imula t ion is p r e s u m a b l y med ia t ed b y n o r e p i n e p h r i n e re leased at

the neuro-ef fec tor j u n c t i o n w h i c h in turn act ivates adenyla te cyclase pro-

duc ing inc reased phosphory la se and g lucose -6 -phospha tase act ivi ty. In accor-

dance w i t h the v i e w that per iphera l au tonomic nerves can regulate me tabo l i c

even t s in i nne rva t ed t i s sues are the f indings that s y m p a t h e c t o m y or total

denerva t ion of the hear t resul ts in accumula t ion of g lycogen in th i s t i ssue

(40 ,65) . P r e s u m a b l y , the loss of n o r e p i n e p h r i n e from the hear t fo l lowing

degenera t ion of adrenerg ic neu rons s lows the rate of g lycogen b r e a k d o w n and

s t imula tes g lycogen syn thes i s in the m y o c a r d i u m unt i l a n e w and h i g h e r

s teady-s ta te g lycogen level is ach ieved . T h e loss of n o r e p i n e p h r i n e wou ld

decrease adenyla te cyclase act ivi ty resul t ing in lowered intracel lular cyclic

A M P , w h i c h in turn w o u l d appropr ia te ly c h a n g e the ac t iv i t ies o f phosphory l -

ase and g lycogen syn the tase . T h i s m o d e l does no t , h o w e v e r , take in to account

addi t ional me tabo l i c modula tors w h i c h regulate g lycogen m e t a b o l i s m in-

2 1 0 Alfred Heller and Philip C. Hoffmann

eluding 5 ' - A M P , g lycogen , and g lucose . In fact, D a w and Be rne were unab le

to account for the increase in cardiac g lycogen con ten t fo l lowing sympa thec -

tomy on the bas i s of m e a s u r e d changes in the in vitro ac t iv i t ies of phosphory l -

ase and g lycogen syn the tase (9) . Desp i t e the uncer ta in t ies w i th respect to the

m e c h a n i s m s invo lved , it is clear that sympathe t i c denerva t ion of a per iphera l

t i s sue resul ts in long- s t and ing me tabo l i c c h a n g e s in the effector cells .

Des t ruc t ion of the nigrost r ia ta l pa thway b y uni la teral l e s ions t ransec t ing the

med ia l forebra in b u n d l e and the med ia l in ternal capsule resul ts in a loss o f

d o p a m i n e - c o n t a i n i n g ne rve e n d i n g s and thus represents a part ial denerva t ion

of the caudate . S ince in the pe r iphe ry the hear t r e sponds to denerva t ion b y ac-

cumula t ing g lycogen , w e inves t iga ted whe the r , w h e n chronica l ly depr ived of

dopamine rg ic neurona l e n d i n g s , the pos tsynapt ic caudate cellular e l emen t s

would r e spond to th is loss b y accumula t ing g lycogen . Fo l lowing such central

denerva t ion , a long-s tand ing increase in the ipsi lateral level of cauda te gly-

cogen w a s found to occur (Table III) . T h e signif icant increase in g lycogen con-

tent of 0 .82 jLtmoles/gm is res t r ic ted to the ipsi lateral caudate w h e r e it r epresen ts

a 3 0 % inc rease relat ive to the control s ide caudate w h i c h had g lycogen levels

comparab le to those in n o n l e s i o n e d an imals (34) . G lycogen did not s ignif i-

cant ly accumula te in o the r ipsi la teral areas o f b ra in desp i te the w idesp read

and subs tan t ia l loss of no rmal m o n o a m i n e con ten t in these areas.

T h e m e a s u r e m e n t of the in vivo s teady-s ta te g lycogen levels in b ra in is a dif-

ficult p rob lem. G lycogen levels decrease rapidly fol lowing the onse t of anoxia

w h i c h results from decapi ta t ion (44) . Es t ima tes of reg ional " ze ro t i m e " s teady-

Table III Effect of Unilateral Diencephalic Lesions on Regional Glycogen Levels

in Rat Brain

Glycogen Content (fxmoles/gm)

Side Difference (lesion

Brain Area N Lesion Control minus control) Significance''

Caudate n 4.41 0.48 3.58 0.43 + 0.82 0.29 P < 0.02

Cortex l i 5.49 0.94 5.74 ± 1.02 - 0 . 3 8 0.43 NS Septum n 4.55 0.42 5.02 0.58 - 0 . 4 7 0.53 NS Amygdala n 5.27 0.58 4.61 0.45 + 0.66 0.54 NS Hippocampus n 4.64 0.67 4.66 ± 0.80 - 0 . 0 3 0.47 NS Cerebellum n 5.37 0.70 4.48 0.57 + 0 . 8 9 0.53 NS Brainstem 10 3.91 ± 0.49 3.59 •±2 0.53 + 0 . 1 0 0.22 NS

" Reprinted with permission from Hoffmann et al. (34). b Values represent means ± S.E. for the number (N) of animals indicated. Data were obtained approximately 6 months postoperatively on rats anesthetized with chloral hydrate and perfused with paraformaldehyde. c Paired sample comparison with two-tailed Student's Mest. NS = P > 0.05.


state levels of g lycogen can b e dis tor ted b y the rapid enzymat i c degrada t ion of

g lycogen resul t ing from anoxia . T h e va lues for g lycogen in T a b l e III w e r e o b -

ta ined from the b ra ins of an imals perfused w i t h paraformaldehyde to p reven t

g lycogen b r e a k d o w n secondary to decap i ta t ion (23) . T h e resul ts o b t a i n e d w i t h

th is m e t h o d are in good ag reemen t w i th the l i terature va lues for rapidly frozen

m o u s e b ra in (54) . There fo re , it s e e m s l ikely that the e levated g lycogen va lues

on the l e s ion s ide w h i c h is deple ted of d o p a m i n e represent a r ea sonab le ap-

p rox ima t ion to the in vivo s teady-s ta te caudate levels .

To de t e rmine if g lycogen m e t a b o l i s m in the caudate o f the l es ion and con-

trol s ides b e h a v e d s imi lar ly unde r cond i t ions in w h i c h the g lycogen levels

were c h a n g i n g over short per iods of t ime , g lycogen levels were man ipu la t ed

in uni la teral ly l e s ioned rats b y a l lowing g lycogen to decrease as the result of

1 5 - 3 0 seconds of anox ia after decap i ta t ion , and converse ly , to inc rease as the

result o f barb i tura te anes thes i a (44 ,54) . Unfor tuna te ly , pa ra formaldehyde per-

fusion does not a l low for the de te rmina t ion of c o m p o u n d s such as g lucose and

lactate, the precursor and end-product , respec t ive ly , of g lycogen m e t a b o l i s m ,

s ince they are l eached out dur ing perfus ion and the para formaldehyde also in-

terferes w i th m o n o a m i n e de te rmina t ions . Therefore , rapid freezing in sol id-

l iqu id n i t rogen s lush w a s used desp i te the fact that f reezing renders the b ra in

unsu i t ab le for accurate regional d i ssec t ion . A crude " c a u d a t e " sample from

frozen b r a in s can b e o b t a i n e d b y use of a b a n d saw gu ided b y ex ter ior land-

marks . C o m p a r i s o n of the d o p a m i n e concen t ra t ions in these samples w i t h

those found in carefully d i ssec ted samples of caudate sugges ted that the frozen

samples con ta ined approximate ly 5 0 % caudate b y we igh t . Desp i t e the cons id -

erable d i lu t ion b y noncauda te e l emen t s and the decreased g lycogen levels on

bo th the l es ion and control s ide due to the 1 5 - 3 0 - s e c o n d anoxic pe r iod in ter-

v e n i n g b e t w e e n decap i ta t ion and f reezing, g lycogen levels were found to b e

e levated in the " c a u d a t e " samples from the l es ion s ide b y 0.5 ^ m o l e s / g m or

3 1 % (Table IV) . T h e level of g lucose w a s inc reased b y 7 2 % on the l e s ion s ide .

H o w e v e r , the abso lu te level of g lucose is on ly abou t 1 0 % of that of g lycogen

so that the pe rcen tage increase represents only 0 .05 umoles /gm of g lucose . Lac-

tate levels were vir tual ly ident ica l in the " c a u d a t e " from the l e s ioned and

n o n l e s i o n e d s ide .

Cauda te g lycogen levels were acutely e levated b y barb i tu ra te admin i s t r a -

t ion. L e s i o n e d an imals we re anes the t i zed w i t h p h e n o b a r b i t a l for a pe r iod of 2

hours pr ior to f reezing 1 0 - 3 0 seconds after decapi ta t ion . Cauda te samples

from the les ion s ide of these anes the t i zed an imals s h o w e d e leva t ions in b o t h

the g lycogen and glucose levels of 0 .41 ^ m o l e s / g m and 0 .31 umoles /gm, respec-

t ively, compared to the control s ide despi te the fact that g lycogen levels on

bo th s ides were h i g h e r than those in unanes the t i zed rats (Table IV) . Glucose

levels on the control s ide inc reased b y a lmost 4-fold as the result of anes thes ia ,

but the les ion s ide w a s e levated a lmost 5-fold. A s in the unanes the t i z ed

an imal , the lactate levels did not vary s ignif icant ly from s ide to s ide as the


Table IV The Effect of Unilateral Diencephalic Lesions on "Caudate" Levels of Glycogen, Glucose, Lactate, and Monoamines after 15-30 Seconds of Anoxia"

Side

Lesion ( L ) Control ( C ) L — C % Change

Unanesthetized Rats

DA Oig/gm) 0.05 ± 0.04 2.71 0.25 - 2 . 6 5 ± 0.25 - 9 7

Glycogen (yumoles/gm) 2.13 ± 0.13 1.62 0.14 + 0.51 ± 0.17 + 3 1

Glucose (/^moles/gm) 0.15 ± 0.03 0.12 0.03 + 0 . 0 5 ± 0.02 + 72 Lactate (/nmoles/gm) 5.40 ± 0 . 1 6 5.30 0.17 + 0.12 ± 0.22 -Total lactate equivalents (/xmoles/gm) 9.95 ± 0.28 8.72 0.34 + 1.09 ± 0.43 + 14

Anesthetized Rats

DA (jag/gm) 0.21 ± 0.13 2.32 0.25 - 2 . 1 1 ± 0.31 - 8 8

Glycogen (/xmoles/gm) 3.12 ± 0 . 1 4 2.71 0.15 + 0 . 4 1 ± 0.16 + 15

Glucose (/xmoles/gm) 0.72 ± 0.16 0.43 0.09 + 0 . 3 1 ± 0.11 + 72 Lactate (/xmoles/gm) 6.11 ± 0.54 6.71 0.57 - 0 . 5 9 ± 0.55 -Total lactate equivalents (/umoles/gm) 13.78 ± 0.61 13.12 0.54 + 0 . 6 8 ± 0.74 -

" Values represent means ± S.E. Data were obtained approximately 90 days postoperatively on unanesthetized rats or rats anesthetized for 2 hours with phenobarbital. After decapitation, the heads were kept at room temperature for 15-30 seconds and then frozen in a slush of solid-liquid nitrogen. A coronal section containing most of the caudate nucleus was cut and analyzed.

result of the les ion . T h e s e results sugges t that wha teve r effect the l es ion is

p roduc ing in the caudate , it is of such a na ture that shor t - te rm changes in

g lycogen m e t a b o l i s m are not affected b y it.

T h e f inding that caudate lactate levels are unaffected b y the l es ion in e i ther

unanes the t i zed or anes the t i zed an imals p roved to b e useful. S i n c e only gly-

cogen and glucose w h i c h are precursors of lactate appear to b e e levated in

assoc ia t ion wi th the l es ion , w h i l e the product , lactate, is not affected, it is pos -

s ib le to avoid p rob l ems in the m e a s u r e m e n t of these labi le c o m p o u n d s b y

a l lowing the t i ssue to glycolyze all of its g lycogen and g lucose to lactate (44)

and de te rmin ing the latter as a measu re of total ca rbohydra te reserves . T h i s

exper imenta l des ign has the added advantage that it al lows for the accurate

d i ssec t ion of the t i ssue . W h e n the total ca rbohydra te reserves of the caudate in

uni lateral ly l e s ioned rats were e x a m i n e d in th is m a n n e r , they were found to

b e 3 ^Ltmoles/gm (or 2 9 % h igher ) in the caudate from the les ion s ide w h i c h suf-

fered a 7 8 % loss in d o p a m i n e compared to the control s ide (Table V ) . M o r e -

over , the sample cons t i tu t ing the r e m a i n i n g t e lencepha lon s h o w e d no s ide- to-

s ide differences in total ca rbohydra te reserves desp i te a 7 1 % decrease in the

pr inc ipa l ca t echo lamine in these areas , no rep ineph r ine . T h u s , th is exper i -

m e n t also sugges ted that ca rbohydra te reserves , mos t l ikely in the form of

g lycogen , were chronical ly and unilateral ly e levated in the caudate fol lowing

8. Neuronal Control of Neurochemical Processes 2 1 3

p lacemen t of uni la tera l d iencepha l i c l es ions w h i c h result in a loss of d o p a m i n e

from th is s tructure b y direct denerva t ion . S i n c e o ther t e lencepha l ic areas s h o w

no demons t r ab l e changes in g lycogen levels assoc ia ted w i th loss of ca techol -

and indo lea lky lamines , it s e ems reasonab le to focus a t ten t ion on the dopa-

m i n e loss in the caudate desp i te the fact that these l es ions also reduce caudate

n o r e p i n e p h r i n e (31) and se ro ton in (50 ,52) .

T h e s ingular r e sponse of the caudate to m o n o a m i n e dep le t ion could b e a

reflect ion of a u n i q u e me tabo l i c ac t ion of d o p a m i n e in the caudate . T h e d e m -

ons t ra t ion that h o m o g e n a t e s of the rat caudate con ta in an adenyla te cyclase

w h i c h is ac t ivated 2-fold specifically b y d o p a m i n e at low concen t ra t ions sup-

ports the concep t that d o p a m i n e does play a u n i q u e regulatory role in the

caudate . In contrast , h o m o g e n a t e s from ce rebe l lum have a h igh basa l adenyl -

ate cyclase act ivi ty w h i c h is no t ac t ivated b y d o p a m i n e , bu t b y no rep i -

n e p h r i n e (41) . T h e u n i q u e r e spons ivenes s of caudate adenyla te cyclase to dopa-

m i n e has recent ly b e e n conf i rmed b y the demons t r a t ion of inc reased cyclic

A M P levels in the caudate , bu t no t in the ce rebe l lum, fo l lowing sys temic ad-

min i s t ra t ion of the precursor of d o p a m i n e , L -dopa, to rats (19) . T h u s , at least a

fraction of caudate adenyla te cyclase act iv i ty appears to b e m o r e sens i t ive to

d o p a m i n e . If cyclic A M P serves to regulate g lycogen m e t a b o l i s m , it is poss ib l e

that the dopamine rg i c nigrostr ia ta l inpu t to the caudate has a regulatory me ta -

bol ic effect on the cells w h i c h it innerva tes . T h e me tabo l i c a l tera t ions seen fol-

lowing denerva t ion of the caudate provide a useful m o d e l for examina t i on of

the role of the dopamine rg i c nigrost r ia ta l p a t h w a y in regula t ion of me tabo l i c

even ts and thei r re la t ion to cell act ivi ty.

Se lec t ive des t ruc t ion of specific neurona l sys t ems b y the p l acemen t of d is -

crete l e s ions in the central ne rvous sys tem fol lowed b y n e u r o c h e m i c a l ana lyses

has p rov ided a powerful tool for the ident i f ica t ion of neu rona l sys t ems e s sen -

tial for the regula t ion and m a i n t e n a n c e of neuro t ransmi t t e r s in b ra in . T h e

abi l i ty to ident i fy such pa thways and to p roduce select ive a l terat ions in neuro-

Table V The Effect of Unilateral Diencephalic Lesions on Caudate and Telencephalic Total Carbohydrate Content and Monoamine Levels"

Lactate

% of Control Side

DA NE

Caudate Lesion side + 2 9 - 7 8 -Remaining

Telencephalon + 2 - - 7 1 Lesion side

" Samples were incubated for 10 minutes following killing of the animals to allow glycogen and glucose to be metabolized anaerobically to lactate.


t ransmi t te r con ten t in specific ana tomica l r eg ions has already p rov ided infor-

ma t ion on the role of chemica l t ransmit ters in the central ne rvous sys tem.

H o w e v e r , the m e c h a n i s m s under ly ing al terat ions in t ransmi t te r con ten t m a y

b e m o r e complex than those occur r ing fo l lowing removal o f per iphera l

neu rons . The re is reason to be l i eve , on the bas i s of ana tomica l and n e u r o c h e m -

ical compa r i sons , that at least in s o m e areas o f t e lencepha lon the loss o f

m o n o a m i n e s fol lowing les ions of the media l forebra in b u n d l e is not ent i re ly

a t t r ibutable to direct denerva t ion , bu t ra ther is secondary to a d is rupt ion of

normal neurona l control o f m o n o a m i n e b i o s y n t h e s i s m e d i a t e d across poly-

neurona l sys tems ( 2 5 , 2 9 , 3 0 , 4 7 ) . H o w e v e r , in the case of the dopaminerg ic

nigrost r ia ta l p ro jec t ion , it i s c lear that des t ruc t ion of th i s sys t em results in a

direct denerva t ion of the caudate . It is pos s ib l e , therefore , to depr ive these

central neu rons chronica l ly of a part o f the i r normal neurona l inpu t and thus

s tudy the r e sponse of the pos t synap t ic e l emen t s to such depr iva t ion . T h i s

mode l of direct central denerva t ion has p rov ided ev idence that d o p a m i n e m a y

serve to regulate ca rbohydra te m e t a b o l i s m in the neos t r i a tum.

It should b e e m p h a s i z e d that the effects of central l e s ions can lead to w ide -

spread and ex tens ive loss of b io logica l ly act ive subs tances in areas far d is tant

from the les ion . T h u s , cons ide ra t ion m u s t b e g iven to the poss ib i l i ty that

phys io log ica l and behav io ra l c h a n g e s assoc ia ted wi th neurologica l and psych i -

atric d isorders m a y ar ise from rest r ic ted pa tho logy in groups of neu rons qui te

d is tant from reg ions of b ra in s h o w i n g the p r imary funct ional d i s tu rbances .

ACKNOWLEDGMENT

This work was supported in part by research Grant No. MH-04954 from the National Institute of Mental Health.

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8A Discussion: L-Dopa-Induced Improvement of

Conditioned Response Inhibition

ANNE KITSIKIS Department of Physiology, School of Medicine,

Laval University, University City, Quebec, Canada

Dr. Hel ler ' s w o r k m o s t cer ta in ly opens the w a y to a w i d e area of research

conce rn ing the funct ional s igni f icance of b ra in d o p a m i n e . For t hose such as

myse l f w h o s e a t ten t ion is d i rec ted toward the re la t ion of striatal d o p a m i n e to

behav io r , the poss ib i l i t y of p reven t ing ca t echo lamine deve lopmen t in an ima l s

undoub ted ly prov ides a useful tool; especia l ly as s o m e of our p rev ious exper i -

m e n t s have s h o w n that the p resence of striatal d o p a m i n e is no t equa l ly impor -

tant for the per formance of all behav io ra l tasks ; cats wi th a nonexpe r imen ta l l y

i nduced d o p a m i n e def ic iency were found unab le to reach cr i ter ion level per-

formance on a spat ia l de layed r e sponse task, a l though they per fo rmed a s imul -

t aneous v i sua l d i sc r imina t ion perfect ly (9) . T h e resul ts repor ted b y Dr . Hel ler

conce rn ing the me tabo l i c effects p roduced b y l e s ion of the med ia l forebra in

b u n d l e no t on ly reveal ano ther fruitful exper imen ta l approach for those in ter-

es ted in the funct ional s igni f icance o f b ra in d o p a m i n e bu t also cons t i tu te a

w a r n i n g aga ins t any too has t i ly conc luded corre la t ions b e t w e e n n e u r o c h e m -

ical and behav io ra l data.

Before d i scuss ing Dr . B u c h w a l d ' s paper , I w o u l d l ike to p resen t s o m e of our

o w n resul ts w h o s e in te rpre ta t ion call u p o n e lec t rophys io log ica l data. T h e find-

ings I am go ing to repor t are part of a project a i m e d to evaluate the funct ional

s ignif icance of d o p a m i n e for b e h a v i o r w h i c h has b e e n s h o w n to involve the

caudate nuc leus . T h e ques t i on w e asked ourse lves w a s w h e t h e r the impa i r ed

per formance of cer ta in behav io ra l tasks , after bi la teral caudate l e s ions , could

b e accoun ted for, at least par t ia l ly , b y a d o p a m i n e def ic iency s ince mos t b ra in

d o p a m i n e is concen t ra ted in the s t r ia tum.

In the first s tage of our expe r imen t s w e were able to s h o w that in bo th

2 1 9

220 Anne Kitsikis

normal and d o p a m i n e deficient ca ts , inc reas ing striatal d o p a m i n e th rough

L -dopa admin i s t r a t ion , p roduces a differential effect on the per formance of

two behav io ra l tasks; spat ial de layed r e sponse b e i n g s ignif icant ly improved

b y L -dopa wh i l e s imul t aneous v isua l d i sc r imina t ion per formance r e m a i n s es -

sent ia l ly unaffected (8) . S i m u l t a n e o u s b i o c h e m i c a l s tud ies per formed b y Dr.

Andree R o b e r g e revealed that th is L -dopa- induced faci l i tat ion of delayed

response per formance co inc ided wi th a s ignif icant inc rease of dopamine

in the caudate nuc leus (9) . T h e impor tance of th is a m i n e was further de-

mons t ra ted w h e n lower ing of b ra in d o p a m i n e , th rough a -methy l ty ros ine ,

impa i r ed de layed r e sponse per formance as was expec ted and left v isua l dis-

c r imina t ion per formance unaffected (7) . D o p a m i n e therefore s eems to b e func-

t ional ly s ignif icant for the per formance of cer ta in behav iora l tasks and not

others . I then dec ided to inves t iga te , in col labora t ion wi th Miche l Br ie re , the

effect of L-dopa on the per formance of four more behav io ra l tasks h o p i n g that

those w h o s e per formance w a s improved b y L-dopa wou ld reveal cer ta in

c o m m o n character is t ics and thus he lp us to de t e rmine the aspect of b e h a v i o r

w h i c h is affected b y L -dopa admin is t ra t ion . I wou ld l ike to stress the fact that

L -dopa did not improve lea rn ing bu t on ly per formance . T h e four tasks w e

chose we re a spat ial a l ternat ion, a success ive v isual d i sc r imina t ion , and two

go no -go tasks . L -dopa h a d n o effect w h a t s o e v e r on the per formance of e i ther

spatial a l ternat ion or success ive v isua l d i sc r imina t ion , bu t i m p r o v e d signifi-

cantly the per formance on b o t h go no-go tasks .

Before d i scuss ing resul ts , I wou ld l ike to desc r ibe brief ly the exper imenta l

se tup and the four behav io ra l tasks. All an imals we re t ra ined in a w o o d e n

c h a m b e r e q u i p p e d wi th a o n e - w a y screen in front of w h i c h is p laced a m o b i l e

tray wi th one or two carved-out food wel ls , d e p e n d i n g on the task to b e per-

formed. Fo r the spat ial a l te rnat ion task , the screen is ra i sed and the an imal

p resen ted wi th the tray on w h i c h two food wel ls are covered w i t h ident ica l

w o o d e n b locks . An ima l s had to learn that left and r ight food wells were ba i ted

al ternately. In success ive v isua l d i sc r imina t ion ( V D ) , an imals learned to as-

socia te a f lashing l ight wi th food in the r igh t -hand s ide well and a con t inuous

l ight w i t h food in the left wel l . T h e two go no-go tasks w h o s e per formances

were improved b y L-dopa we re a de layed r e sponse and a v isual d i sc r imi -

na t ion . In the de layed r e sponse (DR) task, an ima l s w e r e s h o w n a s ing le food

well w h i c h was e i ther ba i t ed or left u n b a i t e d b y the inves t iga tor . T h e screen

was then lowered for 5 seconds after w h i c h it w a s ra ised . O n go tr ials , w h e n

the well w a s ba i t ed , the correct r e sponse was to uncover the food wel l and re-

tr ieve the reward . O n no-go trials w h e n the wel l w a s not ba i t ed the an imal

had to refrain from uncove r ing the food wel l for 7 seconds , after w h i c h it

r ece ived a p iece of mea t from the inves t iga tor . In go no -go v isua l d i sc r imi -

na t ion , an imals we re p re sen ted a s ing le food wel l as soon as the sc reen w a s

lifted. T h i s t ime a b lack cover s ignif ied that the wel l was ba i t ed and a b e i g e

o n e that it w a s not ba i t ed .

L-Dopa and Conditioned Inhibition 221

L - D O P A P L A C E B O ERRORS

6 l

0 T7 1 1 1 1 TT 1 1 1 1

0* 4 6 8 24 HOURS 0* 4 6 8 24 HOURS

FIGURE 1. ( A ) Effect of L-dopa on the performance of a successive visual discrimination ( V D ) and

a spatial alternation ( S A ) task. Graphs represent the average number of errors (ordinate) made by

6 V D cats ( 2 7 experiments) and 6 S A cats ( 1 8 experiments) just before ( 0 ) and 4 , 6 , 8 , and 2 4 hours

(abscissa) after 3 0 mg/kg of L-dopa per os ( | ) . ( B ) Number of errors made by the same cats in 6 V D and 8 S A placebo experiments carried out in the second or third week of training.

L -Dopa w a s admin i s t e r ed to an imals once a w e e k , b e g i n n i n g on the third

day of t ra in ing . Each L -dopa expe r imen t cons i s t ed of a control s e s s ion at 0

hour fol lowed b y the admin i s t r a t ion of 30 mg/kg of L -dopa per os. T e s t i n g w a s

then repea ted 4 , 6 , 8, and 24 hours after L -dopa admin i s t ra t ion . As s h o w n in

F igure 1 A , a s ingle dose of L -dopa had n o effect on the per formance of e i ther

spat ial a l ternat ion or success ive v isua l d i sc r imina t ion ; the n u m b e r of errors

r e m a i n i n g u n c h a n g e d th roughou t L -dopa expe r imen t s .

T h e effect of L -dopa on the per formance of the v isua l d i sc r imina t ion and

delayed r e sponse , go no -go tasks is i l lustrated in F igure 2 in w h i c h the results

of no -go and go trials have b e e n calculated separate ly . It i s in te res t ing to no te

that L -dopa s ignif icant ly i m p r o v e d (P < 0 . 0 0 1 , V D task; P < 0 . 0 1 , D R task)

the pe r fo rmance of all cats on no-go trials b e t w e e n 4 and 8 hours after i ts ad-

min i s t ra t ion . T h i s i m p r o v e m e n t is par t icular ly s t r ik ing w h e n c o m p a r e d to the

per formance in p l acebo expe r imen t s in w h i c h an imals ind i sc r imina te ly un-

covered the food wel l on all tr ials . A look at the per formance dur ing go trials

reveals a s l ight increase in the n u m b e r of errors m a d e b e t w e e n 4 and 8 hours .

T h i s inc rease is not s tat is t ical ly s ignif icant , except at 4 hours (P < 0 .05) on the

V D task.

O n e of the q u e s t i o n s ra ised b y these resul ts and to w h i c h w e t r ied to find

an a n s w e r w a s w h a t aspec t o f b e h a v i o r is actually i m p r o v e d b y L -dopa ad-

min i s t r a t ion? U p to date , w e have s tud ied the effect of L -dopa on the perform-

ance of six behav io ra l tasks ; three we re i m p r o v e d b y L -dopa and 3 r e m a i n e d

u n c h a n g e d . Essen t ia l ly two k inds of r e sponses are per formed in these six

tasks ; e i ther an ins t rumenta l " a p p r o a c h " r e sponse or an act ive suppres s ion of

that same r e sponse w h i c h w e call a " n o a p p r o a c h " r e sponse . T h e c o m m o n

character is t ic of tasks w h o s e per formance was improved b y L -dopa is that they

all involve e i ther delay or suppres s ion of the " a p p r o a c h " r e sponse , thus

4 -

2 -

A B

222 Anne Kitsikis

V D D R

N O - G O

ERRORS

5 i .PLACEBO — . . . . P L A C E B O

2 4 HOURS ol 4 6 8 2 4 HOURS

FIGURE 2. Effect of L-dopa on the performance of go no-go visual discrimination (VD) and delayed response (DR) tasks. Graphs (full line) represent the mean error score (ordinate) made in the 5 no-go (A) and 5 go (B) trials of each 10 trial session, by 7 VD cats (14 experiments) and 6 DR cats (14 experiments) just before (0) and 4, 6, 8, and 24 hours (abscissa) after 30 mg/kg of L-dopa per os ( | ) . The broken line represents the average number of errors made in 7 VD and 6 DR placebo experiments carried out in the second or third week of training.

sugges t ing that L-dopa m a y improve r e sponse i nh ib i t i on . T a b l e I classifies the

six tasks inves t iga ted accord ing to the type of r e sponse invo lved , e i ther " a p -

p r o a c h " or " n o app roach . " Each of these r e sponses m a y b e e i ther direct or

de layed, direct m e a n i n g that the r e sponse is in i t ia ted as soon as the screen is

ra ised. N o n e of the tasks r equ i r ing direct " a p p r o a c h " re sponses w a s affected

Table I Classification of Six Behavioral Tasks According to the

Type of Response Required

Approach No Approach

Direct Delayed Direct Delayed

Simultaneous VD Spatial DR" VD: no-go" DR: no-go" Successive VD Spatial alternation VD: go trial

" Performance improved by the administration of 30 mg/kg of L-dopa per os.

A

4-

3-

2-

1-

T T 1 1 1 1 r r 1 1 1 1

0* 4 6 8 24 HOURS 0? 4 6 8 24 HOURS

G O R

^ P L A C E B O

ERRORS

L-Dopa and Conditioned Inhibition 223

b y L -dopa. T h e r e m a i n i n g three r e sponses , spat ial de layed r e sponse , and

no -go trials o f v isua l d i sc r imina t ion and de layed r e sponse tasks all r equ i red

p e r m a n e n t or de layed suppress ion of the " a p p r o a c h " r e sponse and we re all

i m p r o v e d b y L -dopa admin i s t r a t ion .

A n impor tan t i s sue in the in terpre ta t ion of our resul ts is to de te rmine

w h e t h e r L -dopa admin i s t r a t ion i m p r o v e d cond i t i oned r e sponse i n h i b i t i o n

th rough ac t ion on a specific neural m e c h a n i s m necessa ry for th is type of

b e h a v i o r or if L -dopa s imply depressed m o t o r act ivi ty . At the p resen t stage of

expe r imen t s , our data are no t conc lus ive on this po in t a l though an examina -

t ion of r e sponse la tenc ies t ends to favor an act ive r e sponse i n h i b i t i o n .

It s e e m s that a part ial exp lana t ion of our results m a y b e found in e lec-

t rophys io logica l data such as those of Dr . B u c h w a l d w h i c h are a i m e d to

disclose synapt ic in f luences exer ted b y the caudate nuc leus p ro jec t ion n e u r o n s

upon target e l emen t s in the p u t a m e n , pa l l idum, and d iencepha l i c s t ructures .

Th i s c i rcui t ry is cer ta in ly invo lved in the effects I o b t a i n e d th rough L -dopa ad-

min is t ra t ion .

O u r results s h o w that oral admin i s t ra t ion of 30 mg/kg of L -dopa in cats ,

w h i c h s ignif icant ly inc reases the level of d o p a m i n e in the caudate (9) , also

i m p r o v e s the per formance of cond i t i oned i n h i b i t i o n . Cou ld there b e a causal

re la t ion b e t w e e n inc reased d o p a m i n e act ivi ty in the caudate and improved

r e sponse i n h i b i t i o n ? W e already k n o w that the overt effect o f caudate s t imula-

t ion on en topeduncu la r cells is a long- las t ing i n h i b i t i o n e i ther m o n o s y n a p -

tically e l ic i ted , as p roposed b y Y o s h i d a , R a b i n , and A n d e r s o n (12) , o r fol low-

ing in i t ia l exc i ta t ion as s h o w n th is m o r n i n g b y B u c h w a l d (2) . I w o n d e r

w h e t h e r dur ing the genera l d i scuss ion Dr . B u c h w a l d w o u l d l ike to m a k e a

c o m m e n t on the difference b e t w e e n h i s resul ts s h o w i n g E P S P - I P S P s e q u e n c e s

in pall idal n e u r o n s and those of Y o s h i d a p o i n t i n g out that I P S P s occur w i thou t

p reced ing exc i ta t ion . If therefore caudate output to pall idal and nigra l cells is

i n h i b i t o r y as ind ica ted b y e lec t rophys io logica l date , w e m a y ask w h e t h e r in

our e x p e r i m e n t s the L -dopa- induced increase of striatal d o p a m i n e e n h a n c e d

the effect iveness of the caudate i nh ib i t o ry output . T w o h y p o t h e s e s conce rn ing

this i s sue re ta in our a t ten t ion , b o t h of w h i c h find suppor t in the l i terature.

D o p a m i n e m i g h t e i ther exert an i n h i b i t i o n on inh ib i t o ry in t e rneurons or ex-

cite exci ta tory in t e rneu rons act ing on caudate output e l emen t s . B o t h of these

eventua l i t ies wou ld lead to r e in fo rcement of caudate i n h i b i t o r y output to

pall idal cells and c o n s e q u e n t l y disfaci l i tate the ventrolateral nuc leus of the

tha lamus and the m o t o r cor tex. It appears r easonab le to cons ide r in t e rneurons

as the target of d o p a m i n e ac t ion s ince w e already k n o w that i n t e rneu rons con-

st i tute the major part of the caudate ' s neurona l popula t ion , no t on ly from

K e m p and Powel l ' s ana tomica l data (6) bu t also b e c a u s e w e learned today from

B u c h w a l d that on ly 3 to 5 % of caudate n e u r o n s could b e i nvaded an-

t idromical ly from the pa l l idum or the n ig ra (2) . T h e p r e d o m i n a n c e of i n h i b i -

tory in t e rneurons s e e m s l ikely , on account of the poor spon t aneous back -

224 Anne Kitsikis

ground firing seen in caudate cells (3 ,10 ) , a l though exci ta tory e l emen t s mos t

cer ta inly exist . It should , h o w e v e r , b e e m p h a s i z e d that Dr . Purpura rarely o b -

ta ined IPSPs in the caudate nuc leus after specific or centre m e d i a n tha lamic

s t imula t ion (10) . I w o n d e r i f Dr . Purpura or Dr . B u c h w a l d could specula te on

their conf l ic t ing resul ts , n a m e l y pure E P S P s in Purpura ' s inves t iga t ion and

E P S P - I P S P s e q u e n c e s in B u c h w a l d ' s work . T h e exact na ture of d o p a m i n e ac-

t ion r ema ins obscu re as b o t h exci ta tory and inh ib i to ry effects have b e e n o b -

served in different ne rve cells of the caudate nuc leus after mic ro ion tophore t -

ical appl ica t ion of d o p a m i n e ( 1 , 4 , 5 , 1 1 ) . H o w e v e r , desp i te lack of knowledge

conce rn ing the prec ise ac t ion of d o p a m i n e on caudate in te rneurons , w e

suggest that in our expe r imen t s the L -dopa- induced d o p a m i n e increase in the

s t r ia tum reinforced caudate i n h i b i t o r y output to the pa l l idum and thus facili-

tated cond i t i oned r e sponse i n h i b i t i o n .

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6. Kemp, J . M. , and Powell, T. P. S. The structure of the caudate nucleus of the cat: Light and electron microscopy. Philos. Trans. R. Soc. London [Biol. Sci.], 1971, 262: 383^101.

7. Kitsikis, A., and Roberge, A. G. Behavioral and biochemical effects of a-methyltyrosine in cats. Psychopharmacologia, 1973, 31: 143-155.

8. Kitsikis, A. , and Roberge, A. G. Behavioral and biochemical effects of L - D O P A in cats. Isr. J. Med. Sci., 1973, 9: 17-23.

9. Kitsikis, A., Roberge A. G., and Frenette, G. Effect of L - D O P A on delayed response and visual discrimination in cats and its relation to brain chemistry. Expl. Brain Res., 1972, 15: 305-317.

10. Purpura, D . P., and Malliani, A. Intracellular studies in the corpus striatum. I. Synaptic po-tentials and discharge characteristics of caudate neurons activated by thalamic stimulation. Brain Res., 1967, 6: 325-340.

11. York, D . The inhibitory action of dopamine on neurons of the caudate nucleus. Brain Res., 1970, 20: 223-249.

12. Yoshida, M., Rabin, A., and Anderson, M. Monosynaptic inhibition of pallidal neurons by axon collaterals of caudato-nigral fibers. Expl. Brain Res., 1972, 15: 333-347.

8B Discussion: Acetylcholine and the

Caudate

J O H N B L A S S




After all the d i scus s ion of the impor t ance of d o p a m i n e in the s t r ia tum, let

m e put in a w o r d on the " c h o l i n e r g i c " s ide . The re are two reasons for th ink-

ing ace ty lchol ine m a y b e impor tan t in the caudate . Firs t , there is a lot there .

T h e bes t avai lable chemica l marke r for " c h o l i n e r g e n i c i t y " is p r o b a b l y the en-

z y m e w h i c h m a k e s ace ty lchol ine , cho l ine acetyl t ransferase. U s i n g our o w n

data for the cat, w h i c h are typical o f results o b t a i n e d b y m a n y peop le , the

levels of this e n z y m e in the caudate are abou t 0 .17 /xmoles/minute pe r g ram

t i ssue , abou t 6-fold h i g h e r than in mos t areas of cat b ra in gray mat te r and

20-fold h i g h e r than in the area of cat ce rebe l lum we e x a m i n e d . M e a s u r i n g the

actual levels of ace ty lchol ine in b ra in r eg ions is harder to do. T h e chemica l

m e a s u r e m e n t s are accurate e n o u g h , bu t the levels d e p e n d on h o w the an imal

is ki l led. The re m a y , i ndeed , b e a pool of ace ty lchol ine in the b ra in w h i c h

turns over in a s econd or so . A l though the exact values are n o w b e i n g reeval-

uated in several l abora tor ies , it does appear sure that the caudate con ta ins

more ace ty lchol ine than any o ther reg ion of the b ra in . In one expe r imen t at

U . C . L . A . , Dr s . L e v i n e , Hull , B u c h w a l d , and J e n d e n found a level of ace-

ty lchol ine in a cat caudate of abou t 0.5 nmoles /gm t i ssue (0.5 FJM). As far as I

k n o w , that is the h ighes t level that has b e e n found in any b io log ica l sys tem.

T h e s econd , and m o r e direct , r eason to feel that ace ty lchol ine is impor tan t is

that d ropp ing a little b i t of it on to the caudate causes profound c h a n g e s in the

an imal ' s b e h a v i o r (1) . O f course , for m a n y years , drug t rea tment of basa l

gangl ia d isorders d e p e n d e d on c o m p o u n d s act ive on cho l ine rg ic s y s t e m s , and

there is n o w a good deal of specula t ion abou t the role of " i m b a l a n c e s "

b e t w e e n chol inerg ic and dopamine rg i c sys t ems in var ious d i sease s tates .

225

226 John Blass

The control of ace ty lchol ine m e t a b o l i s m in the caudate r ema ins a knot ty

p r o b l e m on b o t h a phys io log ica l and a b i o c h e m i c a l level . S t r ipp ing even large

amoun t s of cortex did not c h a n g e the level of cho l ine acetyl t ransferase in cat

caudate in recent expe r imen t s w e conduc ted ; thus , axon te rmina ls of cort ical

neurons do not appear to b e a major por t ion of the chol inerg ic s tructures in

the caudate . In a recent report from G l o w i n s k i ' s labora tory in Par i s , however ,

l es ions in the m i d b r a i n t e g m e n t u m w e r e found to reduce ace ty lchol ine in the

caudate (2) .

T h e me tabo l i c control o f ace ty lchol ine syn thes i s is also no t clear. T h e syn-

thet ic e n z y m e , cho l ine acetyl t ransferase , is p resen t in a severalfold excess . T h e

concen t ra t ions of the reactants (namely , acetyl C o A and chol ine) and of the

products ( C o A and ace ty lchol ine) are a round e q u i l i b r i u m ; the sugges t ion has

b e e n m a d e that the syn thes i s is control led b y m a s s ac t ion . T h e r e is a h i g h af-

finity up take sys t em for cho l ine . I n h i b i t i n g th is sys t em (wi th h e m i c h o l i n i u m )

does reduce syn thes i s . T h e m a i n phys io log ica l precursor for the acetyl C o A

used for ace ty lchol ine syn thes i s is pyruvate . Recen t ly , G i b s o n and J o p e in m y

labora tory found that even relat ively mi ld i m p a i r m e n t of pyruvate ox ida t ion

caused a propor t iona l decrease in acetylchol ine syn thes i s . T h e s e expe r imen t s

were done wi th m i n c e s of rat b ra in . T h e result is qu i t e surpr is ing s ince the

fluxes are all wrong : less than 0 . 5 % of the pyruvate b u r n e d b y b ra in goes to

ace ty lchol ine , so that even w i t h 9 0 % i n h i b i t i o n of pyruvate ox ida t ion , there is

still a 20-fold excess of acetyl C o A for ace ty lchol ine syn thes i s . Never the les s ,

the resul t s e e m s real and has he ld up , even though the m e c h a n i s m r e m a i n s to

b e s tudied .

T h e s e resul ts do sugges t that there m a y b e an in te rac t ion b e t w e e n ca rbohy-

drate m e t a b o l i s m and ace ty lchol ine . In th i s v o l u m e , Dr . Hel ler has sugges ted

that there m a y well b e an in te rac t ion b e t w e e n d o p a m i n e and carbohydra te

me tabo l i sm . T h e obv ious specula t ions rest on wha t Dr . Pop jak has referred to

as " a few flecks of subs tance on a rag ing v o i d / ' T h e corre la t ions of phys io log-

ical and n e u r o c h e m i c a l data that Dr . Hel ler p resen ted depend on , a m o n g other

th ings , detai led unde r s t and ing of the e n z y m o l o g y and control of ca-

t echo lamine m e t a b o l i s m . If control o f ace ty lchol ine m e t a b o l i s m b e c o m e s as

wel l unders tood , it wou ld b e cha l leng ing to try to follow Dr . Hel ler ' s p rece-

dent and a t tempt to correlate phys io log ica l and n e u r o c h e m i c a l data abou t that

n e u r o h o r m o n e . A n d the caudate wou ld aga in appear to b e a good place to

look.

REFERENCES

1. Hull, C. D. , Buchwald, N. A., and Ling, G. Effects of direct cholinergic stimulation of forebrain

structures. Brain Res., 1 9 6 7 6 : 22-35 . 2. Jones, B . E . , Guyenet, P., Cheramy, A., Gauchy, C., and Glowinski, J . The in vivo release of

acetylcholine from cat caudate nucleus after pharmacological and surgical manipulation of dopaminergic nigrostriatal neurons. Brain Res., 1973, 64: 355-369.

9 Dopamine-Containing Neurons of the

Substantia Nigra and Their Terminals in the Neostriatum

VIRGINIA M. TENNYSON, CATHERINE MYTILINEOU, RICHARD HEIKKILA, ROBERT E. BARRETT,

GERALD COHEN, LUCIEN COTE, PHILIP E. DUFFY, and LUIS MARCO

Department of Pathology, Division of Neuropathology, and Department of

Neurology, College of Physicians and Surgeons,

Columbia University, New York, New York

D o p a m i n e , w h i c h is found in ve ry h i g h concen t ra t ions in the neos t r i a tum

(11) , has b e e n r ecogn ized as a puta t ive neuro t ransmi t t e r in the ex t rapyramidal

mo to r sys tem. T h e f inding of a decreased concen t ra t ion of d o p a m i n e in the

neos t r i a tum of p o s t m o r t e m b ra ins from pa rk inson ian pa t ien ts (27) led to the

in t roduc t ion of L -dopa for the t rea tment o f P a r k i n s o n ' s d i sease ( 2 0 , 2 1 , 9 5 ) .

W i t h the use of the Falck-Hil larp f luorescence mic ro scopy t e c h n i q u e (28) , it

has b e e n s h o w n that d o p a m i n e is found in t iny axonal t e rmina ls in the

neurop i l of the neos t r i a tum, bu t not in cell b o d i e s (12 ,34) . Dah l s t rdm and Fuxe

(23) sugges ted that f luorescent neu rons of the subs tan t i a n ig ra gave r ise to the

d o p a m i n e - c o n t a i n i n g te rmina ls in the neos t r i a tum. T h i s a s s u m p t i o n is sup-

por ted b y the f inding that l es ions in the subs tan t i a n igra or upper m i d b r a i n

result in a r educ t ion of f luorescence and d o p a m i n e con ten t in the ipsi la teral

neos t r i a tum ( 4 , 5 , 6 5 , 7 3 , 7 4 ) . Never the le s s , there w a s cont roversy in i t ia l ly abou t

the ex i s t ence of a n igroneos t r ia ta l pa thway , b e c a u s e the axons l i nk ing the s u b -

s tant ia n igra to the neos t r i a tum do no t f luoresce in the normal adult an imal ,

and the Nau ta s i lver impregna t i on m e t h o d s h o w e d few, if any , degene ra t ing

axons fol lowing les ions m a d e in the subs tan t i a n igra (13 ,29) . R e c e n t modif ica-

t ions of the Nau ta m e t h o d , w h i c h make it pos s ib l e to see degene ra t ion in

ex t remely fine axons , pe rmi t the demons t r a t ion of the n igroneos t r ia ta l path-

w a y th roughou t its course (14 ,65 ,82 ) . T h e n igroneos t r ia ta l pa thway has b e e n

227

228 Virginia M. Tennyson et al.

m a p p e d b y f luorescence m i c r o s c o p y after electrolytic les ions or the in jec t ion of

6 -hydroxydopamine ( 6 - O H - D A ) at var ious po in t s a long the course of the

media l forebra in bund le (92) . F luorescence appears in the por t ion of axons

p rox imal to the l e s ion , e v e n t h o u g h it canno t b e s een in the no rma l an imal . It

is l ikely that e i ther the levels o f d o p a m i n e f lowing d o w n normal axons are so

low that the f luorescence in t ens i ty is no t v i s ib l e to the n a k e d eye , or else the

synthe t ic e n z y m e s are concent ra ted in te rmina l r eg ions unt i l damage act ivates

cell b o d i e s to rep len ish the supply of d o p a m i n e and its syn thes i z ing e n z y m e s .

Desp i t e the in teres t and poss ib l e s ignif icance of neu rons in the subs tan t ia

n igra compac ta , unt i l recent ly little a t ten t ion has b e e n g iven to the e m b r y o n i c

deve lopmen t of these cells and the i r t e rmina ls . T h e s e neurob las t s and thei r

axons w h i c h g row from the m e s e n c e p h a l o n to the p rosencepha lon are f luores-

cent at early s tages of deve lopmen t ( 3 7 - 3 9 , 6 4 , 6 8 , 7 0 , 7 1 , 8 9 ) , as are the i r early

growth cones and te rmina ls w h i c h invade the neos t r i a tum ( 3 7 , 3 8 , 6 2 , 6 3 , 6 8 ,

7 0 , 7 1 , 8 3 , 8 4 ) . T h e f luorescence in the axons forming the n igroneos t r ia ta l path-

w a y d isappears in the per inata l pe r iod (37,71) and r ema ins absen t th roughou t

normal adult l ife.

T h e presen t paper s u m m a r i z e s our f luorescence , electron mic roscop ic , and

b i o c h e m i c a l s tudies on the deve lopmen t of the subs tan t i a n igra and n e o -

s t r ia tum in r abb i t s from early different iat ion to the adult pe r iod (83 ,84 ,89 ) . T h e

ini t ia l s tages of deve lopmen t of the neurob las t s o f the subs tan t i a n igra and the

course the i r axons take " e n r o u t e " to the neos t r i a tum will b e i l lustrated, as

well as matura t iona l changes in these cells . Quan t i t a t ive m e a s u r e m e n t s of en-

dogenous d o p a m i n e concen t ra t ions in the deve lop ing neos t r i a tum wil l b e

compared wi th the up take of rad ioac t ive d o p a m i n e at each s tage. A s a frame

of reference , the pe r t inen t features of the adult subs tan t i a n ig ra and n e o -

s t r ia tum will b e po in t ed out and ev idence conce rn ing the ident i f icat ion of

dopamine -con t a in ing te rmina ls in the neos t r i a tum d iscussed .

ADULT SUBSTANTIA NIGRA

T h e subs tan t ia n ig ra con ta ins s o m e ace ty lchol ines terase (AChE) and signifi-

cant amoun t s of d o p a m i n e , y - aminobu ty r i c acid ( G A B A ) , and the e n z y m e s in-

volved in thei r syn thes i s (19) . N e u r o n s of the subs tan t ia n igra compac ta (Fig-

ure 1A) and the i r t e rmina ls in the neos t r i a tum (Figure I B ) in the adult r abb i t ,

as in o ther spec ies , exh ib i t f luorescence a t t r ibutable to d o p a m i n e . T h e Falck-

Hil larp t e c h n i q u e (28) was used for the fluorescence mic ro scopy s tudies in this

paper . S l ices of r abb i t b ra in were frozen in p ropane cooled b y l iqu id n i t rogen ,

dr ied under v a c u u m , t reated at 80°C w i t h fo rmaldehyde gas at 7 0 % relat ive

humid i ty and e m b e d d e d in paraffin. E igh t -mic rome te r sec t ions were p h o -

tographed us ing K o d a k T r i - X Pan film in a Le i tz ul traviolet mic roscope u s ing

a B G 12 exci ta t ion filter and 510 bar r ie r filter.

9. Neurons of the Substantia Nigra 229

FIGURE 1. A. A moderately intense greenish-yellow fluorescence attributable to dopamine fills

the cytoplasm and some processes (arrow) of the neurons of the substantia nigra, pars compacta.

The neurons of the pars reticulata (r) lack fluorescence. Adult rabbit. X160. B. Dopamine-con-

taining terminals from the neurons of the pars compacta are widely distributed throughout the

neostriatum giving the neuropil an intense diffuse fluorescence. The cell bodies of the neurons

(arrow) lack fluorescence. Caudate nucleus near ependymal surface. Adult rabbit. X140.

A n u m b e r of ul trastructural s tudies have b e e n carr ied out on the genera l

features of the ma tu re subs tan t i a n igra in the rat (43 ,44 ) , m o u s e (7) , cat (42 ,78 ) ,

and m o n k e y (79) . T h e t i ssue for mos t of the e lect ron mic rog raphs i l lustrated in

the present s tudy w a s fixed b y perfusion wi th a mix ture of buffered glu-

tara ldehyde and para formaldehyde , fo l lowed b y postf ixat ion in o s m i u m te-

t roxide , and then rou t ine dehydra t ion and e m b e d d i n g in epoxy res in . T h i n

sec t ions we re pho tog raphed wi th a S i e m e n s E l m i s k o p IA electron mic roscope .

T h e p r e d o m i n a n t neurona l cell type in the pars compac ta of the subs tan t i a

n igra is a m e d i u m s ized ( 1 5 - 2 0 fjum) mul t ipolar ne rve cell w i t h four to six

smoo th dendr i tes w i thou t sp ines and an eccentr ic nuc leus h a v i n g o n e or two

deep info ld ings (44) . A th in astroglial shea th (44) (Figure 2 A , gl) covers the

soma of the n e u r o n except w h e r e it is con tac ted b y synapses (arrow and

crossed ar row) . T h e per ikaryal cy top lasm exh ib i t s several c lumps of wel l -

o rgan ized rough-sur faced endop la smic re t i cu lum, the Niss l subs t ance (er) ,

and an e longa ted Golg i complex (g) that ex tends in to the dendr i tes a long wi th

mic ro tubu le s and r i b o s o m e s (44) . C o m p l e x granules typical of l ipofuscin and

me lan in have b e e n desc r ibed in these neu rons in m a n (22,25) and m o n k e y

(47 ,67 ) , bu t relat ively small ovo id he t e rogeneous dense b o d i e s (b) are found in

lower spec ies . In m a n , the granules are very dense and i r regular in shape (Fig-

ure 2 B ) . T h e y usual ly exh ib i t vacuoles (v) p ro jec t ing from the i r surfaces. T h e s e


FIGURE 2. A. A medium-sized neuron with an eccentric nucleus (N) from the pars compacta exhibits several clumps of endoplasmic reticulum (er), a well-developed Golgi complex (g), mi-tochondria (m), and scattered dense bodies (b). A glial sheath (gl) surrounds the neuron except


vacuoles were p r o b a b l y the former s torage s i tes of l ip id that w e r e r e m o v e d

dur ing the prepara t ive t e c h n i q u e s for e lect ron mic roscopy . At h i g h magni f ica-

t ion , dense , i r regularly shaped mate r ia l (Figure 2 C ) , w h i c h m a y b e t he mel -

an in p i g m e n t , is p resen t on a less dense s tructure. Part ial ly d e p i g m e n t e d

me lan in granules from the subs tan t i a n ig ra o f a pa rk in son i an pa t ien t (25)

s h o w the granular subs t ruc ture (Figure 2 D , ar row) m o r e clearly. T h e me lan in

granule in m a n r e s e m b l e s a l ipofuscin granule in w h i c h m e l a n i n p i g m e n t has

b e e n depos i t ed , w h e r e a s , the granules o f the neu rons o f the subs tan t i a n ig ra

of lower spec ies m o r e c losely r e s e m b l e l y sosomes . The re is cons ide rab le evi -

dence that pe rox ida t ion of fatty acids l ead ing to the format ion of h igh ly reac-

t ive ox idan ts can induce format ion of l ipofuscins (97) . Pe rox id ized unsa tu-

rated fatty ac ids can cause the format ion of c ross - l inked po lymers (15) , w h i c h

in the i r final s tate m a y r ema in as res idual b o d i e s , as p r o p o s e d b y K o e n i g et al.

(60) .

T h e neurop i l of the pars compac ta con ta ins small , r andomly o r i en ted un-

mye l ina ted axons , small mye l ina t ed axons , and a var ie ty of synapt ic j u n c t i o n s

(43) . W h i l e axosomat i c (F igure 2 A , ar row, c rossed ar row) and axoaxon ic j u n c -

t ions are p resen t in the subs tan t i a n igra , m o s t synapses are axodendr i t i c (Fig-

ure 2 E ) . T h e dendr i tes are s tudded w i t h " b o u t o n s en p a s s a g e " so that the sur-

face of the dendr i te is part ial ly or comple te ly su r rounded w i t h synapt ic con-

tacts ( 7 , 4 3 , 5 7 , 6 6 , 7 8 , 7 9 ) . M o s t b o u t o n s have p redominan t ly small clear ves ic les

and form j u n c t i o n s in w h i c h the pre- and pos t synap t ic m e m b r a n e s are of

equa l dens i ty and th i ckness (Figure 2 E , arrow) (43 ,78 ) . T h i s type of t e rmina l ,

w h i c h w a s first de sc r ibed b y Gray (40) as the type 2 synapse , is called the

symmet r ica l synapse in th i s paper . T h e o ther type of j u n c t i o n i s the type 1

synapse (40) , and it forms an a symmet r i ca l j u n c t i o n i n w h i c h the pos t synap t i c

m e m b r a n e is th icker and dense r than the p resynap t ic m e m b r a n e (Figure 2 E ,

c rossed a r row) . T h i s will b e referred to as the asymmet r ica l synapse . At least

where it is contacted by axosomatic terminals. One is filled with small vesicles and forms a sym-

metrical junction (crossed arrow), the other has fewer vesicles and makes an asymmetrical junc-

tion (arrow). A subsurface cisterna is immediately beneath the latter junction. Adult rabbit.

X9000. B . Melanin granules in the normal adult human substantia nigra, pars compacta, are

complex irregularly shaped bodies, which contain very dense material and have protruding

vacuolelike structures (v). The latter probably represent the sites of lipid which have been ex-

tracted by the preparatory techniques. X50,000. C. A high magnification of a portion of a melanin

granule shows that it is filled with small very dense, irregularly shaped material, which has indef-

inite boundaries. Normal adult human. X 120,000. D. A partially depigmented melanin granule

from a parkinsonian patient reveals that the dense material overlies a moderately dense substruc-

ture (arrow). Adult human, x90,000. E. Most of the synapses in the pars compacta take place on

medium-sized dendrites, which contain microtubules (t), mitochondria, and agranular reticulum.

One bouton, which contains small ovoid or flattened clear vesicles and three large dense core

granules, forms a symmetrical junction (arrow). The other, which contains masses of slightly large

ovoid or spherical clear vesicles and some large vesicles with a moderate density, forms an asym-

metrical junction (crossed arrow). Adult rabbit. X30,000.

232 Virginia M . Tennyson et al.

s o m e of the te rmina ls h a v i n g symmet r i ca l j u n c t i o n s or ig ina te from in t r ins ic

neu rons of the neos t r i a tum, s ince they degenera te after l es ions of the caudate

nuc leus and p u t a m e n (42 ,57 ) . S i n c e A C h E is p resen t in the s t r ia tonigral f ibers

(69) , these b o u t o n s m a y b e cho l ine rg ic (43) . A m u c h smal ler percen tage of

b o u t o n s (about 1 0 % ) con ta in s o m e large dense core ves ic les (also cal led large

dense core granules in th is manusc r ip t ) a m o n g the clear ves ic les . T h i s type of

b o u t o n forms a symmet r i c j u n c t i o n s (43 ,78) . Gul ley and S m i t h b e r g (43) pos tu-

lated in the i r s tudies o f the rat that the latter type of b o u t o n m i g h t con ta in

n o r e p i n e p h r i n e , and that ano the r type of a symmet r i ca l synapse , w h i c h has

small clear ves ic les and some large dense core ves ic les , m a y con ta in sero tonin .

S ince b o u t o n s h a v i n g e longa ted ves ic les are a s s u m e d to b e i nh ib i t o ry , te rmi-

nals w i th th is type of e n d i n g on the axon hi l lock are p r e s u m e d to con ta in the

i nh ib i t o ry t ransmi t te r G A B A (43) . T h e large 700- to 1 2 0 0 - A dense core ves ic le

d i scussed in the latter two types of b o u t o n s in the rat appears to b e even m o r e

u b i q u i t o u s in o ther spec ies . R i n v i k and Grofova (78) repor ted that all types of

synapt ic b o u t o n s in the subs tan t i a n ig ra o f the cat could con ta in the large

dense core ves ic le . T h e y are found f requent ly in the r abb i t as wel l . T h e at-

t empt b y Gul ley and S m i t h b e r g (43) to correlate m o r p h o l o g y w i t h the t rans-

mi t te r is p rovoca t ive , b u t it should b e po in ted out that defini t ive expe r imen t s

p rov ing these a s sumpt ions have no t b e e n accompl i shed wi thou t ques t ion as

yet.

ADULT NEOSTRIATUM

T h e f luorescent p re te rmina l axons and b o u t o n s of n e u r o n s of the subs tan t i a

n igra , pars compac ta , t e rmina te in the neurop i l of the neos t r i a tum (Figure I B ) .

T h e t iny axons and te rmina ls are n u m e r o u s and so w ide ly d i s t r ibu ted that the

neuropi l appears diffusely and in t ense ly f luorescent , in contrast to the non-

f luorescent fibers o f the in ternal capsule and the neurona l cell b o d i e s of the

neos t r i a tum (arrow). T h e s ize and shape o f neost r ia ta l n e u r o n s , as well as the

extent of the dendr i t ic a rbor iza t ion and the length of the i r axons , have b e e n

the subjec t o f s tudies u s ing the Golg i impregna t ion m e t h o d ( 3 0 - 3 2 , 5 4 , 5 5 , 5 8 ) .

O v e r 9 6 % of the cells in the caudate nuc leus are m e d i u m - s i z e d Golg i type II

neu rons , about 12 to 18 /mm in d iamete r , and have five or s ix dendr i tes w h i c h

are s tudded wi th sp ines on the i r b r a n c h e s . T h e s e cells are in t r ins ic to the

caudate nuc leus ; the i r axons ramify w i th in the l imi ts of the spread of the

dendr i tes o f the parent cell and form a dense p lexus w i th the dendr i t es of

n e i g h b o r i n g in t r ins ic cells . Less than 1 % of the neu rons are large fusiform

cells , about 22 to 30 /Jim i n s ize , and have large s traight dendr i tes w i t h few

sp ines and long axons . U n l i k e the shor t axons of the Golg i type II n e u r o n s , the

long axons of the large neu rons b e c o m e l ight ly mye l ina t ed and p r o b a b l y g ive

r ise to the th in ly mye l ina t ed radial f ibers that en te r the g lobus pal l idus


(31 ,59) . Collaterals of these fibers m a y lose the i r m y e l i n w h e n they reach the

" c o m b s y s t e m " th rough w h i c h t hey travel to r each the subs tan t i a n ig ra (31) .

Elec t ron mic roscop ic s tudies have also b e e n d o n e on the adult neos t r i a tum

in the cat ( 1 , 2 , 5 5 - 5 7 , 8 7 ) , m o n k e y ( 3 0 - 3 2 , 7 5 ) , rat (35 ,66 ) , m o u s e (7) , and r abb i t

(83 ,84) . T h e m o s t c o m m o n neu ron , the m e d i u m - s i z e d Golg i type II cell , has a

spher ica l nuc leus and a modera t e a m o u n t of clear cy top lasm wi th few inclu-

s ions and scat tered e l emen t s of the endop lasmic re t icu lum that do not form

Niss l b o d i e s . T h e i r dendr i tes have a large n u m b e r of sp ines . T h e pale den-

drit ic cy top lasm con ta ins di lated smooth-sur faced c is ternae and poor ly devel-

oped mic ro tubu le s . T h e largest cells have an i nden t ed nuc leus and a large

amoun t of cy top lasm w i t h m a n y stacks of granular endop la smic re t i cu lum

forming Niss l b o d i e s . T h e mi tochondr i a o f these cells are large and pale w i t h

few cris tae. T h e p re sence of th is type of m i t o c h o n d r i o n in dendr i tes hav ing

consp icuous mic ro tubu le s and few sp ines a ids in ident i fy ing these dendr i t es

as o r ig ina t ing from the large neu rons (55) .

M o s t of the synapt ic t e rmina ls in the neos t r i a tum are smal l axodendr i t i c

sp ine synapses w i t h round clear ves ic les in the axonal b o u t o n and a s y m m e -

trical m e m b r a n e t h i cken ings (Figure 3 A , c rossed ar row) . A m u c h smal ler

n u m b e r of the t e rmina ls have symmet r ica l j unc t iona l contac ts . T h e axonal

b o u t o n in th is case m a y have e i ther r ounded ves ic les or p l e o m o r p h i c ves ic les .

A l though m a n y of the i so la ted synapses m a y b e the t e rmina l b o u t o n s of

axons , at least s o m e of t h e m are sec t ions th rough b o u t o n s " e n p a s s a g e , " i . e . ,

synapt ic var icos i t i es occurr ing a long the course o f the axon p roper (F igures 3 A

and B ) . B o u t o n s " e n p a s s a g e " can b e demons t r a t ed after s i lver impregna t i on

(30 ,54 ,55) and have p rev ious ly b e e n seen b y e lect ron mic ro scopy

( 1 , 2 , 3 0 , 5 6 , 5 7 , 8 3 , 8 4 , 8 7 ) . Un l ike axons in mos t areas of the centra l ne rvous

sys tem, clear ves ic les are f requent ly found all a long the i r course , part icular ly

in those that form synapses " e n p a s s a g e . "

A x o n s fo rming synapses " e n p a s s a g e " can or ig ina te from the cor tex , thal-

a m u s , or m i d b r a i n , s ince they are found in var ious s tages of degenera t ion

after l e s ions of fiber tracts from these respec t ive areas (57) . In add i t ion , s o m e

synapses " e n p a s s a g e " are present a long the axons of n e u r o n s in t r ins ic to the

caudate nuc leus , s ince a large n u m b e r o f t h e m r e m a i n after a c o m b i n e d cor-

tical and tha lamic l e s ion (54) . T h i s conc lus ion is conf i rmed b y the var icos i t i es

" e n p a s s a g e " in F igure 3 B w h i c h w a s found in the caudate nuc leus after sec-

t ion of all of i ts afferent c o n n e c t i o n s (87) .

K e m p and Powel l (57) s tud ied the character is t ics of degenera t ing t e rmina l s

after l es ions in the cor tex , tha lamus , or m i d b r a i n in an a t tempt to desc r ibe the

var ious afferent b o u t o n s in the caudate nuc leus . A large n u m b e r of the te rmi-

nals h a v i n g clear ves ic les and asymmet r i ca l m e m b r a n e t h i cken ings degenera te

after l es ions in the cor tex or tha lamus , up to 40 and 2 5 % , respec t ive ly (57) .

T h e s e te rmina ls con ta in small clear ves ic les (30 ,31 ,53 ) and e n d p redominan t ly

on dendr i t ic sp ines of the Golg i type II n e u r o n s (31) . Large l es ions in the


F IGURE 3. A. The neuropil of the caudate nucleus of the adult rabbit is filled with axodendritic

spine junctions (crossed arrow) and axodendritic junctions. Two axons which form axospinous

junctions "en passage" (*) have clear vesicles along the course of the axon proper. Although most

of the axonal vesicles are clear, a few larger dense core granules are present (arrow). Dendritic

spine apparatus (s), dendrite (d). X24 , 000 . B . An axon with varicosities "en passage" containing

some clear and large dense core granules (arrow) must originate from an intrinsic caudate neuron,

since all of the hodological connections to this nucleus were sectioned. Adult cat. X52,000.

m i d b r a i n produce very sparse , t hough widesp read , degenera t ion of synapt ic

te rmina ls in the caudate nuc leus (57) . T h e degenera t ing te rmina ls obse rved

had asymmet r ica l m e m b r a n e t h i cken ings . M o s t were found in contact w i th

dendr i t ic sp ines .


FETAL DEVELOPMENT

Substantia Nigra, Pars Compacta

A s early as day 14 of ges ta t ion in the rabb i t , the anlage of the subs tan t i a

n igra , zona compac ta , and the area vent ra l i s t egmen t i can b e r ecogn ized in the

midven t ra l r eg ion of the caudal m i d b r a i n (Figure 4 A , arrow). The re are a few

faint ly f luorescent cell b o d i e s (Figure 4 B , ar row) and p rocesses in the mant le

layer. B y day 16 of ges ta t ion (Figure 4 C ) , the f luorescent cells have b e c o m e

more n u m e r o u s (Figure 4 D ) ; and t hey ex tend a lmost to the pial surface dorso-

ventral ly fo rming an inver ted V - s h a p e d f luorescent zone . T h e y are p resen t

th roughou t the caudal th i rd of the midb ra in . T h i s conf i rms ear l ier s tudies

u s ing o the r t e c h n i q u e s w h i c h s h o w that the midven t ra l prol i fera t ion of the

m i d b r a i n is the si te in w h i c h the neu rons of the subs tan t i a n ig ra (18) and of

the area vent ra l i s t egmen t i o r ig ina te (45) . O u r f luorescent data canno t p rov ide

in format ion on the o r ig in of the pars ret iculata. H a n a w a y et al. (45) r ecogn ized

that some nigral n e u r o n s m i g h t arise from the mid l ine anlage , bu t t hey state

that the p r imary si te of o r ig in o f neu rons of the subs tan t i a n ig ra is from the

midd le th i rd of the basa l neu roep i the l i um. S i n c e w e d id no t see any f luores-

cent cells in that loca t ion , the cells s tud ied b y H a n a w a y et al. (45) m a y have

b e e n des t ined only for the pars ret iculata . T h i s sugges t s a less close e m b r y o -

logical r e la t ionsh ip b e t w e e n the cells o f the pars compac ta and pars ret iculata

of the subs tan t i a n ig ra than b e t w e e n neu rons of the pars compac t a and the

area vent ra l i s t egmen t i .

W h e n one compare s the ul traviolet pho tomic rog raph of the midven t ra l p ro-

l iferat ion of the 16-day ges ta t ion r abb i t w i t h the same sec t ion seen in dark-

field u s ing w h i t e l ight to reveal the nuc le i and cellular pack ing in the t i s sue ,

one sees that the f luorescent profiles appear i m m e d i a t e l y vent ra l to the epen -

dymal zone (Figure 4 D , ep) . W e e x a m i n e d b y electron m i c r o s c o p y the reg ion

co r re spond ing to the f luorescent inver ted V - s h a p e d area in l i t te rmate e m -

bryos . E longa ted sp ind le - shaped b ipo la r neurob las t s (Figure 5 A ) are o r i en ted

dorsoventra l ly i m m e d i a t e l y b e n e a t h the e p e n d y m a l zone . T h e y are a l igned in

t a n d e m w i t h the cell b o d y of o n e adjacent to a t ra i l ing p rocess of ano the r as i f

the p reced ing cell w e r e d i rec t ing the course of mig ra t ion of the fol lowing cell.

T h e nucle i of the neurob las t s are e longa ted and often i nden t ed . N u m e r o u s

r i b o s o m e s (r) fill the cy top lasm, bu t there are on ly a few channe l s o f granular

endop la smic re t i cu lum (er) . T h e Golg i complex (g) is small and m a y b e located

nea r the nuc leus o r c lose to the b a s e o f a p rocess . A mode ra t e n u m b e r o f m i -

tochondr ia (m) are scat tered th roughou t the cell b o d y and its p rocess . T h e ear-

l iest p rocesses (p) cons i s t of per ikaryal cy top lasm. M o r e matu re p rocesses in

the neu rop i l (n) h a v e deve loped mic ro tubu le s and neurof i laments . Neu ro -

blas ts c loser to the pial surface appear m o r e mature than the sp ind le - shaped

F IGURE 4. A. A diagram of a transverse section of the midbrain, a parasagittal section of the brain, and of the stage of development of a 14-day fetus. The arrow points to the location of the cells in Figure 4B. Reproduced by permission of / . Comp. Neurol., 1973, 149: 233. B . A fluorescence micrograph showing some fluorescent cells (arrow) and processes in the mantle layer of the basal plate of the caudal mesencephalon of a 14-day fetus. See Figure 4A for orientation. X152. C. A diagram of a transverse section of the midbrain, an oblique parasagittal section of the brain, and of the stage of development of a 16-day fetus. The arrows indicate the location of the fluorescent zones in Figure 4D. The dotted line indicates the course of the fluorescent axons in Figure 6A. Reproduced by permission of /. Comp. Neurol., 1973, 149: 233. D. A fluorescent inverted V-shaped zone is apparent immediately ventral to the stratified ependymal zone (ep). Most of the fluores-cence appears as dots or linear processes, but occasionally fluorescent perikarya can be recog-nized. See Figure 4C transverse section, arrow, for orientation. 16-day fetus. X 1 5 5 . Reproduced by permission of / . Comp. Neurol., 1973, 149: 233.

F IGURE 5. A. A bipolar neuroblast courses ventrally from the ependymal zone. Scattered mi-tochondria (m), polyribosomes (r), and a few cisternae of granular endoplasmic reticulum (er) fill the cytoplasm and immature process (p) of the neuroblast. A Golgi complex (g) is present in the cytoplasm. The other processes in the neuropil (n), which are more mature, have microtubules and a few neurofilaments. 16-Day fetus. X 13,600. B . The globular neuroblasts in the ventral mantle layer are directly adjacent to one another. Their nuclei (N) are eccentric and indented toward the cell center which contains the Golgi complex (g). There is more granular endoplasmic reticulum (er) than previously. The process (p) contains neurofilaments and microtubules. 16-Day fetus. X 13,600. C. A vesicle containing a dense core (arrow) is present close to the Golgi complex. 16-Day fetus. x ! 4 , 8 0 0 .


F IGURE 6. A. A montage of the mesencephalon and diencephalon in the 16-day fetus showing

the presumptive nigroneostriatal pathway. (See Figure 4C for orientation.) This parasagittal sec-

tion was cut obliquely. The cells and processes in the caudal midbrain (arrow head) are close to

midline, but the fibers in the rostral midbrain (arrow) are somewhat lateral. Rostrally the fibers


cells. T h e y are g lobular neurob las t s and are f requent ly direct ly adjacent to one

ano ther and form small g roups (Figure 5 B ) . T h e nuc leus (N) is located eccen-

trically, and it i s i n d e n t e d toward the cell center . T h e Golg i complex (g) is

more deve loped and occup ie s the cell center . Occas iona l ly , a ves ic le w i th a

central dens i ty (Figure 5 C , arrow) is p resen t nea r the Golg i complex . C h a n n e l s

of the granular endop la smic re t i cu lum (Figure 5 B , er) are m o r e n u m e r o u s , b u t

they do not form Niss l b o d i e s as yet . T h e p rocesses (p) o f these cells have

mic ro tubu le s , neurof i laments , and agranular re t i cu lum. Smal l d i ame te r axons

fill mos t of the neurop i l su r round ing the neurob las t s , bu t s o m e larger i m m a -

ture p rocesses are found as well . Wel l -def ined synapt ic j u n c t i o n s have no t

b e e n seen at th i s t ime . Large granular ves ic les are found in s o m e of the

p rocesses in the neurop i l . T h e s e granular ves ic les are too sparse to account for

the large n u m b e r of f luorescent profiles seen in t he inver ted V - s h a p e d zone

(Figure 4 D ) . A l though it is not poss ib l e in e lectron mic rog raphs such as these

to ident i fy any par t icular cell or p rocess as d o p a m i n e - c o n t a i n i n g , a very large

major i ty of the profiles in the l imi ted area occup i ed b y the inver ted V - s h a p e d

zone (Figure 4 D ) are f luorescent b y the Falck-Hil larp t e c h n i q u e . It is h igh ly

p robab le , therefore , that the e lectron mic rog raphs p re sen ted he re i l lustrate the

charac ter is t ics o f d o p a m i n e - c o n t a i n i n g s t ructures . O u r resul ts s h o w that ca te-

c h o l a m i n e - s y n t h e s i z i n g e n z y m e s m u s t b e p resen t in re la t ively undif feren-

t ia ted cel ls , s ince these cells f luoresce.

D u r i n g th i s early pe r iod of fetal l ife, i . e . , day 16 of ges ta t ion , a f luorescent

n igroneos t r ia ta l p a t h w a y (Figure 6A) ex tends from the f luorescent cell b o d i e s

in the midven t ra l prol i ferat ion (arrow head) and runs m o r e laterally in the ros-

tral m i d b r a i n and th rough the lateral h y p o t h a l a m u s toward the tha l amohy-

po tha lamic j u n c t i o n (crossed ar row) . S o m e of these axons are f ibers from the

m e s e n c e p h a l o n to the l i m b i c sys t em, s ince f luorescent axons of neu rob la s t s in

the area vent ra l i s t egmen t i run w i t h the nigros t r ia ta l f ibers to the nuc leus ac-

c u m b e n s sept i and o the r areas of the sep tum. T h e p a t h w a y has g r o w n as far as

the j u n c t i o n of the d i encepha lon w i t h the t e l encepha lon at day 16 of ges ta t ion ,

bu t has no t r eached the neos t r i a tum at th is t ime . It i s in te res t ing to specula te

on the role that d o p a m i n e is p lay ing in these neurob las t s and the i r g rowing

run in the lateral diencephalon. Some course toward the ventral diencephalon (*), others (crossed arrow) are directed toward the telencephalon. The axons are spaced apart and have rather smooth contours, although some varicosities are seen. x260 . Reproduced by permission of / . Comp. Neurol., 1973, 149: 233. B. A transverse section through the rostral portion of the mesencephalon. There is very little fluorescence in the midline (*), but lateral from the midline fluorescent cell bodies (arrow) can be seen. There are many fluorescent axons laterally (crossed arrow), but they are less apparent in this cross section than in longitudinal sections (see Figure 6C). 18-Day fetus. X140. C. A parasagittal section showing the large number of fluorescent axons (arrow) emerging from the cells in the midbrain and coursing rostrally during day 18 of gestation. X140. D. A cross section of the putamen showing the fluorescent axons which have entered at day 18 of gestation. Some rather large profiles (arrow) are present laterally. The caudate nucleus lacks fluorescence. X140. E. A sagittal section of the putamen which gives a longitudinal view of the very thin fluorescent axons and their minute varicosities (arrow). 18-Day fetus. X140.


axons , s ince they have not yet e s t ab l i shed thei r u l t imate connec t i ons . Is it pos -

s ib le that d o p a m i n e acts as a chemotac t i c agen t se rv ing to es tab l i sh c o m m u n i -

ca t ion w i t h distal ly p laced cells? For ins t ance , d o p a m i n e m i g h t b e re leased

in to the neurop i l , w h e r e it can s ignal the d o p a m i n e - s e n s i t i v e neurob las t s

in the neos t r i a tum to r e s p o n d w i t h the re lease of an appropr ia te subs t ance .

T h e latter subs t ance , in turn, m i g h t form a diffusion gradient to attract the

dopamine rg i c axons . T h e re lease of d o p a m i n e from these relat ively undif-

ferent ia ted neurob las t s , therefore , m a y serve as an impor tan t l ink in a feed-

b a c k m e c h a n i s m w h i c h directs the ca techo laminerg ic axons to the i r target in

the neos t r i a tum.

T h e f luorescent anlage ex tends rostrally to inc lude the uppe r m i d b r a i n b y

days 17 and 18 of ges ta t ion . B y this t ime , the f luorescent cells have migra ted

laterally from the m i d l i n e (Figure 6 B , *) and the i r cell b o d i e s are m o r e discrete

(arrow). T h e i r f luorescent axons (Figure 6 C , arrow) run parallel to the pial sur-

face in the m i d b r a i n and course th rough the hypo tha l amus to en te r the pu-

t amen at day 18 of ges ta t ion (Figures 6D and E , ar rows) . T h e neurob las t s con-

t inue to migra te laterally and b y day 20 of ges ta t ion the mature topography is

e s t ab l i shed , i . e . , a f luorescent area vent ra l i s t egmen t i (Figure 7 A , *) l ies

media l to the subs tan t i a n igra , w h i c h cons is t s of a th in semic i rcu lar pars c o m -

pacta (arrow) dorsal to a nonf luorescen t pars ret iculata (r). F luorescen t axons

from the m i d b r a i n can b e t raced to the tube rcu lum olfactor ium, p u t a m e n ,

nuc leus a c c u m b e n s , and sep tum, bu t the caudate nuc leus still lacks f luores-

cence at th is t ime .

A s econd w a v e of mig ra t i on of neurob las t s takes p lace in the subs tan t i a

n igra at day 23 of ges ta t ion (Figure 7 B ) . T h e cells s e e m to b e concen t ra ted in a

f luorescent w e d g e - s h a p e d zone ( inset , ar row) in the rostral por t ion of the

midb ra in . Med ia l ly on ly w i s p y fluorescent profiles (crossed arrow) ind ica te

the si te o f t he se cells w h i c h appear as i m m a t u r e as those at earl ier s tages of

deve lopmen t in the caudal r eg ion of the m i d b r a i n (Figure 4 D ) . T h o s e cells

close to the j unc t i on wi th the zona ret iculata (Figure 7 B , r ) , h o w e v e r , are m o r e

mature (arrow). A th in r im of fluorescent cy top lasm sur rounds the nuc leus

(Figure 7 C , arrow) and shor t p rocesses can b e d i s t ingu i shed . B y day 27 of ges -

ta t ion, the pars compac t a is a fairly compac t semic i rcu lar zone of fluorescent

neu rons (Figure 7 D , ar row) dorsal to the z o n a ret iculata (r). T h e y have a

greater vo lume of cy top lasm than prev ious ly and are m o r e ma tu re . T h e

neu rons of the subs tan t i a n igra , pars compac ta , appear to reach the i r final

deve lopmen t in the early postnata l pe r iod . B y the n in th postnata l day (Figure

7E) , the fluorescent n e u r o n s are as large and as f luorescent as in the adult

(compare F igure 7E w i t h F igure 1 A ) . T h e per ikarya of neona ta l n igra l neu rons

(Figure 7 E , arrow) are ovo id or angular , and e longa ted fluorescent p rocesses

can f requent ly b e seen . T h e nuc leus m a y b e e i ther central ly located or eccen-

tric.

Elec t ron mic roscop ic e x a m i n a t i o n o f the cells o f the subs tan t i a n igra from

days 18 to 20 of ges ta t ion s h o w s that m a n y of t h e m have ma tu red in to mul -

F IGURE 7. A . The fluorescent area ventralis tegmenti (*) is separated laterally from the substantia nigra, which now has a fluorescent pars compacta (arrow) distinct from the nonfluorescent pars reticulata (r). 20-Day fetus. Montage. X 1 2 5 . B . A wedge-shaped zone of fluorescent neurons is present in the rostral midbrain at day 23 of gestation. The outlines of cell bodies (arrow) close to the zona reticulata (r) are easier to distinguish than those located more medially (crossed arrow). X 1 2 5 . (Inset) A diagram of the location of the wedge-shaped zone of migrating cells (arrow). C. The fluorescent cell bodies and proximal processes (arrow) of the more developed cells adjacent to the zona reticulata. 23-Day fetus. X 3 0 0 . D. The pars compacta forms a semicircular zone of rela-tively mature fluorescent cells (arrow) dorsal to the zona reticulata (r) at day 27 of gestation. X 1 2 5 . E . A large number of the fluorescent neurons of the pars compacta (arrow) in the 9-day postnatal rabbit appear to be as large and as mature as those of the adult. X 3 2 5 .

Virginia M. Tennyson et al.

F IGURE 8 . A. An electron micrograph showing a filopodial process which contains a few fila-

ments (f), microtubules, and scattered vesicles (v) in a filamentous matrix. It is apposed to a distal

portion of a perikaryon (N). Mitochondria (m), polyribosomes, and endoplasmic reticulum (er) are

242


t ipolar n e u r o n s . T h e cell b o d i e s are en la rged , e longa ted , and taper in to b road

dendr i t ic p roces ses , some of w h i c h end in g rowth cones . All of the usual

organel les have inc reased great ly in n u m b e r . S o m e neu rons have small col lec-

t ions of granular endop l a smic re t icu lum fo rming small Niss l b o d i e s , o thers

have m o r e r andomly d i sposed endop la smic re t i cu lum (Figure 8 A , er) . T h e

fi lopodia of g rowth c o n e s con t a in ing finely f i lamentous mat r ix mater ia l (f) are

n u m e r o u s in the neuropi l . T h e f i lamentous mater ia l p r o b a b l y cons i s t s of

microf i laments , w h i c h m a y b e r e spons ib l e for the mot i l i ty of g rowth cones

(10 ,96) . It i s uncer ta in w h e t h e r the f i lopodium in F igure 8 A is axonal or

dendr i t ic , s ince b o t h types con ta in f i lamentous mater ia l and few organel les

unt i l t hey b e c o m e m o r e ma tu re (89) . A few axodendr i t i c j u n c t i o n s (Figure 8B)

are p resen t in the neurop i l , bu t they are still sparse . A x o s o m a t i c j u n c t i o n s

have also b e e n seen , bu t t hey are even rarer at th is s tage than the o ther

synapses (88) . M a n y of the t e rmina ls appear to b e i m m a t u r e , s ince t hey are

s o m e w h a t b u l b o u s , have few ves ic les , and still con ta in the f i lamentous ma te -

rial s een in the g rowth cone . A l though n o stat is t ical ana lys i s has b e e n done ,

w e s e e m to find a symmet r i ca l j u n c t i o n s (Figure 8 B , ar row) s o m e w h a t m o r e

f requent ly than symmet r i ca l j u n c t i o n s dur ing th is deve lopmen ta l pe r iod . T h e

clear ves ic les in the axonal b o u t o n s m a y b e small or s l ight ly larger , and the i r

shape m a y b e spher ica l , ovo id , or f lat tened. A few large dense core ves ic les

are found (crossed ar row) .

T h e n e u r o n s at day 25 of ges ta t ion are larger than those o b s e r v e d pre-

v ious ly , and they usual ly exh ib i t Ni s s l b o d i e s (Figure 8 C , er) . T h e Go lg i

complex (g) is wel l deve loped and the o ther organel les have inc reased in

n u m b e r cons i s t en t w i th the inc reased s ize of the cell . Synap t i c t e rmina ls are

more n u m e r o u s , bu t they are sparse compared to the ma tu re s tage. S o m e are

still i m m a t u r e in appea rance , w h e r e a s o thers r e s e m b l e ma tu re t e rmina ls . M o s t

of the types of synapt ic te rmina ls seen in the adult are p resen t b y the late fetal

s tage.

E x a m i n a t i o n of the neu rons of the subs tan t i a n ig ra in the early postnata l

pe r iod b y phase m i c r o s c o p y reveals few de tec tab le differences in the i r mor -

pho logy , as c o m p a r e d to adult neu rons . V e r y dense cy top lasmic granules are

more o b v i o u s in adult neu rons and the i r dendr i tes can often b e fo l lowed for

seen in the neuron. 20-Day fetus. X20,000. Reproduced by permission of / . Comp. Neurol, 1973, 149: 233. B . Two axonal boutons, containing rounded and ovoid vesicles, synapse with an imma-ture dendrite. One junction is cut obliquely but the other is clearly asymmetrical (arrow). A large dense core granule (crossed arrow) is present in one bouton. The dendrite has a few ribosomes, agranular reticulum, and a filamentous matrix. 20-Day fetus. X36,000. Reproduced by permission of / . Comp. Neurol, 1973, 149: 233. C. A young neuron of the substantia nigra has an eccentric nucleus (N), clumps of endoplasmic reticulum (er), a well-developed Golgi complex (g), mi-tochondria, and dense bodies. Two axonal boutons make symmetrical junctional contacts with the basal portion of a process (arrow) as it emerges from the perikaryon. Since the glia are poorly developed at time, axonal processes (ax) are directly apposed to the surface of the perikaryon. 25-Day fetus, x 16,200.

F IGURE 9 . A . A high magnification fluorescence micrograph of the putamen close to the anterior

commissure reveals punctate fluorescence (arrow) and large irregular fluorescent profiles (crossed

arrow). 20 -Day fetus. X365. B . Moderately large (arrow) and small (crossed arrow) fluorescent dots

and interconnecting linear strands are numerous in the midportion of the putamen. 2 2 - D a y fetus.

244

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longer d i s tances . A l so , scat tered mye l ina t ed fibers are more f requent ly found

in the adult neurop i l of the pars compac ta . Ultrastructural ly , the e l emen t s of

the neurop i l are no t as wel l deve loped in the pos tna ta l pe r iod as in the adult ,

bu t the n u m b e r a n d d i s t r ibu t ion of such organel les as the e n d o p l a s m i c re-

t icu lum, Go lg i c o m p l e x , and m i t o c h o n d r i a in neurona l per ikarya appear to b e

s imilar . In older adult r abb i t s , the l y s o s o m e s are s o m e w h a t larger and m o r e

complex than in the postnata l pe r iod .

D e v e l o p m e n t o f F luo re scen t A x o n s in the N e o s t r i a t u m

T h e neurop i l o f the neos t r i a tum in the adult (Figure I B ) i s so in t ense ly and

diffusely f luorescent that it is usual ly not p o s s i b l e to see ind iv idua l axons . At

the early s tages , h o w e v e r , there are so few te rmina ls that t hey are readi ly dis-

t i ngu i shab le f rom the nonf luorescen t neurop i l . T h e f luorescent d o p a m i n e - c o n -

ta in ing axons w h i c h first g row in to the p u t a m e n appear as dots (var icos i t ies)

in cross sec t ion (F igure 6 D , arrow) and as l inear profiles c o n n e c t i n g t iny

var icos i t i es in long i tud ina l sec t ion (Figure 6 E , a r row) . S u b s e q u e n t l y , the

f luorescent profiles b e c o m e large, i r regularly s h a p e d s t ructures (F igure 9 A ,

c rossed a r row) , and t hen w i t h inc reas ing matura t ion , they appear as small

va r icos i t i e s l i nked b y curvi l inear s t rands (Figure 9 B , c rossed ar row) . F ina l ly

the va r icos i t i e s b e c o m e less ev iden t and a curv i l inear h o n e y c o m b pat tern

r ema ins (Figure 9 C , a r row) . W e have a t t empted to correlate the c h a n g i n g

f luorescent i m a g e s w i t h the types of axonal profiles seen in the neurop i l of lit-

termate fe tuses o f the s a m e stage b y e lec t ron mic roscopy : (1) L o n g l inear

profiles s een at the ear l ies t s tage (F igure 6 E , ar row) m a y b e axonal g rowth

cones (Figure 1 0 A ) , w h i c h have f ingerl ike t ips filled w i t h microf i laments (f).

T h i s sugges t s that d o p a m i n e is in the g r o w i n g axonal t ip as it p roceeds

th rough the neuropi l . (2) Large irregularly s h a p e d f luorescent profiles (F igure

9 A , c rossed arrow) m i g h t b e the ex t remely large ves i c l e -con ta in ing axonal b u l b s ,

w h i c h still con ta in the microf i laments character is t ic o f g rowth c o n e s (Figure

10B) . S i n c e w e h a d no t s een th is type of b o u t o n in the adult , w e a s s u m e d that

th i s t e rmina l h a d jus t recent ly m a d e contac t w i t h the dendr i te . I ts b u l b o u s

contours (poss ib ly d a m m e d up axoplasm) w o u l d have b e e n r emode l ed w i t h

matura t ion . (3) Smal l f luorescent var icos i t i es c o n n e c t e d b y curvi l inear profiles

(Figure 9 B , c rossed ar row) could b e exp la ined b y the matura t ion of the large

axonal b u l b s to the very small var icos i t ies and axons w i t h synapses " e n pas -

X365. C. Curvilinear fluorescence in a honeycomb pattern (arrow) is present in the putamen of the 25-day gestation fetus. X365. D. Montage of the neostriatum at day 28 of gestation. The epen-dyma (ep) and subependymal region lack fluorescence, as does the internal capsule (ic). Punctate fluorescent dots are scattered throughout the neuropil of the neostriatum. Fluorescent weblike islands of various size and shape are found in a patchy distribution in the caudate nucleus (arrow) and adjacent to the internal capsule in both caudate and putamen (crossed arrow). The choroid plexus (cp) has a few autofluorescent granules. X150 .

F IGURE 10. A . A growing tip of an axon from the putamen of a 19-day fetus. Microtubules and a collection of vesicles (arrow) are present along the axon proper. The filopodium (f), which has a filamentous ground substance and a few vesicles, is forked. X33,000. Reproduced by permission of Brain Res., 1972, 46: 251. B . A bulbous process, which has a large collection of spheroidal and ellip-soidal vesicles (v) in a filamentous ground substance, exhibits an immature symmetrical junction

246


s a g e " (F igures 1 0 C - E ) that are typical o f the next s tage . (4) In t e rconnec ted

curv i l inear f luorescent profiles (Figure 9C) could b e due to a r ed i s t r ibu t ion of

d o p a m i n e - c o n t a i n i n g ves ic les or a n e w popula t ion of ves ic les f lowing un i -

formly d o w n the axon from the cell b o d y . V e r y th in axons fo rming synapses

" e n p a s s a g e " and h a v i n g ves ic les a long the axon proper (Figures 10F and G)

are c o m m o n at th i s and later s tages . T h e ultrastructural loca l iza t ion of dopa-

m i n e w i t h i n neos t r ia ta l axons has not b e e n comple te ly e s t ab l i shed , never the -

less , there is ev idence that some of the d o p a m i n e m a y b e s tored in the clear

ves ic les (see later sec t ion ent i t led Uptake S tud ies in T i s s u e S l i ces ) .

T h e deve lopmen t of f luorescence does no t occur un i formly in the n e o -

s t r ia tum. A s m e n t i o n e d , the p u t a m e n rece ives the first d o p a m i n e - c o n t a i n i n g

axons at abou t day 18 of ges ta t ion from the caudal por t ion of the midb ra in .

T h e caudate nuc l eus , on the o the r h a n d , does no t s h o w a c o n s p i c u o u s a m o u n t

of f luorescence unt i l days 24 or 25 of ges ta t ion . It is pos s ib l e that s o m e of the

later ma tu r ing neurob las t s in the rostral m i d b r a i n (Figure 7B) m a y b e con-

t r ibu t ing s o m e fibers to the caudate nuc leus . A l though there are t iny f luores-

cent t e rmina ls th roughou t b o t h the caudate nuc leus and p u t a m e n at day 28 of

ges ta t ion , i r regular i s lands of f luorescence (Figure 9 D , ar row, c rossed arrow)

are p resen t in b o t h nucle i . T h e f luorescent i s lands are m o s t cons i s t en t ly found

adjacent to the in terna l capsule (ic) in the areas med ia l and lateral to the cen -

tral por t ion of the pu t amen . W e have e x a m i n e d co r re spond ing areas in l i t ter-

mate e m b r y o s b y e lectron m i c r o s c o p y in an a t tempt to ident i fy the na ture of

the f luorescent i s lands . T h e s e areas con ta ined unusua l ly large n u m b e r s of

g rowth c o n e s , and w e conc luded that these we re the s i tes of i ng rowth of n e w

d o p a m i n e - c o n t a i n i n g axons . R a n d o m e x a m i n a t i o n of the r e m a i n i n g neurop i l

revealed the m o r e mature forms of axons and te rmina ls de sc r ibed in the pre-

ced ing s tage .

T h e i r regular i s lands of f luorescence are s een in the ear ly pos tna ta l pe r iod ,

bu t in add i t ion , ano the r type of f luorescent i s land is p resen t w h i c h is cir-

c u m s c r i b e d b y a nonf luorescen t zone (Figure 11 A , a r rows) . W h e n e x a m i n e d in

dark field u s ing w h i t e l ight , the nonf luorescent zone con ta ins the cell b o d i e s

of neu rons . T h e p re sence of these i s lands sugges t s the poss ib i l i t y o f an ana-

tomical and funct ional o rgan iza t ion w i t h i n the caudate nuc leus no t revealed

(arrow) with only a few associated vesicles. Putamen, 19-day fetus. X42,000. Reproduced by per-mission of Brain Res., 1972, 46: 251. C. Elongated processes containing filamentous ground sub-stance and scattered vesicles (v) are present in the 22-day gestation fetus. It is possible that the density (arrow) is an oblique section through an early junctional contact. X50,000. D. A small ter-minal with clear spherical vesicles makes an asymmetrical synapse (arrow) "en passage" with a dendrite. 22-Day fetus. X36,000. E. A terminal with spherical and ovoid clear vesicles makes a symmetrical synapse (arrow) with a dendrite. 22-Day fetus. x35,200. F. An elongated axonal process with ovoid clear vesicles forms a symmetrical junction (arrow) "en passage" with a dendritic spine. Another varicosity of this axon has clear vesicles and a large dense core vesicle (crossed arrow). 25-Day fetus. X46,000. G. A thin axonal process with clear ovoid vesicles makes asymmetrical junctions (arrows) with two dendrites. 25-Day fetus. X48,000.


F IGURE 11. A. Scattered punctate fluorescence fills the neuropil of the caudate nucleus below the subependymal zone (ep) at the tenth postnatal day. Fluorescent islands in the caudate nucleus show fine punctate and linear fluorescence surrounded by a nonfluorescent zone (arrows). X145. B. Small discrete fluorescent dots (arrow) are evident. Irregular twiglike profiles of a lower inten-sity fluorescence are numerous. Caudate nucleus, postnatal day 9. X420. C. Punctate dots and extremely thin linear fluorescence (arrow) are present in the putamen at the ninth postnatal day. X365. D. The neuropil of the caudate nucleus beneath the narrow nonfluorescent subependymal zone appears intensely and homogeneously fluorescent at the twenty-fifth postnatal day. The cell bodies (arrow) are rather closely packed. X145.

<§>


w i th the usual h i s to logica l p repara t ions , no r b y h i s to f luorescence in the m a -

ture s tage, w h e n the ent i re neurop i l has b e c o m e filled w i th f luorescent axons .

T h e c i r c u m s c r i b e d i s lands , w h i c h appear to b e a cluster o f n e u r o n s enc los ing a

core of d o p a m i n e - c o n t a i n i n g axons , create a g lomerular - l ike pat tern in w h i c h a

local ized axonal inpu t could effect a synch ronous modu la t ion on the dendr i tes

of the c luster of neu rons . W e have not ident i f ied these i s lands b y e lect ron

mic ro scopy as yet , bu t w e h o p e to do so , s ince they m a y b e of in teres t to

neu rophys io log i s t s .

T h e r ema inde r of the neuropi l of the caudate nuc leus is filled w i t h t iny

f luorescent var icos i t i es (Figure 1 1 B , a r row) , w h i c h are part icular ly n u m e r o u s

b e n e a t h the s u b e p e n d y m a l reg ion (Figure 11 A ) . F luorescen t var icos i t i es are

p resen t in the p u t a m e n , as wel l , and the i r p r e sence a long the course of axons

(Figure H C , arrow) is addi t ional ev idence that these d o p a m i n e - c o n t a i n i n g

axons form synapses " e n p a s s a g e . " Thereaf ter , there is a rap id ing rowth of

d o p a m i n e - c o n t a i n i n g te rmina ls and from days 16 to 25 of pos tna ta l life the en -

tire neos t r i a tum b e c o m e s diffusely and in tense ly f luorescent (Figure 1 1 D ) . It

wou ld b e difficult to r ecogn ize th is s p e c i m e n from o n e o f an adult an imal . T h e

major i ty of the synapses in the neurop i l appear mature ul trastructural ly, ex-

cept for the fact that the nonf luorescen t cell b o d i e s are c loser toge ther in the

25-day-o ld rabb i t .

B i o c h e m i c a l S t u d i e s

T h e e n d o g e n o u s levels of d o p a m i n e presen t in the caudate nuc leus and

p u t a m e n (Figure 12) were correla ted w i t h the f luorescence mic roscop ic

s tudies . D o p a m i n e w a s assayed b y the m e t h o d of A n t o n and Sayre (6) . A s ex-

pec ted , e n d o g e n o u s d o p a m i n e concen t ra t ions are ve ry low in the early fetal

pe r iod (days 20 to 22) and increase gradual ly dur ing the prenata l and early

pos tnata l pe r iod . B y the twenty-fif th pos tnata l day, h o w e v e r , w h e n the cau-

date nuc leus appears to b e as f luorescent as that of the adult w h e n e x a m i n e d

microscopica l ly , the e n d o g e n o u s d o p a m i n e concen t ra t ions are on ly abou t half

that found in the adult. T h i s p h e n o m e n o n of a concen t r a t i on -dependen t

q u e n c h i n g has b e e n s tudied b y J o n s s o n (52) , w h o s h o w e d that above a cer ta in

level the eye canno t apprec ia te a further r ise in f luorescence in tens i ty . O u r

b i o c h e m i c a l s tud ies ind ica te that the neos t r i a tum of the r abb i t does no t reach

adult levels even b y the forty-fifth pos tnata l day, a l though the neurop i l is in-

tense ly f luorescent .

S tud ies have also b e e n done on the relat ive uptake and accumula t ion of 3 H -

d o p a m i n e b y fresh t i s sue sl ices of the neos t r i a tum in an effort to assess the

degree of matur i ty of the d o p a m i n e - c o n t a i n i n g axons at each s tage of deve lop-

m e n t (Figure 13) . T h e uptake of 3 H - d o p a m i n e w a s m e a s u r e d b y a modif ica t ion

of the m e t h o d of S h a s k a n and S n y d e r (80) . T h e accumula t ion of 3 H - d o p a m i n e

from days 19 to 28 of ges ta t ion r ises to a va lue of 20 ( t i s sue - to -med ium rat io) ,


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F IGURE 12. Endogenous dopamine levels in the rabbit caudate nucleus and putamen. Repro-duced by permission of Brain Res., 1972, 46: 251.

0 15 0 15 3 0 4 5

PRENATAL POSTNATAL

DAYS F IGURE 13. The uptake of 3 H-dopamine into tissue slices of rabbit neostriatum. The data are expressed as the 3 H-dopamine (disintegrations/minute, dpm) per gram of tissue divided by the 3 H-dopamine (dpm) per ml of medium (tissue to medium ratio, T/M) after 15 min at 37°C. A T/M greater than 1.0 is generally considered to reflect an active uptake process. Reproduced by permis-sion of Brain Res. 1972, 46: 251.

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t hen there i s a rap id inc rease shor t ly after b i r th . B y the twenty-fif th pos tna ta l

day, the up take and accumula t ion of d o p a m i n e in the neos t r i a tum approach

that found i n the adult . A s m e n t i o n e d , th i s is long before e n d o g e n o u s dopa-

m i n e levels approach m a x i m u m values . T h e uptake of 3 H - d o p a m i n e , there-

fore, ach ieves adult va lues m u c h earl ier than the e n d o g e n o u s d o p a m i n e con-

tent. T h e deve lopmen t of the up take m e c h a n i s m in the neos t r i a tum correlates

wel l w i th the morpho log ica l s igns of synapt ic t e rmina l ma tu ra t ion b y e lect ron

mic roscopy .

STORAGE SITES OF DOPAMINE

Neuronal Cell Body

It is wel l e s t ab l i shed b y f luorescence m i c r o s c o p y that d o p a m i n e is p resen t

in neurona l per ikarya of the subs tan t i a n ig ra compac ta , bu t the local iza t ion of

the a m i n e at the ultrastructural level is a cont rovers ia l i s sue . V a n O r d e n et al.

(93) have d i scussed the ev idence that n o r e p i n e p h r i n e in the per iphera l sympa-

thet ic ne rvous sys t em is correla ted w i th small dense core ves ic les , w h i c h were

first de sc r ibed b y R icha rdson (77) and Gri l lo and Palay (41) . S imi l a r ca te-

c h o l a m i n e s torage ves ic les have b e e n sought in the subs tan t i a n igra . L e n n

(61) failed to find a s ignif icant n u m b e r of granular or agranular ves ic les in cate-

c h o l a m i n e n e u r o n s to expla in the h y p o t h e s i s o f a per ikaryal ves icu la r pool

of m o n o a m i n e s . Fuxe et al. (36) conc luded that a ves icu la r pool o f m o n o a m i n e s

ex is t s , b u t that it is r ep resen ted b y agranular ves ic les found in the per inuc lear

area of the n e u r o n nea r the Golg i complex . B a k (7) , on the o the r h a n d , as-

s u m e d that d o p a m i n e w a s present in large dense core granules in the per i -

karya and in axonal b o u t o n s , because he found a decrease in these granules

after t r ea tment o f m i c e w i th reserp ine , w h i c h deple tes ca t echo lamines and

i n d o l a m i n e s . It shou ld b e p o i n t e d out that it is a fo rmidable task to d o c u m e n t

such a decrease in granules w i thou t ques t i on b y electron m i c r o s c o p y , b e c a u s e

the granules are relat ively sparse in the cell b o d i e s , even in un t rea ted an imals .

Gul ley and W o o d (44) , moreove r , no t ed that there are fewer dense core ves -

icles in the per ikarya o f the z o n a compac t a ( the f luorescent n e u r o n s ) than in

the cell b o d i e s of the zona ret iculata (most ly nonf luorescen t neu rons ) . Gro -

fova and R i n v i k (42) a t t empted a sys temat ic quan t i t a t ive ana lys is o f the ef-

fects of r e se rp ine t rea tment on the axonal b o u t o n s in the neurop i l of the s u b -

stant ia n igra ret iculata . T h e clear and dense core ves ic les w e r e coun ted in 100

b o u t o n s from 16 cats . T h e y repor ted a s ignif icant reduc t ion of dense core

ves ic les c o m p a r e d to no rma l an imals from 5 to 49 hours after r e se rp ine treat-

men t , w i t h a m i n i m u m at 24 hours . A s d i scussed later, it is pos s ib l e that the

dense cores deple ted in the i r s tudy are the s torage s i tes of se ro ton in .

Elec t ron mic roscop ic au torad iographic s tud ies after the in t ravent r icular in -


j e c t ion of ca t echo lamines have b e e n d o n e in an a t tempt to e lucidate the

p rob lem. Si lver gra ins due to up take of t r i t ia ted n o r e p i n e p h r i n e w e r e u b i q u i -

tously d i s t r ibu ted th roughou t the neurona l per ikarya and dendr i t es in the

subs tan t ia n igra (24 ,72 ,81 ) . Descar r i es and Droz (24) sugges ted that exogenous

ca t echo lamines are b o u n d to a macromolecu la r complex in the per ikarya ra ther

than to preformed synapt ic - l ike ves ic les . So te lo (81) suppor ted th is h y p o t h e s i s

and s h o w e d that the large d e n s e core ves ic les in nigral n e u r o n s have a h i g h

ac id phospha ta se act ivi ty , and thus , those nea r the Golg i complex are related

to p r imary l y sosomes , no t to s torage s i tes of m o n o a m i n e s . T h e axonal b o u t o n s

in the neurop i l o f the subs tan t i a n ig ra s e e m to have less affinity for exogenous

ca techo lamines than the neurona l per ikarya . In two s tud ies , in w h i c h 3 H -

n o r e p i n e p h r i n e w a s used , there was no concen t ra t ion of labe l over axonal

b o u t o n s (24 ,81 ) , bu t in ano the r inves t iga t ion , s o m e 3 H - d o p a m i n e - and 3 H -

norep inephr ine - l abe l ed nerve te rmina ls we re obse rved in axodendr i t i c syn-

apses (72) . M o r e consp i cuous , howeve r , w a s the labe l ing of axodendr i t i c ter-

mina l s in the subs tan t i a n ig ra after in jec t ions o f rad ioac t ive se ro ton in and its

precursors (72) . S i lver gra ins we re found over b o u t o n s con ta in ing clear v e s -

icles and large dense core ves ic les . It is pos s ib l e that the large dense core

granules in axonal b o u t o n s that w e r e dep le ted b y rese rp ine (7 ,42) m a y b e

storage s i tes of se ro ton in ra ther than d o p a m i n e . T h i s wou ld b e in accord wi th

f luorescence mic roscop ic f indings (33) that the z o n a compac t a o f the s u b -

s tant ia n ig ra h a s a ve ry sparse popu la t ion of ca t echo lamine te rmina l s , bu t

there is a m a s s of dense ly packed , very fine se ro ton in te rmina ls in the zona re-

t iculata.

A x o n s in the Neos t r i a tum

T h e neurop i l of the neos t r i a tum con ta ins a w i d e var ie ty of t e rmina ls from

var ious sources . A l though a t tempts have b e e n m a d e to ident i fy the d o p a m i n e -

con ta in ing axons , the i r character is t ics have no t b e e n fully demons t ra ted wi th

cer ta in ty b y e lectron m i c r o s c o p y as yet . O u r f luorescence mic roscopy s tudies

of the deve lop ing neos t r i a tum lead us to conc lude that the mature d o p a m i n e -

con ta in ing axon is th in and has small var icos i t i es w h i c h form synapses " e n

p a s s a g e . " After a l es ion in the m i d b r a i n , K e m p and Powel l (57) i l lustrated

degenera t ing axons forming synapses " e n p a s s a g e , " bu t th is type of t e rmina l

o r ig ina tes in o the r par ts o f the b ra in as wel l . All o f the junc t iona l contac ts of

degenera t ing extr ins ic te rmina ls in the caudate nuc leus w h i c h they o b s e r v e d

we re asymmetr ica l . In an au torad iographic s tudy, Hat tor i et al. (46) p re sen ted

o ther ev idence sugges t ing that n igros t r ia ta l t e rmina ls in the neos t r i a tum e n d

p redominan t ly in a symmet r i ca l synapses . Severa l days after the s tereotaxic in-

j ec t i on of 3 H - l e u c i n e in to the zona compac ta of the subs tan t i a n igra of the rat,

7 5 % of the s i lver gra ins i n the neos t r i a tum w e r e located over synapses w i t h

modera te ly p l eomorph ic ves ic les and asymmet r i ca l j u n c t i o n s , w h e r e a s only


2 5 % w e r e over t e rmina l s w i t h h igh ly p l eomorph i c ves ic les and symmet r i ca l

j u n c t i o n s . S ince in t raventr icular in jec t ion of 6 - h y d r o x y d o p a m i n e caused

degenera t ion of on ly the former type of ne rve e n d i n g , it w a s a s s u m e d that

dopamine rg i c n igros t r ia ta l b o u t o n s in the neos t r i a tum con ta in modera te ly

p l eomorph ic ves ic les and m a k e asymmet r ica l synapt ic contac ts (46) .

T h e ul trastructural local iza t ion in the neos t r i a tum of the va r ious puta t ive

neuro t ransmi t t e r s , d o p a m i n e , ace ty lchol ine , y - aminobu ty r i c ac id , se ro ton in ,

and n o r e p i n e p h r i n e , ha s no t b e e n e s t ab l i shed . B i o c h e m i c a l s tud ies have in -

d ica ted that at least a por t ion of the d o p a m i n e m a y b e s tored in ves ic les (94) .

A l though s o m e inves t iga tors have sugges ted that the large dense core granules

m a y b e d o p a m i n e s torage s i tes (7 ,66 ) , o thers ( 1 , 2 , 9 , 3 0 , 3 5 , 5 5 , 8 3 , 8 4 ) have

po in t ed out that the clear ves ic les are b y far the m o s t a b u n d a n t type of ves ic le

in the neos t r i a tum. T h e large dense core ves i c l e s , moreove r , can b e found in

pos t synap t ic dendr i t es (83) , w h i c h are no t though t to con ta in d o p a m i n e . T h e

dense core ves ic les r ema in in axons in the neurop i l (Figure 3B) after all of the

hodolog ica l c o n n e c t i o n s to the caudate nuc leus , inc lud ing nigros t r ia ta l f ibers ,

have b e e n sec t ioned and d o p a m i n e f luorescence is absen t (87) . Fur the r ev i -

dence w h i c h sugges t s that d o p a m i n e m a y b e p resen t in s o m e clear ves ic les is

the f inding that s i lver gra ins due to in t ravent r icular in jec t ions of labe led

n o r e p i n e p h r i n e are local ized over ne rve e n d i n g s c o n t a i n i n g pr imar i ly the

agranular type of ves ic le (3) .

Uptake Studies in Tissue Slices

T h e i n c u b a t i o n of fresh t i s sue in the p re sence o f " fa lse t r ansmi t t e r s , " such

as a - m e t h y l n o r e p i n e p h r i n e , 5 - h y d r o x y d o p a m i n e ( 5 - O H - D A ) , 6 - O H - D A , and

6 ,7 -d ihydroxy te t r ahydro i soqu ino l ine ( 6 , 7 - d i O H T I Q ) , forms dense cores i n v e s -

ic les of ca t echo lamine rg ic b o u t o n s of the per iphera l ne rvous s y s t e m (48 ,85 ,90 )

and central ne rvous sys t em (48 ,76) . S i n c e 6 - O H - D A resul ts in des t ruc t ion of

t e rmina ls after a pe r iod o f t i m e (91) , the i n c u b a t i o n pe r iod w i t h th i s s u b s t a n c e

should b e kept as shor t as poss ib l e . 6 , 7 - d i O H T I Q , w h i c h is a condensa t i on

product of d o p a m i n e wi th me thy l or e thyl a ldehyde , m a y b e re levant to the

b io log ica l effects of a lcohol i nges t ion ( 1 6 , 1 7 , 8 5 ) . T h e t e c h n i q u e of i n c u b a t i n g

fresh t i s sue in the p re sence of false t ransmi t te rs has also b e e n e m p l o y e d to

give addi t iona l data c o n c e r n i n g the morpho log ica l charac ter i s t ics o f the dop-

a m i n e - c o n t a i n i n g axons in the neos t r i a tum (48 ,50 ,84 ) . A l though there is con-

s iderable d a m a g e to m o s t of the b r a in t i s sue , the axonal b o u t o n s are relat ively

wel l p reserved . After i n c u b a t i o n , some b o u t o n s can b e v i sua l i zed o w i n g to

the uptake and s torage of the e x o g e n o u s t ransmi t te r b y small ves ic les . The re is

format ion of a dense prec ip i ta te in the ves ic les due to the reac t ion of N a M n 0 4

( the fixative) w i t h the t ransmi t te r (49) . T h i s i s a c o n v e n i e n t m e t h o d to " t a g "

the ves ic les for e lec t ron mic roscopy . T h e a m o u n t of reac t ion product in the

ves ic les , therefore , is d e p e n d e n t no t on ly on the abi l i ty o f the ves ic les to store


F IGURE 1 4 . A. No precipitate is present in the vesicles of the boutons after incubation in Krebs-

Ringer solution for 20 minutes. Adult rabbit putamen. X60,000 . B. A dense precipitate is present

in the vesicles (arrow) of some varicosities, but not in others after incubation in 5-OH-DA and

DMI for 20 minutes. A vesicle (v) containing dense material appears to be fusing with the surface

membrane. Adult rabbit putamen. X60 , 000 . C. A dense precipitate is present in the vesicles


the exogenous a m i n e , bu t also on the chemica l reac t ion b e t w e e n the a m i n e

and the pe rmangana t e . It shou ld b e p o i n t e d out that at the h i g h concen t ra -

t ions of false t ransmi t te rs n e e d e d to carry out these s tud ies (5 X 1 0 ~ 4 M ) ,

ves ic les w h i c h s tore n o r e p i n e p h r i n e or se ro ton in , a long wi th those w h i c h

s tore d o p a m i n e , m a y incorpora te the subs t ance and b e c o m e tagged . In s o m e

of our expe r imen t s , w e have inc luded d e m e t h y l i m i p r a m i n e (DMI) in the

m e d i u m w i t h 5 - O H - D A . At the concen t ra t ion used in th is s tudy ( 1 0 ~ 5 M ) ,

D M I is a po ten t i n h i b i t o r of n o r e p i n e p h r i n e uptake in to the hypo tha l amus

(51 ,80) and se ro ton in up take into the neos t r i a tum and hypo tha l amus (80) , bu t

is a very m u c h w e a k e r i n h i b i t o r of d o p a m i n e uptake in to the neos t r i a tum (51).

In our e xpe r imen t s on the neos t r i a tum, the addi t ion of 1 0 ~ 5 M D M I to the

m e d i u m w a s i n t e n d e d to b lock the accumula t ion of 5 - O H - D A in to nor-

e p i n e p h r i n e and se ro ton in te rmina ls , bu t no t in to d o p a m i n e - c o n t a i n i n g ter-

mina l s .

T h e con ten t s o f the small ves ic les o f all o f the axonal b o u t o n s in the n e o -

s t r ia tum of the r abb i t r e m a i n clear w h e n the t i ssue is i ncuba t ed in K r e b s - R i n g e r

solut ion (Figure 1 4 A ) , b u t a va r iab le n u m b e r of dense depos i t s form in ves ic les

of s o m e of the b o u t o n s w h e n the t i ssue is i n c u b a t e d in 5 - O H - D A (Figure 1 4 B ,

arrow) and 6 - O H - D A (Figure 1 4 C , ar row) . I ncuba t i on in 6 , 7 - d i O H T I Q (Fig-

ure 1 4 D , ar row) or d o p a m i n e (Figure 1 4 E , arrow) resul ts in less depos i t in the

ves ic les and fewer t agged b o u t o n s , w h i c h m a k e s ident i f ica t ion of s o m e ter-

mina l s uncer ta in . In the case of d o p a m i n e , it is poss ib l e that the reac t ion

wi th pe rmangana t e is a l imi t ing factor. In our expe r i ence , 5 - O H - D A is m o r e

sat isfactory for t agg ing the ca t echo l amine -con t a in ing var icos i t i es than the

o the r subs t ances m e n t i o n e d , b e c a u s e it s e e m s to tag a larger n u m b e r of

b o u t o n s and there is more prec ip i ta te in the ind iv idua l ves ic les .

W e have coun ted abou t 7800 var icos i t i es in the neos t r i a tum of the rabb i t

after i n c u b a t i o n in 5 - O H - D A (86) . T h e n u m b e r of b o u t o n s h a v i n g small

ves ic les w i t h dense precipi ta te var ied from 2 .9 to 1 4 . 6 % wi th a m e a n of 8 . 9 % .

(arrow) of some varicosities, but not others after incubation in 6-OH-DA for 10 minutes. Very

small clear vesicles (v) are aligned close to the presynaptic membrane of an asymmetrical axo-

spinous junction (crossed arrow). Adult rabbit putamen. X60 , 000 . D. A few dense particles are

found in the vesicles (arrow) of some boutons after incubation in 6,7-di-OHTIQ for 15 minutes.

Adult rabbit caudate nucleus, x60,000. E. A few particles are barely discernible in the vesicles

(arrow) of some boutons after incubation in dopamine for 20 minutes. The varicosity is closely ap-

posed to a dendrite with a very large mitochondrion (m) and makes a symmetrical contact (crossed

arrow) with a thin process, which runs out of the plane of section. Adult rabbit putamen.

X60,000. F. A varicosity having small vesicles with a dense precipitate (arrow) is closely apposed

to dendritic processes with large mitochondria (m) and makes a symmetrical contact (crossed

arrow) with one of the dendrites. Adult rabbit putamen, incubated in 5-OH-DA and DMI for 20

minutes. X28 , 200 . G. A long thin axon has vesicles with a dense precipitate after incubation in

5-OH-DA for 15 minutes. Adult rabbit caudate nucleus, x28 ,200 . H. Varicosities "en passage"

have vesicles with a dense precipitate after incubation in 5-OH-DA for 15 minutes. Adult rabbit

caudate nucleus. X28 ,200 .


Hokfel t (48) repor ted a range of 10 to 1 9 % wi th a m e a n of 1 6 . 4 % in over 2500

bou tons coun ted in the rat neos t r i a tum after i ncuba t i on in a -me thy l -

no rep ineph r ine . T h e s e values s e e m small cons ide r ing the in t ense d o p a m i n e

f luorescence obse rved in the neos t r i a tum, bu t are p robab ly r easonab le f igures,

s ince it i s k n o w n that the nigrost r ia ta l f ibers are the smal les t inpu t to the

neos t r i a tum (57) . W e have found also that the tagged b o u t o n s are scat tered

th roughout the sec t ion . T h i s is in k e e p i n g wi th the f indings of Carpen te r and

Pe te r (14) in w h i c h degenera t ing fibers impregna t ed wi th s i lver we re d is t r ib-

u ted fairly even ly over large areas of the neos t r i a tum.

T h e character is t ics of the tagged ves ic les and the i r b o u t o n s can b e apprec i -

ated readi ly b y c o m p a r i n g the tagged b o u t o n s in F igures 14B and 14C wi th ad-

j acen t non t agged b o u t o n s . T h e r e are fewer ves ic les in the tagged var icos i t i es ,

and they tend to b e s l ight ly larger than the smal les t ves ic les seen in non tagged

bou tons . T h e tagged ves ic les m a y b e p l eomorph ic and appear ovo id or

e longated (Figure 1 4 B , arrow) or they m a y b e m o r e spher ica l (F igure 14C ,

arrow). A l though the ves ic les m a y b e close to the surface m e m b r a n e (Figure

1 4 B , v ) , they are no t character is t ica l ly a l igned in rows aga ins t the p resynapt ic

m e m b r a n e oppos i t e a j unc t iona l contact , as are the very small ves ic les in F ig -

ure 14C (v). F igure 14B (v) sugges t s the poss ib i l i ty that the conten ts of ves ic les

of d o p a m i n e - c o n t a i n i n g te rminals m a y b e ex t ruded a long the ent i re surface of

the var icos i ty , ra ther than b e i n g res t r ic ted to a d iscre te po in t a long a j u n c -

t ional spec ia l iza t ion , as is a s s u m e d for m a n y synapses .

T h e tagged ves ic les are usual ly p resen t in small- to modera t e - s i zed var icos -

i t ies (F igures 1 4 B - F ) b u t t hey are also found a long the course o f ve ry th in

axons (Figure 1 4 G ) . S o m e o f the tagged var icos i t i es can b e ident i f ied as var icos-

i t ies " e n p a s s a g e " (Figure 1 4 H ) , as has b e e n no ted in o ther s tudies (48 ,84) .

Occas iona l ly tagged b o u t o n s are seen direct ly apposed to dendr i tes hav ing

large mi tochondr i a (Figure 1 4 E , 1 4 F , m ) and even m a k i n g a symmet r i ca l j u n c -

t ional contac t w i t h t h e m (Figure 1 4 F , c rossed arrow). S i n c e K e m p and Powel l

(55) ident i f ied these dendr i tes as o r ig ina t ing from the sparse popula t ion of

large neu rons w h i c h p r o b a b l y s e n d the i r axons to the subs tan t ia n igra , F igure

14F m a y b e morpho log ica l ev idence suppor t ing the ex i s t ence of the n ig roneo -

striatal loop sys tem. T h i s sys t em could b e a chol inerg ic s t r ia tonigral pa thway

and migh t represent a f eedback m e c h a n i s m on the d o p a m i n e - c o n t a i n i n g

n e u r o n s in the subs tan t i a n ig ra (8) .

In ag reemen t w i th the f indings of Hokfel t (48) , w e have only occas iona l ly

obse rved a wel l -def ined junc t iona l contac t assoc ia ted w i th the t agged var icos -

i t ies . M o s t of the j unc t i ons encoun te red are symmet r i ca l contacts (Fig-

ures 14E and F , c rossed a r rows) , b u t it i s no t k n o w n w h e t h e r th i s represen ts

a symmet r ica l synapt ic j u n c t i o n or mere ly an adhes ion p laque b e t w e e n the

var icos i ty and a dendr i te . If the j u n c t i o n is a symmet r ica l synapse , the f inding

of b o u t o n s con ta in ing the i nh ib i t o ry t ransmi t te r , d o p a m i n e , wou ld b e in

k e e p i n g wi th the hypo thes i s der ived from s tudies of o the r areas of the b ra in

that symmet r i ca l synapses are i n h i b i t o r y (26) .


S i n c e w e have s een on ly a smal l pe rcen tage o f j u n c t i o n s assoc ia ted w i t h

over 2450 tagged b o u t o n s e x a m i n e d , w e do no t k n o w w h e t h e r it is u n c o m m o n

for these va r icos i t i e s to form typical synapt ic j u n c t i o n s , or w h e t h e r w e have

failed to find t h e m b e c a u s e of the s ampl ing p r o b l e m s inhe ren t i n e lec t ron

mic roscopy . Cer ta in ly i f the j unc t iona l r eg ion is small , t hey wil l b e sec t ioned

and found less readi ly . M o r e o v e r , symmet r i ca l j u n c t i o n s are not as obv ious as

a symmet r i ca l j u n c t i o n s , part icular ly after th i s t e c h n i q u e . S i n c e the t e c h n i q u e

of i ncuba t i on o f fresh b ra in t i ssue in the p re sence of false t ransmi t te rs before

fixation causes des t ruc t ion of m o s t o f the t i s sue o the r than te rmina ls , it is pos -

s ib le that s o m e junc t i ona l contac ts are des t royed as wel l . Neve r the l e s s , a s y m -

metr ica l j unc t iona l contac ts (Figure 1 4 C , c rossed arrow) assoc ia ted w i th non -

tagged b o u t o n s are f requent ly obse rved . T h i s larger type of b o u t o n , w h i c h

m a k e s an a symmet r i ca l axosp inous synapt ic j u n c t i o n and has large n u m b e r s

of the smal les t s ized ves ic l e s , has neve r b e e n tagged in our expe r imen t s . Ev i -

dence from o ther s tud ies ind ica te that the lat ter t e rmina ls p r o b a b l y or ig ina te

from cort ical or tha lamic n e u r o n s (31 ,53) . W e have seen on ly a few " in t e r -

rupted j u n c t i o n s , " a type of synapt ic contact desc r ibed b y K e m p and Powel l

(56) . F r o m l e s ion s tud ies , w e have sugges ted that b o u t o n s h a v i n g in te r rupted

j u n c t i o n s are in t r ins ic t e rmina ls (87) . A s expec ted , no tag was found in the

ves ic les of th i s type of synapt ic j u n c t i o n .

If further s tud ies p rove that the symmet r i ca l contac ts i l lustrated in F igures

14E and F are a d h e s i o n areas ra ther than synapt ic j u n c t i o n s and the var icos -

i t ies can b e demons t r a t ed u n e q u i v o c a b l y to b e d o p a m i n e con ta in ing , it

wou ld p rov ide in te res t ing ev idence that m i g h t b e a r on the m o d e of re lease of

d o p a m i n e in the neos t r i a tum. It wou ld sugges t that the d o p a m i n e - c o n t a i n i n g

var icos i t ies in the neos t r i a tum m a y b e s imi la r to those in the per iphera l

ne rvous sy s t em, w h i c h do no t have j u n c t i o n a l spec ia l iza t ions . U n l i k e the ter-

mina l s at the m o t o r e n d plate , w h e r e ace ty lchol ine is re leased and its effects

are exer ted at specif ic s i tes , i . e . , the j unc t iona l complex , n o r e p i n e p h r i n e in the

sympa the t i c va r icos i t i e s of the i r is m a y b e re leased a long the en t i re surface of

the var icos i ty to spread diffusely in to the extracellular space b e t w e e n the

var icos i ty and the myoep i the l i a l cel ls . If d o p a m i n e te rmina ls re lease d o p a m i n e

diffusely in to the neu rop i l o f the neos t r i a tum, o n e w o u l d expec t the neu ro -

t ransmi t te r to have a w idesp read effect ra ther than a discre te effect l imi t ed to a

small por t ion of the m e m b r a n e of an adjacent cell .

It should b e po in t ed out , howeve r , that our inab i l i ty to demons t r a t e j u n c -

t ions assoc ia ted w i th a s ignif icant pe rcen tage of the b o u t o n s that take up false

t ransmit ters is no t in accord w i t h the a t tempts of o thers (46 ,57) to ident i fy the

d o p a m i n e - c o n t a i n i n g t e rmina l s . A s m e n t i o n e d , K e m p and Powel l (57) have

repor ted that all afferent te rmina ls to the caudate nuc leus have asymmet r i ca l

m e m b r a n e t h i c k e n i n g s , and they i l lustrate a s y n a p s e " e n p a s s a g e " fo rming an

asymmet r i ca l j u n c t i o n , w h i c h is degene ra t ing after a large l e s ion in the m i d -

bra in . T h e y s tate , h o w e v e r , that there were very few degenera t ing te rmina ls

avai lable for s tudy b y e lec t ron m i c r o s c o p y after th i s l es ion . It is pos s ib l e ,


moreover , that degenera t ing symmet r ica l j u n c t i o n s m a y b e more difficult to

ident i fy or t hey m a y degenera te in such a w a y that the j unc t i on is no longer

present . Hat tor i et al. (46) also ind ica ted that d o p a m i n e - c o n t a i n i n g te rmina ls

ended in a symmet r i ca l j u n c t i o n s , s ince 7 5 % of the te rmina ls that are labeled

wi th s i lver gra ins after the in jec t ion of 3 H - l e u c i n e in to the subs tan t i a n igra

compac ta we re of th is type . T h e r e m a i n i n g labe led j unc t i ons we re symmet r i ca l

and h a d h igh ly p l eomorph ic ves ic les .

S ince there are relat ively few nigrostr ia ta l t e rmina ls in the neos t r i a tum c o m -

pared to those from o ther areas (57) , s ampl ing the t i ssue is a cons ide rab le

p rob lem in all o f the e lect ron mic roscop ic s tudies d i scussed . It is also poss ib l e

that the different p rocedures are reveal ing a he t e rogeneous popula t ion of ter-

mina l s . As stated prev ious ly , the h i g h concen t ra t ion of false t ransmit ters

n e e d e d for the s tudies i l lustrated in F igures 1 4 B - H could label n o r e p i n e p h r i n e

and se ro ton in t e rmina l s , as wel l as d o p a m i n e te rmina ls . W e have a t t empted to

add specif ici ty to our ident i f ica t ion of the d o p a m i n e te rmina l , howeve r , b y

the use of D M I in mos t of our expe r imen t s (51 ,80) . A n y one of the three t rans-

mit ters could have b e e n p resen t in s o m e of the te rmina ls i l lustrated after large

les ions of the m i d b r a i n (57) , as wel l as after the in jec t ion of 3 H - l e u c i n e into the

subs tan t ia n ig ra (46) , s ince the or ig in of the n o r e p i n e p h r i n e and se ro ton in ter-

mina l s in the neos t r i a tum is no t cer ta in . B o t h types of axons course th rough

the m i d b r a i n (33) , and they m a y run close to the d o p a m i n e - c o n t a i n i n g axons ,

part icularly in the lateral hypo tha l amus (5) . T h u s , large les ions of the m i d -

bra in and in jec t ions of subs t ances in to the m i d b r a i n could b e expec ted to ef-

fect n o r e p i n e p h r i n e and se ro ton in te rmina ls in the neos t r i a tum, as wel l as

d o p a m i n e - c o n t a i n i n g var icos i t i es .

SUMMARY

T h e ultrastructural and f luorescence h i s tochemica l character is t ics of the ma-

ture r abb i t subs tan t i a n igra and neos t r i a tum have b e e n r ev iewed as a frame of

reference for the deve lopmenta l s tudy. B i o c h e m i c a l inves t iga t ions were re-

por ted on neos t r ia ta l d o p a m i n e concen t ra t ions and the relat ive uptake and ac-

cumula t ion of 3 H - d o p a m i n e b y this t i ssue from fetal to adult s tages , to p rov ide

quant i ta t ive data for corre la t ion wi th the f luorescence informat ion .

T h e deve lopmen t of the neu rons of the subs tan t i a n ig ra and thei r axons

w h i c h project to the neos t r i a tum has b e e n p resen ted from thei r appearance at

day 14 of ges ta t ion to thei r matura t ion in early pos tna ta l life. T h e ini t ia l

b ipo la r neurob las t s , w h i c h develop in the mid l ine of the caudal m e s e n -

cepha lon , are f luorescent as soon as t hey emerge from the e p e n d y m a l zone .

The i r f luorescent axons , w h i c h form the n igroneos t r ia ta l pa thway , reach the

te lencepha lon at day 16 of ges ta t ion and ramify ex tens ive ly in the p u t a m e n b y

day 2 0 , bu t do not en te r the caudate nuc leus unt i l several days later. S o m e of


the early fluorescent axonal profiles in the p u t a m e n are ex t remely large. E lec-

t ron mic roscop ic s tudy of th is s tage sugges t s that the large fluorescent profiles

m a y co r re spond to axonal g rowth cones or early synapses .

A dis t inct subs tan t i a n igra , pars compac ta and ret iculata , can b e r ecogn ized

b y fluorescence m i c r o s c o p y b y day 20 of ges ta t ion . Elec t ron m i c r o s c o p y re-

veals that the y o u n g neu rons are mul t ipolar w i th n u m e r o u s deve lop ing den-

dri tes , s o m e of w h i c h exh ib i t early synapt ic j u n c t i o n s . T h e s u b s e q u e n t matu-

ra t ion of t hese cells and the neurop i l is de sc r ibed .

T h e fluorescent axons of the subs tan t i a n ig ra g row into the p u t a m e n and

caudate nuc leus in a n o n u n i f o r m m a n n e r fo rming fluorescent i s lands

th roughou t the neos t r i a tum in late fetal life. Occas iona l ly , m i n u t e b e a d e d

fluorescent axons are found. T h e s e profiles m i g h t co r respond to s o m e of the

axons w i t h var icos i t i es " e n p a s s a g e " revealed b y electron mic roscopy .

In an a t tempt to ident i fy further the d o p a m i n e - c o n t a i n i n g axon , the ultra-

structure of adult neos t r i a tum incuba t ed in 5 -hyd roxydopamine w a s repor ted .

Axona l var icos i t i es " e n p a s s a g e " con ta in ing a dense " t a g " in the ves ic les we re

found. M o s t of the tagged b o u t o n s d id no t exh ib i t synapt ic contac ts . T h e pos -

s ib le s igni f icance of these f indings as related to d o p a m i n e secre t ion are d i s -

cussed .

ACKNOWLEDGMENTS

The authors wish to express their gratitude to the following persons for their excellent assis-tance in their respective disciplines: Mrs. Mary Budininkas-Schoenebeck, Dr. Domingo Espiritu, Mr. Moshe Rosen, Miss Laura Yahr, and Miss Susan Bassett (electron microscopy); Mrs. Irene S. Tar (fluorescence microscopy); Mrs. Gizella Goldfinger and Miss Nancy Rogers (dopamine assay); Miss Dorothy Dembiec and Miss Felicitas Cabbat (dopamine uptake studies).

These studies were supported by the Clinical Center for Research in Parkinson's and Allied Diseases, Grant No. NS-05184 from the United States Public Health Service, and the Muscular Dystrophy Associations of America, New York, New York.

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33: 1-12.

9A Discussion: Nigrostriatal Projections and the

"Dopamine Receptor"

ANTHONY M. ADINOLFI Mental Retardation Research Center and

Neuropsychiatric Institute,



R e g i o n a l d i s t r ibu t ion s tudies of e n d o g e n o u s d o p a m i n e levels in the centra l

ne rvous sys t em reveal that approx imate ly 8 0 % of the total b r a in d o p a m i n e is

concen t ra ted in the caudate nuc leus , p u t a m e n , and subs tan t i a n ig ra (15) .

The re exis ts a fundamenta l difference in the cytological d i s t r ibu t ion of dopa-

m i n e of the nigros t r ia ta l p ro jec t ion in that the n igra l a m i n e is concen t ra t ed in

cell b o d i e s w h i l e striatal d o p a m i n e is p resen t in ve ry fine var icose ne rve fibers

th roughou t the neurop i l (4 ,5 ) . Dr . T e n n y s o n has demons t r a t ed clearly that

dur ing fetal and early pos tnata l deve lopmen t o f the n igros t r ia ta l p a t h w a y

there i s a p rogress ive inc rease in the n u m b e r and in t ens i ty of f luorescent

profiles in the neos t r i a tum w h i c h co r responds w i t h the appearance of en pas-

sant axodendr i t i c synap t ic contac ts seen b y e lect ron mic roscopy . Dr . T e n n y s o n

and he r co-workers also repor ted that dur ing th is pe r iod there is a gradual

increase in the levels of e n d o g e n o u s d o p a m i n e and in the capac i ty of n e o -

striatal t i s sue to accumula te t r i t ia ted d o p a m i n e (23) . Nigra l s t imula t ion and

ion tophore t i c appl ica t ion of d o p a m i n e (9 ,16) evoke faci l i tatory and depressan t

r e sponses a m o n g caudate un i t s , i nd ica t ing that d o p a m i n e r g i c m e c h a n i s m s are

an in tegral part of the cauda te n e u r o n firing pa t te rns . T h e p rec i se form w h i c h

in te rac t ions a s s u m e at r ecep tor levels n e e d s to b e def ined. T h e fo l lowing dis-

cuss ion focuses on the p r o b l e m of ident i fy ing n igra l dopamine rg i c ne rve end-

ings a long neu rona l surfaces w i t h i n the cauda te nuc leus .

T h e caudate nuc leus and p u t a m e n e x h i b i t an apparen t structural h o m o g e n e -

ity w h e n e x a m i n e d b y l ight and e lect ron mic roscopy . T h e average d iamete r of

striatal n e u r o n s is 12 to 14 pum w h e n m e a s u r e d in sec t ions s ta ined wi th

265

266 Anthony M. Adinolfi

to lu id ine b lue (1 ,2) and w i t h t h ion in (19) , or impregna t ed b y the Golg i

m e t h o d (19) . T h e s e m e d i u m - s i z e d n e u r o n s c o m p r i s e approx imate ly 9 6 % of

the total cell popula t ion and are charac ter ized , b y Golg i impregna t ion , as in-

t e rneurons w i t h m e d i u m - l e n g t h , sp ine- laden dendr i tes , and a s ingle short

axon w i t h var icose collaterals . Elec t ron mic roscop ic s tud ies (1 ,2 ,20 ) on the

synapt ic o rgan iza t ion w i t h i n the striatal neurop i l no t ed that axonal profiles

are th in (mean d iamete r of 630 n m ) , f ine ly-myel ina ted , or unmye l ina t ed and

form mul t ip le synapt ic contac ts en passant on cons t i tuen t neu rons . M o s t axon

te rminals con ta in spher ica l 500 A synapt ic ves ic les and synapse a symmet -

rically on dendr i t ic sp ine s , or to a l esser extent , on distal dendr i tes .

T h e sources of the synapt ic profiles w i t h i n the caudate nuc leus have b e e n

de t e rmined from s tudies no t ing te rmina l degenera t ion fo l lowing discre te le-

s ions of e i ther the cerebra l cor tex , tha lamus , m i d b r a i n , o r the caudate nuc leus

i tself (21) . All afferent fibers to the caudate nuc leus form mul t ip le a s y m m e t -

rical synapt ic contac ts on dendr i t ic sp ines and distal dendr i tes . C o m p a r i s o n

of the dens i ty o f the degene ra t ion fol lowing les ions of the different pa thways

to the caudate nuc leus ind ica tes that the largest inpu t s arise in the ipsi la teral

cerebra l cor tex and the tha lamus . A b o u t 30 to 4 0 % of the e n d i n g s w h i c h form

asymmet r i ca l j u n c t i o n s ar ise in the cerebra l cor tex and abou t 20 to 2 5 % in the

tha lamus . Pro jec t ions from the contralateral cerebral cor tex and the m i d b r a i n

account for on ly a few percen t o f the total a symmet r ica l synapses .

T h i s sugges t s that s l ight ly less than 5 0 % of the a symmet r i ca l contac ts in the

caudate nuc leus represent in t r ins ic connec t i ons . After an in t r ins ic l e s ion of the

caudate nuc leus , degene ra t ing te rmina ls are found synaps ing asymmet r ica l ly

on distal dendr i t ic profiles and sp ines as wel l as synaps ing symmetr ica l ly on

neurona l soma , p rox imal dendr i t es , and axonal in i t ia l s egmen t s . O b s e r v a t i o n s

on pers i s t ing ne rve t e rmina l s in chronica l ly isola ted caudate nuc leus b y Dr .

T e n n y s o n (24) conf i rmed th is d i s t r ibu t ion o f in t r ins ic connec t iv i ty . T h u s , all

symmet r ica l contac ts are of in t r ins ic o r ig in , wh i l e a symmet r i ca l j u n c t i o n s m a y

b e e i ther in t r ins ic or ex t r ins ic .

T h e p r o b l e m is to d i s t ingu i sh those a symmet r i ca l axodendr i t i c and axo-

sp inous synapt ic contac ts w h i c h represent n igrost r ia ta l p ro jec t ions or, more

s imply , to d i s t ingu i sh dopamine rg i c receptor s i tes w i t h i n neost r ia ta l neurop i l .

Di rec t v i sua l iza t ion of m o n o a m i n e s tores wi th the e lectron mic roscope de-

pends on the format ion of an e lec t ron-dense prec ip i ta te b y reac t ing the a m i n e

wi th a h e a v y meta l . In the test t ube , b o t h noradrena l ine and d o p a m i n e react

w i th po t a s s ium pe rmangana t e to the s ame extent to form prec ip i ta tes b y

reduc ing the pe rmangana t e to m a n g a n e s e d iox ide . B y us ing p o t a s s i u m

pe rmangana t e as a fixative for e lec t ron mic roscopy small granular (500 A, dense core) and large granular (1000 A, dense core) ves ic les are v i sua l ized in

bo th central and per iphera l noradrenerg ic ne rve te rmina ls . H o w e v e r , small

granular ves ic les are no t s een in dopamine rg i c ne rve te rmina ls in

permangana te - f ixed caudate nuc leus s ince the e n d o g e n o u s d o p a m i n e is

9 A. Nigrostriatal Projections and the "Dopamine Receptor" 267

presen t free (not granule b o u n d ) or in an eas i ly re leased form in the caudate

nuc leus (14) .

Recen t ly , a d o p a m i n e - s e n s i t i v e adenyla te cyclase has b e e n ident i f ied in the

super io r cervical gang l ion (17) , in the i n n e r re t ina (8) , and in the caudate

nuc leus (18) . T h e s e s tudies sugges t that th is e n z y m e represen t s a " d o p a m i n e

r ecep to r " and that the cyclic A M P genera ted b y adenyla te cyclase ac t ion m a y

med ia t e c a t e c h o l a m i n e - i n d u c e d changes in the exc i tab i l i ty o f pos t synap t ic

cells (13 ,22) .

Unfor tuna te ly , no m e t h o d for the ultrastructural loca l iza t ion of b r a i n adenyl -

ate cyc lase has b e e n repor ted . H o w e v e r , cycl ic nuc leo t ide p h o s p h o d i e s t e r a s e

(PDE) , w h i c h ca ta lyzes the m e t a b o l i s m of cyclic A M P , has b e e n v i sua l ized cy-

tochemica l ly at pos t synap t ic dendr i t i c s i tes in the molecu la r layer of the cere -

bral cor tex (11) . Dr . S. Y . S c h m i d t and I have used th is m e t h o d to demons t ra t e

P D E act ivi ty at deve lop ing synapses in the molecu la r layer of i m m a t u r e cere-

bral cortex (3) .

Loca l iza t ion o f P D E act iv i ty is b a s e d on the in situ p rec ip i ta t ion of re leased

inorgan ic p h o s p h a t e w i t h lead accord ing to the fo l lowing reac t ion s e q u e n c e :

P D E cata lyzes the hydro lys i s of cyclic A M P to 5 ' - A M P w h i c h , in the p re sence

of e x o g e n o u s 5 ' -nuc leo t idase , is m e t a b o l i z e d to a d e n o s i n e and inorgan ic

= 10-1

i—i—i i i 1 • — i 2 5 10 15 20 Adult

Postnatal days

F IGURE 1. Measurements of the postnatal rise in phosphodiesterase (PDE) activity in mouse cere-

bral cortex, using a method (7) for the assay of enzymatically released inorganic phosphate. Cour-

tesy of Dr. S. Y. Schmidt, Harvard University.

nm

ole

s cA

MP

h

yd

roly

ze

d/m

g p

rote

in/m

in.


phospha te . T h e fol lowing control expe r imen t s we re per formed to check the

val id i ty of the cy tochemica l local iza t ion: (a) 50 m M theophy l l ine was added

to the s econd i n c u b a t i o n mix ture to i n h i b i t P D E act ivi ty , (b) cyclic A M P w a s

omi t t ed from the s econd i n c u b a t i o n mix tu re , (c) 5 ' -nuc leo t idase w a s omi t t ed

from b o t h i ncuba t i on mix tu res , (d) 5 ' - A M P was used as the subs t ra te in the

p resence or a b s e n c e of 5 ' -nuc leo t idase , and (e) the first i ncuba t i on mix ture

con ta ined only T M S buffer. In th i s s tudy the reac t ion is cons ide red specific

w h e n : (a) cyclic A M P is r equ i red as subs t ra te , (b) the local iza t ion cannot b e at-

t r ibuted to e n d o g e n o u s 5 ' -nuc leo t idase act iv i ty , and (c) theophy l l ine i nh ib i t s

the reac t ion .

B i o c h e m i c a l assay of deve lop ing cerebra l cor tex (Figure 1 ) , u s ing the m e t h o d

of Breckenr idge and J o h n s t o n (7) , s h o w s that P D E act iv i ty is p resen t at b i r th

and inc reases to adult levels dur ing the first pos tnata l m o n t h . It is dur ing th is

pe r iod that large n u m b e r s of n e w synapt ic contacts are fo rming in the

neuropi l . W h e n e n z y m e act ivi ty is local ized cy tochemica l ly in the superficial

neocor tex of m i c e sacrif iced at 5-day intervals from b i r th to 35 pos tna ta l days ,

react ion product is found cons i s ten t ly at or nea r the reg ion of e m e r g i n g post-

F IGURE 2. Cytochemical localization of PDE activity in the molecular layer of 15-day-old mouse

occipital cortex. The reaction product, lead phosphate, accumulates postsynaptically in dendritic

profiles (arrows), x 60,000.

9A. Nigrostriatal Projections and the ''Dopamine Receptor" 269

synapt ic p a r a m e m b r a n o u s dens i t i e s a long dendr i t ic profiles. I m m a t u r e (Fig-

ure 2) and ma tu re (Figure 3) synapt ic contac ts con ta in e l ec t ron-dense ac-

cumula t ions of lead p h o s p h a t e pos tsynapt ica l ly . W e sugges ted that such s i tes

are capab le of cyclic A M P m e t a b o l i s m at all pos tna ta l ages .

S i n c e the ex i s t ence of mul t ip le forms of cycl ic 3 ' , 5 ' - nuc l eo t i da se p h o s p h o -

dies terase has b e e n repor ted for a w i d e var ie ty of t i s sues (6) , inc lud ing the

cerebra l cor tex , it i s impor t an t to cons ide r the k ine t i c p roper t ies o f th i s en -

zyme . B i o c h e m i c a l s tud ies (25) o n the subcel lu lar d i s t r ibu t ion of k ine t ica l ly

d is t inct P D E enzyma t i c ac t iv i t ies have s h o w n that , in rat b r a in , the e n z y m e

wi th a l ow Km va lue for cyclic A M P is local ized in a par t iculate or m e m b r a n o u s

fract ion, w h i l e the less specific e n z y m e w i t h a lower affinity for cycl ic A M P is

soluble . T h e h i g h Km so lub le P D E act ivi ty in r abb i t b r a in s h o w s no corre la t ion

wi th m o n o a m i n e con ten t or w i t h cellular dens i ty (7) . T h e par t iculate act iv i ty is

assoc ia ted wi th synap tosoma l f ract ions ( 1 0 , 1 2 , 2 5 ) , and the P D E act iv i ty that is

de tec table at pos t synap t i c s i tes b y cy tochemica l m e a n s m a y represen t a por t ion

o f the l o w Km par t icula te act iv i ty w h i c h has surv ived the expe r imen ta l p roce-

F IGURE 3. Visualization of PDE activity in the molecular layer of 35-day-old mouse occipital cortex. The reaction product (arrows) is localized at axodendritic synaptic junctions near the post-synaptic paramembranous densities, x 58,000. Courtesy of Brain Research, Elsevier Publishing Co., Amsterdam.


dures . W e sugges t that the l ow Km P D E act ivi ty can b e corre la ted , in part, w i th

m o n o a m i n e content . H o w e v e r , the he te rogene i ty of pos s ib l e neuro t rans -

mi t ters ac t ing at cort ical synapses m a k e s exceed ing ly difficult the corre la t ion

of P D E act ivi ty w i t h pos t synap t i c s i tes b e l o n g i n g to any s ingle group of axonal

end ings .

Pe rhaps a be t te r m o d e l to tes t th is hypo thes i s can b e found in the caudate

nucleus . T h e r e , p ro jec t ions from the subs tan t i a n ig ra and adjacent m i d b r a i n

on to dendr i t ic sp ines and dendr i t es represen t the sole source of d o p a m i n e .

Man ipu la t ion of th is sys t em dur ing its deve lopmen t w h i c h results in a signifi-

cant al terat ion in the striatal levels of cyclic A M P and related e n z y m e s should

provide s t rong ev idence for the ex i s t ence of the " d o p a m i n e r ecep to r . " Fo r the

neuroana tomis t , cy tochemica l local iza t ion of phosphod ie s t e r a se and , be t te r

still, of adenyla te cyclase wil l then afford a r eady m e a n s of see ing these

receptors w i t h i n the neos t r i a tum.

REFERENCES

1. Adinolfi, A. M. The organization of synaptic junctions in cat putamen. Brain Res., 1971, 32: 53-67.

2. Adinolfi, A. M., and Pappas, G. D. The fine structure of caudate nucleus of the cat. / . Comp. Neurol, 1968, 133: 167-184.

3. Adinolfi, A. M , and Schmidt, S. Y. Cytochemical localization of cyclic nucleotide phospho-diesterase activity at developing synapses. Brain Res., 1974, 76: 21-31 .

4. Anden, N. E. , Carlsson, A. , Dahlstrom, A., Fuxe, K. , Hillarp, N.-A., and Larsson, K. Demon-stration and mapping out of nigro-neostriatal dopamine neurons. Life Sci., 1964, 3: 523-530 .

5. Anden, N. E. , Dahlstrom, A., Fuxe, K. , Larsson, K., Olson, L . , and Ungerstedt, U. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Scand., 1966, 67: 313-326.

6. Appleman, M. M., Thompson, W. J . , and Russell, T. R. Cyclic nucleotide phosphodiesterases. In Advances in Cyclic Nucleotide Research. Vol. 3. (P. Greengard and G. A. Robison, Eds.). Raven, New York, 1973: 65-98.

7. Breckenridge, B . , and Johnston, R. E. Cyclic 3',5'-nucleotide phosphodiesterase in brain. /. Histochem. Cytochem., 1969, 17: 505-511.

8. Brown, J . H., and Makman, M. H. Stimulation by dopamine of adenylate cyclase in retinal homogenates and of adenosine-3',5'-cyclic monophosphate formation in intact retina. Proc. Natl. Acad. Sci. USA, 1972, 69: 539-543.

9. Connor, J . D. The nigro-neostriatal pathway: The effects produced by iontophoretic dopa-mine. Res. Publ. Assoc. Res. Nerv. Ment. Dis., 1972, 50: 193-206.

10. De Robertis, E. , Arnaiz, G. R., and Alberici, M. Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. / . Biol. Chem., 1967, 242: 3487-3493.

11. Florendo, N. T., Barrnett, R. J . , and Greengard, P. Cyclic 3',5'-nucleotide phosphodiesterase: Cytochemical localization in cerebral cortex. Science, 1971, 173: 745-747.

12. Gaballah, S., and Popoff, C. Cyclic 3' ,5'-nucleotide phosphodiesterase in nerve endings of developing rat brain. Brain Res., 1971, 25: 220-222.

13. Greengard, P., McAfee, D. A., and Kebabian, J . W. On the mechanism of action of cyclic AMP and its role in synaptic transmission. In Advances in Cyclic Nucleotide Research. Vol. 1. (P. Greengard and G. A. Robison, Eds.). Raven, New York, 1972: 337-355.

9A. Nigrostriatal Projections and the "Dopamine Receptor" 271

14. Hokfelt, T. G. M. In vitro studies on central and peripheral monoamine neurons at the ultra-structural level. Z. Zellforsch. Mikrosk. Anat. 1968, 91 : 1-74.

15. Hornykiewicz, O. Dopamine and extrapyramidal motor function and dysfunction. Res. Publ. Assoc. Res. Nerv. Ment. Dis., 1972, 50: 3 9 0 ^ 1 5 .

16. Hull, C. D. , Bernardi, G., and Buchwald, N. A. Intracellular responses of caudate neurons to brain stem stimulation. Brain Res., 1970, 22: 163-179.

17. Kebabian, } . W. , and Greengard, P. Dopamine-sensitive adenyl cyclase: Possible role in synaptic transmission. Science, 1971, 174: 1346-1349.

18. Kebabian, J . W. , Petzold, G. L. , and Greengard, P. Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the "dopamine receptor." Proc. Natl. Acad. Sci. USA, 1972, 69: 2145-2149.

19. Kemp. J . , and Powell, T. P. S. The structure of the caudate nucleus of the cat: Light and elec-tron microscopy. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 383-401.

20. Kemp, J . , and Powell, T. P. S. The synaptic organization of the caudate nucleus. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 4 0 3 ^ 1 2 .

21. Kemp, J . , and Powell, T. P. S. The site of termination of afferent fibres in the caudate nucleus. Philos. Trans. R. Soc. Lond. [Biol. Sci.], 1971, 262: 413-427.

22. Rail, T. W. , and Gilman, A. G. The role of cyclic AMP in the nervous system. Neurosci. Res. Program, Bull., 1970, 8: 221-323.

23. Tennyson, V. M., Barrett, R. E. , Cohen, G., Cote, L. J . , Heikkila, R., and Mytilineou, C. The developing neostriatum of the rabbit: Correlation of fluorescence histochemistry, electron microscopy, endogenous dopamine levels and (3H) dopamine uptake. Brain Res., 1972, 46: 251-285.

24. Tennyson, V. M., and Marco, L. A. Intrinsic connections of caudate neurons. II. Fluorescence and electron microscopy following chronic isolation. Brain Res., 1973, 53: 307-317.

25. Thompson, W. J . , and Appleman, W. J . Multiple cyclic nucleotide phosphodiesterase activi-ties from rat brain. Biochemistry, 1971, 10: 311-316.

10 Effects of Caudate Nuclei Removal in Cats.

Comparison with Effects of Frontal Cortex Ablation

JAIME R. VILLABLANCA and ROBERT J. MARCUS Mental Retardation Research Center and Department of Psychiatry,



INTRODUCTION

There are several impor tan t cond i t ions w h i c h should b e m e t in any exper i -

men ta l s tudy of the neuro logica l and gross behav io ra l effects of caudate nuc le i

ab la t ion: Firs t , mos t of the caudate t i s sue m u s t b e r e m o v e d . S e c o n d , damage

to adjacent s t ructures , par t icular ly the dorsolateral frontal cor tex, should b e

kept to a m i n i m u m . Final ly , the ab la t ion should b e done in o n e surgical s tage.

T h e m a i n r easons for m e e t i n g these cr i ter ia are respect ively : (a) there are in-

d ica t ions that the caudate t i ssue m a y have a large funct ional compensa to ry

capabi l i ty ( 2 , 5 1 , 7 4 ) ; (b) it has b e e n repeatedly sugges ted that the frontal cor tex

m a y have s o m e funct ions in c o m m o n w i t h the caudate nuc le i ( 5 , 1 6 , 2 3 ) ; and (c)

it has b e e n s h o w n that mul t i s taged l es ions of a g iven b r a in area m a y induce

processes m a s k i n g the effects w h i c h are seen fo l lowing a one - s t age , total

les ion ( 1 , 1 9 , 6 2 ) . Fu r the rmore , the an imals should b e m a i n t a i n e d in good

heal th for a long e n o u g h pe r iod in order to b e sure that any t rans i tory effects

w h i c h usual ly fol low ex tens ive b ra in surgery (1 ,27 ,75) have b e e n a t tenuated or

are no longer present . F ina l ly , an adequa te h i s to logica l report of the l es ions

should b e p rov ided .

S ince it appears that the above cond i t ions have b e e n on ly part ial ly fulfilled

in the caudate ab la t ion s tudies per formed to date in adult a n i m a l s — w h i c h we

k n o w of ( 2 , 1 5 , 2 1 , 2 4 , 3 2 , 3 3 , 3 7 , 4 2 , 5 1 , 5 3 , 5 6 , 6 1 , 7 2 - 7 4 ) — w e th ink that the full

273

274 Jaime R. Villablanca and Robert J. Marcus

potent ia l of the les ion m e t h o d has no t b e e n used in s tudy ing the funct ions of

the caudate nuc le i . T h e presen t expe r imen t s are an a t tempt toward ach iev ing

th is goal .

A m e t h o d is desc r ibed for pe r fo rming a vir tual ly comple te , one-s tage re-

mova l of the caudate nuc le i (avoid ing dorsolateral cort ical damage) and for

m a i n t a i n i n g the an ima l s in good heal th indef ini te ly . T h r e e o the r exper imenta l

groups were s tud ied concomi tan t ly . Cats w i t h uni la teral r emova l o f the cau-

date nuc leus we re s tud ied for the purpose of d i sc los ing any marked lateral ized

neurologica l funct ion and for a s sess ing the m a g n i t u d e of the funct ional c o m -

pensa t ion b y caudate t i s sue . A n i m a l s w i t h bi la tera l ab la t ion of the frontal cor-

tical areas were s tud ied so as to evaluate any frontal cort ical con t r ibu t ion to

the effects of the caudatal r emova l s ince m a n y reports on s imi lar ab la t ions

(28 ,40 ,47 ,70 ,85 ) e i ther vague ly ind ica te that the caudate nuc le i m i g h t also have

b e e n damaged or a l together fail to clarify th i s impor tan t poin t . F inal ly , a

group of sham-ope ra t ed cats w a s s tudied .

T h e neuro logica l , gross behav io ra l , and EEG-po lyg raph i c effects o f these

les ions are p resen ted here and d i scussed compara t ive ly . Severa l o ther s tud ies

of s imi lar exper imen ta l adult cats o r k i t tens have b e e n or are b e i n g per formed

e i ther in our o w n labora tory ( 4 8 , 4 9 , 7 7 , 7 9 - 8 1 ) or in co l labora t ion wi th o ther

labora tor ies of the U C L A M e n t a l Re ta rda t ion Cen te r (44 ,57) . It is expec ted that

a cohe ren t p ic ture wil l e m e r g e of the bas i c defect(s) resul t ing from the a b s e n c e

of wha teve r the caudate nuc le i con t r ibu te to central ne rvous funct ion.

M E T H O D S

T h e expe r imen t s w e r e per formed in 37 adult male (unless o the rwi se stated)

cats: twelve ( two females) w i t h bi la teral r emova l of the caudate nuc le i (BAc) ,

e leven (one female) w i t h uni la tera l r emova l of the caudate nuc leus (UAc) , ten

(two females) w i th bi la tera l r emova l of the frontal cort ical areas (BFr) , and four

sham-opera ted ( S h O ) cats .

Surg ica l P rocedures

All surgery w a s per formed unde r r ig id asept ic t e c h n i q u e us ing pen toba r -

bi ta l anes thes i a w i t h modera t e hypo the rmia . T h e s teps to ablate the caudate

nuc leus were as fol lows: T h e b o n e of the mid l ine of the ca lvar ium w a s re-

m o v e d (an area of abou t 4 0 m m an t e r io r -pos t e r i o r ly and abou t 13 m m on each

s ide of the midsagi t ta l p lane) spar ing the sagittal s inus . T h e dura w a s o p e n e d

bi lateral ly. T h e sagit tal s inus w a s l igated and cut jus t in front of the coronal

suture; the under ly ing falx w a s cut at th is level and the two ends were gent ly

d isp laced away from the sec t ion . A spatula w a s s tereotaxical ly pos i t i oned

b e t w e e n the h e m i s p h e r e s and w a s used b o t h to ind ica te the an te r ior marg in

10. Effects of Caudate Nuclei Removal in Cats 275

of the under ly ing h e a d of the caudate nuc leus and to retract gent ly the h e m i -

sphere . U n d e r v i sua l control and us ing a th in glass p ipe t te for asp i ra t ion , a

small o p e n i n g w a s m a d e in the gyrus c ingul i (area 24) j u s t b e l o w the mid l ine

p ro longa t ion of the crucia te su lcus (Figure 1 ) . T h e corpus ca l losum w a s p e n e -

trated at th i s s i te t h e r e b y ga in ing access to the lateral vent r ic le and expos ing

the dorsomedia l aspects of the caudate . A t th is po in t , the softer cons i s t ency of

the caudate t i s sue enab l ed the suc t ion s t rength to b e reduced to a level w h i c h

was sufficient to aspi ra te the caudate w i thou t affecting n e i g h b o r i n g fiber

s t ructures , par t icular ly the in terna l capsule . A s imi lar p rocedure w a s repea ted

on the contralateral s ide for bi la teral ab la t ion . In the S h O cats , all the above

procedures we re per formed except for the actual asp i ra t ion of the caudate

t i ssue .

For ab la t ion of the frontal cort ical a reas , a m o r e an te r ior b o n e flap w a s

r e m o v e d and m o s t o f the b r a in t i s sue in front o f the h e a d of the cauda te

nuc le i—ind ica ted b y a need le s tereotaxical ly pos i t i oned at A 22 on top of the

b ra in—was r e m o v e d b y asp i ra t ion (Figure 5 ) . In all an imals , the crania l b o n e

defect w a s covered w i t h cranioplas t ic mater ia l .

In 30 cats (11 B A c , n i n e U A c , s ix B F r , and four S h O ) record ing e lect rodes

we re implan ted dur ing the s a m e surgical s e s s ion as fol lows: b i la tera l ep idura l

sc rews (uni - or b ipolar ) in frontal, par ie ta l , and occipi ta l cort ical areas; t r ipolar

e lec t rodes ( twis ted , 0 .01- in . s ta in less-s tee l w i r e s , insu la ted except for the t ips) ,

uni lateral ly in the ventra l h i p p o c a m p u s (A 7 .5 , L 1 0 . 4 , H-5 .0 , ) and p o n t i n e re-

F IGURE 1. Schematic view of the medial surface of the cat brain showing the extent of the pene-

tration lesion (stippled area) in order to expose the caudate nucleus.


t icular format ion (P 5, L 2 . 0 , H - 6 . 0 ) . T h e s tereotaxic coord ina tes of Sn ide r and

N i e m e r (69) were used for all p l acemen t s . In addi t ion , b ipo la r s i lver wi res

were implan ted in the nucha l musc les for E M G record ing and a un ipo la r screw

lead was affixed in the roof o f each orbi t for m o n i t o r i n g eye m o v e m e n t s .

Final ly , one sc rew was affixed in the mid l ine of the frontal b o n e and ano ther

in the b o n e over ly ing the ce rebe l lum as reference and g round leads , respec-

t ively. T h e e lect rodes were affixed to the top of the cranioplas t ic b o n e cover

wi th an a m p h e n o l str ip connec to r and dental cement .

Pos topera t ive Care

All cats were closely m o n i t o r e d dur ing the acute pos topera t ive per iod . T h e

B A c cats were kept for 1 to 4 days in specia l cages (75) in order to m o n i t o r and

control the rectal t empera tu re and to collect u r ine (to assess the hydra t ion

state) . T u b e feed ing w a s ins t i tu ted w h e n an an imal w a s aphag ic for m o r e than

1 day; th i s was supp l emen ted w i t h parentera l fluids d e p e n d i n g on the state of

hydra t ion .

T e s t i n g and R e c o r d i n g Procedures

NEUROLOGICAL TESTING

A bat tery of neuro logica l tests was appl ied per iodica l ly to all cats ; the m a i n i t ems assessed were

a. l ocomot ion (circl ing, obs t ina te p rogress ion , gai t coord ina t ion and speed , s t epp ing up or down)

b . pos ture ( spon taneous , w h e n he ld b y the scruff of the n e c k or h a n g i n g

b y the pelvic girdle) and postura l reflexes ( r ight ing from s ide or in

free fall, M a g n u s reflexes)

c. musc le tonus of the l i m b s (r igidi ty , spast ic i ty)

d. segmenta l l i m b reflexes (stretch reflex, flexor wi thdrawal , l i m b sup-

por t ing reac t ions)

e. l i m b p lac ing reac t ions : the tacti le or contact p lac ing reac t ion ( C P R ) ,

was tes ted fo l lowing the m e t h o d desc r ibed b y Bard (4) as wel l as the

v isual and propr iocep t ive p lac ing r e sponses ; in addi t ion , the cat ' s ab i l -

i ty to wa lk on a p lank w a s assessed

f. gross somat ic sens ib i l i t y

g. p resence of abno rma l " i n v o l u n t a r y " m o v e m e n t s

h. pupi l lary and ocular ac t iv i t ies , in part icular , the o r i en t ing of the eyes

toward the sources of v isual and/or acoust ic s t imula t ion and the pers i s -

tence of t racking ob jec t s in a hor izonta l or ver t ical pendular m o t i o n


RECORDINGS

R e c o r d i n g s we re done wi th the an imals in a sound-a t t enua ted c h a m b e r

wi th a o n e - w a y obse rva t ion w i n d o w and unde r d i m , a l though un in te r rup ted ,

i l lumina t ion . T h e freely m o v i n g an imals were connec t ed to the po lygraph

(type B D y n o g r a p h recorder , B e c k m a n Ins t ruments ) t h rough a counter -

w e i g h t e d cab le sys t em and s l ip-r ing a s sembly . T h e cats had un l imi t ed access

to wa te r and food.

Ove r t m o t o r act ivi ty and s leep-wakefu lness we re evaluated b y m e a n s of

24 -hou r record ing se s s ions conduc ted on or a round the fifth pos topera t ive

day, every 1 0 - 1 5 days dur ing the first 3 m o n t h s , and every 1 0 - 3 0 days thereaf-

ter for up to 6 m o n t h s . Shor te r record ing se s s ions were conduc ted dur ing the

first pos topera t ive days to evaluate early E E G changes . T h e poly graphic cri-

ter ia used to ident i fy m o t o r act ivi ty dur ing wakefu lness we re a de synch ron -

ized E E G , a h igh ly act ive E M G , as wel l as m o v e m e n t artifacts in the records .

Moreove r , the cats were f requent ly obse rved , no t on ly dur ing the day, bu t

also dur ing parts of the n ight . T h e t ime spen t in qu ie t wakefu lness (ocular

m o v e m e n t s and a less act ive E M G ) was not cons ide red as m o t o r act ivi ty . T h e

cr i ter ia u sed to score the s leep-wakefu lness s tates and the co r r e spond ing re-

sults o f th i s part o f the s tudy have b e e n repor ted e l sewhere (49 ,80 ) ; h o w e v e r ,

part of that in format ion wil l b e used to c o m p l e m e n t cer ta in aspects of the

present report .

BEHAVIORAL OBSERVATIONS

T h e an imals w e r e o b s e r v e d m o v i n g freely in the labora tory , w h e r e the

spon taneous or the / / m o t i v a t e d , , b e h a v i o r could b e a s ses sed , and in the

res t r ic ted e n v i r o n m e n t of the record ing c h a m b e r as desc r ibed a b o v e . Par t icu-

lar e m p h a s i s w a s devoted to the eva lua t ion of the fo l lowing i t ems :

a. level of behav io ra l arousal and genera l mo to r act ivi ty; three cri ter ia

were u s e d for a s se s smen t : (1) the a m o u n t of m o t o r act ivi ty d i sp layed

b y the an imal in the record ing cage ; th i s , ident i f ied as desc r ibed

above , w a s expressed as a pe rcen tage of the 24 -hou r record ing se s s ion

for each an imal ; an average of the pe rcen tages for all an ima l s i n each

record ing sess ion was calculated and s h o w n , i ndependen t l y for each

exper imen ta l g roup , b y m e a n s of the bars in F igure 9. F inal ly , an

average o f the pe rcen tages for all s e s s ions , o r average cumula t ive per-

cen tage , w a s also calculated for each exper imen ta l group (Figure 8 ) . (2)

pe rcen tages of the s leep-wakefu lness s tates for each group of an imals ;

(3) an e s t ima te of the an ima l s ' ac t iv i ty w h i l e free in the l abora to ry or in

the i r o p e n - v i e w h o m e cages (cons ide red as a " r e a c t i v i t y " es t imate )


b . the an ima l s ' b e h a v i o r w h e n p resen ted wi th v isual , audi tory , tact i le ,

and olfactory cues

c. genera l awareness and gross b e h a v i o r in re la t ion to the inves t iga tor ,

o ther an imals (cats or m ice ) or ob jec t s , b o t h s ta t ionary and m o v i n g ;

d. g r o o m i n g or feed ing b e h a v i o r

e. pur r ing and voca l iza t ion

At each neuro logica l or behav io ra l tes t ing sess ion , the p resence or absence

of the above i t ems were no ted , de sc r ibed , and i f appl icab le , quant i f ied ( sub-

jec t ive ly) o n a th ree -po in t scale. T h e neuro logy and b e h a v i o r of cats in all

g roups we re evaluated before surgery for purposes of compar i son . All re levant

aspects of the obse rva t ions were recorded on 8- and 1 6 - m m film, still pho-

tography , and on the D y n o g r a p h recorder .

Histology Procedures

At the end of the expe r imen t s (or before , in case of se r ious in tercurrent ill-

nes ses ) the an imals w e r e sacrif iced, the b ra in s w e r e perfused w i t h 1 0 % buf-

fered formal in and e x a m i n e d macroscopica l ly and microscopica l ly emp loy ing

the W e i l and Niss l s ta ins . T o calculate the a m o u n t o f caudate t i ssue r emoved ,

the l e s ions in each b ra in were recons t ruc ted on representa t ive d iagrams of the

cat b ra in taken from the atlas b y S n i d e r and N i e m e r (69) at five regular ly

spaced A - P levels t h roughou t the l e s ioned areas . S u p e r i m p o s e d on these

d iagrams w a s a gr id c o m p o s e d of 1 0 - m m squares . B y coun t ing the n u m b e r of

squares covered b y each l e s ion , it w a s poss ib l e to c o m p u t e a numer i ca l i ndex

and to calculate a pe rcen tage for the a m o u n t of caudate removal . T h e es t imates

were conserva t ive , s ince any caudate t i ssue r emnan t s were not cons ide red

as ab la ted , and yet they f requent ly revealed gl ios is or degenera t ive cell

changes unde r c loser mic roscop ic examina t ion . Fo r eva lua t ing the damage to

noncauda te s t ructures , the atlas for the cat b ra in b y R e i n o s o - S u a r e z (59) was

e m p l o y e d s ince it p rov ides comple te frontal sec t ions of the forebrain .

RESULTS

Survival

T e n of the 12 B A c cats we re m a i n t a i n e d for an average of over 6 m o n t h s

w i th three of t h e m l iv ing for over 1 year . T h e r e m a i n i n g two cats d ied dur ing

the first pos topera t ive m o n t h due to p n e u m o n i t i s and l ymphosa rcoma , respec-

t ively. All the cats in the o ther groups we re m a i n t a i n e d for an average of over

3 m o n t h s , w i th at least two in each group b e i n g m a i n t a i n e d for over 6 m o n t h s .


A n a t o m y

T h e b r a in s of ten B A c cats have b e e n s tudied thus far. A n average of 8 4 % of

caudate t i s sue w a s r e m o v e d in these an imals (86 and 8 2 % for the left and r ight

caudate nuc leus , respec t ive ly) . A n example is s h o w n in F igure 2 . In four cats

the remova l w a s b e t w e e n 95 and 1 0 0 % comple te and in on ly one an ima l w a s it

less than 6 0 % . T h e r e w a s no t any cons tan t loca t ion for the caudate r e m n a n t s ,

a l though they were usual ly found in the mos t ventra l and lateral areas o f the

caudate h e a d and in the m o s t caudal part of the b o d y . T h e caudate tail w a s not

direct ly l e s ioned . In all b ra ins the dorsolateral and frontal cort ical areas were

intact as seen in the sample s h o w n in F igure 3 . In four cat b ra ins o the r areas

did not sus ta in any apparen t l e s ions (except for cort ical or callosal pene t ra t ion

s i tes . ) Add i t iona l damage in the r e m a i n i n g s ix b ra ins w a s uni la teral , s l ight

(except for o n e case w i t h a large l es ion in the fornix and in the area septal is

bi lateral ly a n d o n the ventra l aspects of o n e frontal po le ) , and no t sys temat -

ically repea ted [ inc luding l es ions to the reg ion l i m b i c a an ter ior ( two cases ) ,

area septal is ( three cases) fornix (two cases ) , an ter ior c o m m i s s u r e (one case) ,

and nuc leus p ro tha lamicus ( two cases ) ] . D a m a g e to the in te rna l capsule w a s

neg l ig ib le , except in two cases (small l e s ion of rostral and lateral aspec ts ,

respect ive ly) .

In seven U A c b ra ins avai lable for s tudy, there w a s an average amoun t of

9 5 % remova l of the caudate nuc leus . Add i t iona l , s l ight , uni la teral l e s ions we re

found in five of these b ra in s bu t w i th m o r e i nvo lvemen t of lateral (capsular)

aspects than in B A c cat b ra ins . A represen ta t ive sample is s h o w n in F igure 4 .

Four b r a i n s of cats w i t h frontal pole ab la t ion have b e e n e x a m i n e d . T h e ex-

tent of the areas r e m o v e d i s s h o w n in F igure 5 . Brief ly , the areas bi la teral ly

abla ted were dorsal crucia te and per icrucia te cor tex , gyrus p roreus , mos t of

the sulcus presy lv ius , anter ior one-four th to one- th i rd of the sulcus coronal i s ,

an ter ior one-four th to one- th i rd of sulcus orbi ta l is a long wi th the rostral as-

pects of gyrus coronal i s and orbi ta l i s ; finally, gyrus frontalis , rec tus , and an-

ter ior one- th i rd to one -ha l f of the per icruc ia te areas in the media l as-

pect of the h e m i s p h e r e . T h e a m o u n t of the ab la t ion was fairly cons tan t for the

dorsolateral aspects w i t h var ia t ions from cat to cat w i th respect to the ex tent of

ventra l and mid l ine r emova l s . Caudal ly , the extent of the ab la t ion w a s con-

stantly kept j u s t in front o f the caudates w i th n o o p e n i n g to the ven t r ic les , as

s h o w n in F igure 4 D .

T h e b ra ins of two S h O cats have b e e n e x a m i n e d ; in b o t h there was a cort ical

les ion in the gyrus c ingul i bi la teral ly (area of pene t ra t ion) , e n c o m p a s s i n g the

caudal one -ha l f of area 2 4 , the rostral part of area 2 3 , and ex tended in a tr ian-

gle over area 31 (Figure 1) . T h e caudal one- th i rd of the crucia te sulcus w a s also

l e s ioned . T h e corpus ca l losum w a s perforated i m m e d i a t e l y lateral to the cor-

tical pene t ra t ion . T h e r e w a s n o damage to the caudate o r to the m i d l i n e sub -

cortical s t ructures .


F IGURE 2. Histological sections of the brain of a cat with bilateral removal of the caudate nuclei.

Weil-stained sections at approximately: A, A 18.0 (Snider and Niemer's atlas, 69); B , A 15.0; C, A

13.0; and D, A 8.0.

A

B

F IGURES 2 C and D.


D

C


F IGURE 3. Dorsal view of a bilateral acaudate cat brain to show lack of damage to dorsolateral cor-tical areas (pia and vessels over marginal gyri were peeled away postmortem since they firmly adhered to tissue formed, in the long-term cats, under the cranioplastic prosthesis).

N e u r o l o g y and G r o s s B e h a v i o r

BILATERAL ACAUDATE CATS

T h e obse rva t ions wil l b e p re sen ted unde r the h e a d i n g s of: (a) ear ly post-

opera t ive per iod ; (b) " c o m p u l s o r y a p p r o a c h i n g " syndrome ; (c) o ther neuro -

behav io ra l f indings .

EARLY POSTOPERATIVE PERIOD. (Arbi t rar i ly es t imated to cover the first 3 0 post-

opera t ive days) . T h e cats we re able to s tand and walk b y the first to th i rd post-

operat ive day. D u r i n g the in i t ia l 2 to 6 days , they f requent ly s h o w e d bi lateral

w e a k n e s s , uns tab le pos tu re , and occas iona l dev ia t ion of the head and b o d y

toward o n e s ide . D u r i n g the first m o n t h they exh ib i t ed p r o n o u n c e d

moto r act ivi ty as d o c u m e n t e d b y the act ivi ty m e a s u r e m e n t s (Figure 9) and b y

a s ignif icant reduc t ion of s leep (80). M o s t of the B A c cats t ended to b u m p into

ob jec t s for the first 2 to 4 pos topera t ive days . O t h e r s igns o f reduced aware-

ness were present for 2 to 7 days inc lud ing : ly ing or s leeping over food or

wate r b o w l s , s t i ck ing the i r feet in to water , ignor ing a mot ion le s s rat or cat,

etc. The re w a s no spon taneous ea t ing or d r ink ing b y any of the an imals for an

average of 8 days (a l though wi th large ind iv idua l var ia t ions ) , even t hough

canned food (p re sumab ly m o r e pala table) w a s offered to t hem. A n average of 7

addi t ional days e lapsed before the feeding b e h a v i o r was efficient e n o u g h to

sus ta in the cats in good hea l th . There fore , t ube feeding was ins t i tu ted for as

long as the ea t ing d e r a n g e m e n t las ted.


"COMPULSORY APPROACHING" SYNDROME(CAS). A complex b e h a v i o r , w h i c h

w e call " c o m p u l s o r y a p p r o a c h i n g " s y n d r o m e in order to different iate it f rom

other descr ip t ive n a m e s used in the l i terature ( 3 , 7 , 5 3 , 5 4 , 7 0 , 7 1 ) , w a s p resen t in

all B A c cats . W h a t p r o b a b l y w a s i ts ear l iest mani fes ta t ion occurred as soon as

the cat e m e r g e d from the surgical anes thes i a and cons i s ted of the head and eyes

t racking pers i s ten t ly any ob jec t m o v i n g , hor izonta l ly or vert ical ly, in the cat ' s

v isual field. T h e po lygraphic record ing of th i s even t from the orbi t leads

y ie lded coun t s of over 200 m o v e m e n t s , w i thou t in te r rup t ion (dur ing the first 7

to 10 pos topera t ive days) ; later they t ended to decrease in ampl i tude and per-

s i s t ence , bu t in four cats they lasted for over a m o n t h . T h e head m o v e m e n t s

d i sappeared first; later , the eye m o v e m e n t s b e c a m e irregular. Irregular

t racking, h o w e v e r , pe rs i s ted for over 2 m o n t h s in a few cats .

As soon as the cats could s tand and walk , the m a i n features of the C A s were

mani fes ted , and cons i s t ed of the cat approach ing and fol lowing a m o v i n g

person , cat, or ob jec t (F igures 6 D - F ) . T h i s b e h a v i o r appeared to b e s tereo-

typed in the sense that the an imal wa lked very close to the m o v i n g ob jec t ,

usual ly w i t h the h e a d down , adher ing marked ly to any d i sp lacemen t and

changes of speed (to the po in t of even runn ing w h e n necessa ry ) , and per-

s is t ing a lmost indef ini te ly . The re were o ther features o f the C A s w h i c h d e m -

onst ra ted i ts s te reo typed character and pe rvas iveness . T h u s , it w o u l d occur

unde r unusua l cond i t i ons , such as w h e n the cat was repea tedly p u s h e d away

from the inves t iga tor (F igures 6 A - C ) ; in th i s case , he w o u l d re turn as m a n y

t imes as he was p u s h e d away . Al so , it occur red w h e n the cat w a s no t c o m -

pletely recovered from anes thes ia or w a s s ick and even w h e n b e i n g m o u n t e d

b y ano the r cat. A n o t h e r s t r ik ing character is t ic w a s the t endency of the cat to

actually c o m e in to close phys ica l contact w i th the ob jec t it w a n t e d to ap-

proach; th is w a s mani fes ted by : (1) dis t ress o f the an imal , expressed b y

vocal iza t ion or s t ruggl ing, w h e n he w a s p reven ted from approach ing the o b -

jec t or pe r son ; (2) the t endency to grasp or grab closely m o v i n g ob jec t s w h i c h

he w a s p reven ted from approach ing ; (3) the t e n d e n c y of even j u m p i n g at o b -

jec ts or c l i m b i n g walls (Figure 7A) in order to g rab or grasp t hem. Final ly , the

approach ing b e h a v i o r could b e s topped b y the inves t iga tor or an aggress ive

an imal sudden ly th rea ten ing the cat; in the latter s i tua t ion , the B A c an imal

wou ld not retreat bu t wou ld s imply s top app roach ing—"f reeze"—and wa i t

unt i l the th rea ten ing s i tua t ion d i sappeared . T h e C A s , therefore , lacked the

f lexibi l i ty and o the r concomi tan t s of the b e h a v i o r of a hung ry or playful in-

tact cat w h i c h tends to follow occas ional ly .

A l though visual cues we re the mos t effective in e l ic i t ing the C A s , acoust ic

and tacti le s t imul i were also adequa te . T h u s , a sudden no i s e could induce a

change in d i rec t ion of an approach ing cat toward the source of the n o i s e , or a

sound e m a n a t i n g from the source of the v isua l cue could e n h a n c e the fol low-

ing behav io r . Tac t i le s t imul i to the h e a d or any part of the b o d y resul ted in the

cat t ouch ing or l i terally " s t i c k i n g " to the source w h e t h e r it w a s the inves -


F IGURE 4. Histological sections of the brain of a cat with unilateral removal of the caudate nucleus. Weil-stained frontal sections at approximately: A, A 16.0 (electrolytic lesion in nonab-lated caudate was produced in a terminal experiment); B , A 13.5; C, A 11.5; and D, A 9.0.

A

B

F IGURES 4C and D.


C

D


A B C

D

F IGURE 5. Schematic views of the rostral half of the cat telencephalon to show: A, lateral; B , dorsal; and C, midline extension of the frontal removal. D is a Weil-stained parasagittal section of a frontal lesioned cat brain; note intactness of the caudate nucleus and rostral ventricular wall.

t igator or an objec t ; thus , if the pelvic or shoulder girdle we re touched , the cat

wou ld br i sk ly r ise (Figure 7C) (a p h e n o m e n o n w h i c h , in the b e g i n n i n g , w a s

in terpre ted as an exaggera ted pos i t ive suppor t ing reac t ion) . F inal ly , a phe -

n o m e n o n s imi lar to the root ing r e sponse , as desc r ibed for k i t tens (20) , could

easi ly b e e l ic i ted in B A c cats b y firmly p lac ing the fingers a round the an imal ' s

snout : the cat wou ld s t rongly p u s h forward m a i n t a i n i n g muzz le contact wi th

the m o v i n g h a n d (Figure 7 D ) .

In addi t ion to the actual approach ing b e h a v i o r jus t desc r ibed , o ther features

of the C A s were (a) a marked doci l i ty , adding to the s imilar i ty of all B A c cats ,

no mat te r h o w different they were preopera t ive ly ; (b) repet i t ive and marked ly

exaggera ted k n e a d i n g or t reading m o v e m e n t s of the fo re l imbs , part icular ly

after contac t ing the ob jec t fol lowed. In three o f the cats , these m o v e m e n t s

were so ex t remely exaggera ted that they occurred wi th the an imal in any pos-

ture, i . e . , su spended b y the scruff o f the n e c k or in a p rone pos i t ion (Figures 7

E , F ) , we re ex t remely s low and sus ta ined , and pers i s ted even in the a b s e n c e of

the inves t iga tor ; (c) pur r ing , often w i th repet i t ive voca l iza t ion; (d) some

degree of hyper reac t iv i ty in the p resence of the inves t iga tor or any o ther

v isua l or audi tory cues . In the i r h o m e cages , the cats t ended to b e in pe rma-

nen t m o v e m e n t , usual ly facing the front, m o v i n g up and down , or go ing to


the corners a t t empt ing to escape and approach . W h e n ou ts ide thei r cages ,

they approached and e n g a g e d in r u b b i n g , t read ing , voca l iza t ion , and purr ing .

In genera l , t hey w o u l d b r i sk ly or ien t toward m o v i n g targets or toward the

source of any n o i s e or sound . T h e s e act ivi t ies overt ly dec l ined w h e n the cat

was in the sound-a t t enua ted record ing c h a m b e r .

T h e t ime-course of the C A s w a s not the s a m e in all cats . In all the ma les ,

the changes r e m a i n e d marked for as long as they surv ived , a l though there w a s

a var iab le dec l ine in those cats l iv ing for over 5 m o n t h s . S u c h dec l ine w a s ap-

parent ly related to the a m o u n t of caudate t i s sue r e m o v e d . T h u s , w h i l e the C A s

w a s still s t rong in four cats that had a lmost 1 0 0 % of the caudate nuc le i

r e m o v e d and l iv ing for over 10 m o n t h s , it dec l ined marked ly in the final days

of the cat hav ing 7 4 % of the caudate nuc le i ab la ted and l iv ing for 225 days and

was a lmost gone b y the fifth m o n t h in a cat h a v i n g only 5 4 % of the caudate

r emoved . In the two females , the s y n d r o m e decreased w i t h i n the th i rd post-

opera t ive m o n t h ( the caudate removal in the on ly female b ra in ava i lab le , to

date, w a s 7 9 % ) .

In all cats the s igns pe rs i s t ing longes t were the v isua l and tact i le aspects of

the approach wi th the exaggera ted t reading m o v e m e n t s and purr ing .

OTHER NEUROBEHAVIORAL FINDINGS

Sensory-motor functions. Pe r iod ic neuro logica l examina t i ons revealed surpr is-

ing ly little s enso ry -moto r deficit in the chron ic B A c cats . A l though , oc-

cas ional ly , s l ight a symmet r i e s in s t re tch reflexes and tonus pers i s ted after 15

days , these did no t fol low any cons tan t pat tern and d i sappeared w i t h i n a

mon th . W i t h d r a w a l reflexes and gross somat ic sens ib i l i ty appeared normal .

N o postura l abnormal i t i e s pers i s ted for m o r e than 10 to 15 days , and the

r igh t ing ref lexes, i nc lud ing a qu ick , perfect r igh t ing in free fall, we re normal .

T h e p ropr iocep t ive p lac ing r e sponse w a s presen t in all l i m b s dur ing the days

i m m e d i a t e l y fo l lowing the ab la t ion . V i sua l p lac ing usual ly recovered w i t h i n

the first 15 days . R e c o v e r y of the contac t p lac ing reac t ion (CPR) occurred

w i t h i n the first days in three cats and recovery w a s in progress in all cats b y 20

to 30 days . Usua l ly o n e or b o t h l i m b s on e i ther s ide wou ld b e g i n to exh ib i t a

coarse , s low, or hypermet r i c C P R w h i c h could no t b e e l ic i ted , as in the con-

trols, from all aspects of the ex t remi ty (dorsal , cub i ta l , and radial) . B y the sec-

ond m o n t h , the C P R w a s comple te ly recovered in all cats . W h i l e the C P R was

still absen t or imperfec t , o ther abno rma l m o t o r even ts related to i ts a b s e n c e

were obse rved , such as inab i l i ty or difficulty in p lank wa lk ing , i nadequa te

lifting of l i m b s w h e n wa lk ing on an u n e v e n surface, s l ipp ing of the p a w s over

edges , and le t t ing a l i m b hang over an edge .

In br ief , after 1 to 2 m o n t h s little or no m o t o r abnorma l i t i e s were de tec tab le

in B A c cats except for, pe rhaps , a hard to define lack of " e l e g a n c e . " E v e n

complex mo to r pe r fo rmances such as ca tch ing a m o u s e , adop t ing defens ive

pos tures , and j u m p i n g up or d o w n , appeared to b e normal .


F IGURE 6. Single frame pictures (from a 16-mm movie) showing a chronic cat with bilateral removal of the caudate nuclei: A, B , and C, being pushed away and returning to the investigators; D, closely following a box; E, a ball; or F , an investigator.

A

B

C


FIGURES 6 D - F .

D

E

F


FIGURE 7. Still camera pictures showing a cat with bilateral removal of the caudate nuclei: A, at-tempting to climb a wall to approach the investigator's hand; B , rubbing against the investigator; C, displaying an exaggerated "pseudo" positive supporting reaction (tactile approach); D, dis-playing a "rooting" type of reaction; E and F, performing markedly exaggerated kneading move-ments of the forelegs while being held in awkward postures.

10. Effects of Caudate Nuclei Removal in Cats

F IGURES 7 D - F .

291

F


Amount of motor activity. T h e data on locomotor act ivi ty for all the groups are

s h o w n in F igures 8 and 9. T h e average cumula t ive pe rcen tages (which inc lude

the values for all 11 record ing sess ions ) were h i g h e r for all three exper imen ta l

groups compared to the S h O cats (Figure 8) wi th the B A c cats man i fes t ing the

h ighes t act ivi ty ( 2 7 . 1 % ) level . M o r e o v e r , the hyperac t iv i ty in the B A c an imals

w a s the m o s t pers i s ten t as s h o w n b y the average pe rcen tages (Figure 9 ) . T h e

act ivi ty of the U A c cats w a s b e l o w ( 2 3 . 4 % ) that of the B A c cats . Fur the rmore ,

in six out of 11 ind iv idua l s e s s ions , the U A c values we re closest o f all l es ion

groups to the S h O va lue (Figure 9 ) . T h e B F r cats we re b e t w e e n the B A c and

U A c an imals w i t h respect to b o t h total cumula t ive pe rcen tages of locomotor

act ivi ty (Figure 8) and pe rs i s t ence of the change (Figure 9 ) .

Other observations. S e v e n cats we re tes ted per iodica l ly for the i r reac t ion to a

m o u s e . D u r i n g the first m o n t h , on ly one cat wou ld kill the rodent . T h e others

w o u l d e i ther fol low, eventua l ly p a w i n g , jus t as at any ob jec t , or even let the

m o u s e walk on the i r b o d y wi thou t a t tacking. After 2 m o n t h s the s a m e cats

wou ld readi ly attack and kill a m o u s e .

S i g n s of a l terat ions in sexual act ivi ty w e r e no t i ceab le . Unfor tunate ly , sexual

act ivi ty has on ly b e e n s tudied in the last five B A c cats (four males and one

female) . T w o of the ma les e x h i b i t e d hypersexua l i ty , expressed as an eager ten-

dency to m o u n t o the r ma le or female cats . T h e female cat d isp layed wha t ap-

F IGURE 8. Comparison between average cumulative percentages of motor activity in cats with

unilateral caudate nucleus removal, bilateral caudate nuclei removal, frontal cortical ablation,

and sham operation. The bars illustrate the averages, for each of the four groups, of all individ-

ual percentages in all 24-hour recording sessions shown in Figure 9. The vertical lines represent

the standard deviations.

AVERAGE CUMULATIVE MOTOR A C T I V I T Y

[] SHAM-OP

g UN I LATERALS

| BlLATERALS

FRONTALS

0

Avg

.%

(24

-hr

rec

ord

ing

s )


I

SHAM 0 5

a UN I LATERALS • BILATERALS a FRONTALS

35 45 115 170 60 75 90 DAYS AFTER LESION

FIGURE 9. Effect of unilateral or bilateral caudate nuclei removal and of bilateral frontal cortical ablation upon the amount of motor activity of cats. The bars illustrate the average percentages of time spent by the animals (five with unilateral caudate removal, five with bilateral caudate removal, five with frontal cortical ablation, and three sham-operated) in motor activity during 24-hour recording session on the postoperative days indicated in the abscissa. The vertical lines represent the standard deviations.

peared to b e p e r m a n e n t estrus b e h a v i o r , cons i s t ing of lordos is ( spon tane -

ously , or after gent le s t roking of the b a c k ) , dev ia t ion of the tail away from the

s ide s t roked, and h i n d l i m b b e n d i n g wh i l e t reading markedly .

UNILATERAL ACAUDATE CATS

T h e mos t r emarkab le f inding in these cats was the relat ive a b s e n c e of any

neuro logica l i m p a i r m e n t or dev ian t behav io r .

NEUROLOGY. Pos topera t ive recovery in these cats w a s uneventful . T h e y

s tood and wa lked b y the first to s econd day. Ini t ia l ly , five cats e x h i b i t e d

co n t r a l a t e r a l ^ a s l ight pares i s , w h i c h las ted for 2 to 4 days (except in o n e cat

w h o man i fes t ed a m a r k e d pares i s bu t w h o s e les ion enc roached u p o n the

whi te mat te r of the frontal a reas) . T h e s ame cats t ended to wa lk or rotate

toward the s ide ipsi la teral to the les ion ; th is las ted for 1 to 2 days and was ac-

c o m p a n i e d b y a t e n d e n c y to devia te the head ipsi lateral ly.

T h e a b s e n c e of the C P R on the s ide contralateral to the caudate ab la t ion w a s

a cons tan t and relat ively long- las t ing f inding. T h e C P R w a s recovered in all

cats b e t w e e n 15 to 20 days , except in the o n e cat w i th frontal pole damage .

H o w e v e r , it took abou t 50 days to recover in four cats , two of w h i c h h a d 1 0 0 %

caudate remova l , the r e m a i n i n g two still b e i n g al ive.

Av

g.%

of

mo

tor

ac

tiv

ity

(24

-hr

rec

ord

ing

s)

o

o

o

o

o

o

I 1

1 1

1 1

1 1

1 1

1


BEHAVIOR. N o n e of the U A c cats ever exh ib i t ed the C A s . S i x an imals

s h o w e d very t ransi tor i ly (up to the th i rd pos topera t ive day) , w e a k v isua l

t racking m o r e marked ly toward the s ide of the l es ion . Thereaf ter , these an-

imals b e h a v e d l ike S h O cats . T h e y exh ib i t ed a s l ight , incons tan t hyperac t iv i ty

(see B A c cats and F igure 9) bu t no s leep reduc t ion (80) . B y the third pos topera-

t ive day they b e g a n to eat efficiently.

CATS WITH FRONTAL CORTICAL ABLATION

NEUROLOGY. All the cats exh ib i t ed modera te pares i s of all four l i m b s and

hypes thes i a las t ing from 6 to 12 days and dec l in ing s lowly thereafter . Mos t of

the an imals exh ib i t ed an exaggera ted ex tensor tonus of the l i m b s , part icularly

in the fo re l imbs , such that the i r hype rex t ended fore l imbs w o u l d t ight ly ad-

here to the thorax w h e n the cat w a s he ld b y the scruff of the neck . T h i s

decreased or d i sappeared over a pe r iod of abou t 1 m o n t h . T h e C P R w a s abol -

i shed ini t ia l ly in all four l i m b s ; i ts apparent recovery after 3 to 4 m o n t h s is

present ly b e i n g further s tudied and d o c u m e n t e d .

BEHAVIOR. E igh t cats did not exh ib i t any of the e l ements of the C A s except

for the p re sence , in four an ima l s , o f the v isua l t racking desc r ibed above for

the B A c cats ; h o w e v e r , th is b e h a v i o r was less marked and lasted for a shor ter

per iod (an average of 15 days) than in the B A c cats . I ndeed , these an imals

were t imid and t ended to run away and h ide as soon as p laced u p o n the floor

or counters in the lab . T h e b ra ins for w h i c h the ex tent of the ab la t ion has b e e n

verif ied and desc r ibed above b e l o n g e d to an ima l s in th is g roup .

T w o cats , w h i c h are current ly a l ive , t ransi tor i ly exh ib i t ed s o m e of the fea-

tures of the C A s ; h o w e v e r , these coex is ted w i t h aggress ion as desc r ibed

be low. Bo th cats approached the inves t iga tor , bu t no t ob jec t s , for 13 and 30

days , respect ive ly , bu t d i sp layed little o f the s te reo typed act ivi ty s een in B A c

cats. T h e cat e x h i b i t i n g the v isua l approach ing for 30 days did not s h o w any

o ther form of approach or any t endency toward exaggera ted t reading or pur-

r ing. T h e o ther an imal t ended later, w h e n the v isua l fol lowing had disap-

peared , to develop marked purr ing and tacti le approach . T h e s e two cats h i s sed

and at tacked w h e n hand led and part icularly w h e n touched , even gent ly , on

the back ; three inves t iga tors have b e e n b i t t en several t imes b y o n e of t hem.

T h e B F r an imals started ea t ing and were ea t ing efficiently b y 4 and 7 days

after surgery, respect ive ly . T h e y e x h i b i t e d m o t o r hyperac t iv i ty a l though less

marked than B A c cats (see B A c cats and F igure 9) and a modera te , sus ta ined ,

reduc t ion in the amoun t o f s leep (49) .

SHAM-OPERATED CATS

N o p e r m a n e n t neuro logica l or behav io ra l abnormal i t i e s we re found in these

an imals . T h e C P R w a s al tered, coarse , or ab sen t in one or m o r e l i m b s for a few


days i m m e d i a t e l y after surgery bu t w a s fully recovered in all an ima l s b y the

ten th day.

E l e c t r o e n c e p h a l o g r a m

The E E G w a s essent ia l ly no rmal in all the cats . In s o m e B A c ca ts , dur ing the

first w e e k , i sola ted burs t s of h igh vol tage s low w a v e s occas ional ly in ter rupted

the d e s y n c h r o n i z e d cort ical E E G dur ing arousal , par t icular ly over fron-

topar ie ta l areas . N o such a b n o r m a l burs t s w e r e o b s e r v e d in U A c or B F r cats .

Thereaf ter , b o t h the cort ical and subcor t ica l E E G appeared to b e normal in all

B A c cats as s h o w n b y an adequa te d isplay of all the E E G events character is t ic

of the E E G s l eep -wak ing s tates and a good E E G react iv i ty to barb i tu ra tes and

a m p h e t a m i n e s (marked sus ta ined d e s y n c h r o n y of the E C o G ) .

DISCUSSION

General Effects of Caudate Nuclei A b l a t i o n

SURVIVAL

To our k n o w l e d g e , there have not b e e n any pub l i ca t ions repor t ing resul ts in

chronica l ly m a i n t a i n e d cats fo l lowing bi la teral , s imu l t aneous r emova l of the

caudate nuc le i in the a b s e n c e of dorsolateral cort ical damage . Met t le r and col-

laborators ( 2 4 , 5 1 , 5 3 , 5 4 ) d id per form caudate nuc le i ab la t ion in cats in w h i c h

cortical areas had b e e n prev ious ly r e m o v e d . H o w e v e r , the i r an imals d ied

w i t h i n 3 to 5 days of the caudatal l e s ions . I l l -defined me tabo l i c d i s tu rbances in

w h i c h the s t r ia tum w a s though t to b e specifically invo lved (54) w e r e b l a m e d

for the dea ths . Sat is factory , ch ron ic survival w a s o b t a i n e d later (73 ,74) in cats

w i t h subto ta l ab la t ion of the cauda tes after r emova l of aspects of the dorsola-

teral cor tex in a prepara tory s tage . Fox et al. (21) also repor ted an " e x t r e m e l y

h igh mor ta l i ty" in an unsuccessfu l a t tempt to per form, one - s t age , b i la tera l ,

caudate l e s ions , and recen t ly Har ik and Mor r i s (26) found that cats w i t h large

caudate l e s ions could no t b e m a i n t a i n e d b e y o n d 3 days .

T h e p resen t resul ts suppor t ne i the r the above f indings no r the concep t of

caudate nuc le i par t i c ipa t ion in vital me tabo l i c p rocesses s ince our B A c cats

could b e kept al ive indef in i te ly b y us ing the rou t ine t e c h n i q u e s w h i c h w e

have p rev ious ly deve loped for the care of cats w i th b ra in l e s ions (75) . In fact,

the acaudate cats we re not any m o r e difficult to m a i n t a i n than the B F r cats .

COGNITIVE DEFICIT

T h e p resen t resul ts also fai led to dupl icate the obse rva t ions b y Met t l e r and

col laborators (24 ,51 ,53 ,54 ) that bi la teral removal of the cauda tes p roduces such

296 Jaime R. Villablanca and Robert J . Marcus

a profound deficit in the cat as to warran t desc r ib ing t h e m as " rea l vege tab le s ,

comple te ly out of contac t , w a n d e r i n g a imles s ly" (Hea th , in 54 ) . In the acute

pos topera t ive pe r iod , there we re mani fes ta t ions of lack of awareness and faulty

re la t ionsh ip w i th the e n v i r o n m e n t ; bu t , s ince the s igns d i sappeared soon af-

terward , it is difficult to conce ive of t h e m as b e i n g specific effects of caudate

funct ion deficit. Ra the r , they m i g h t b e cons ide red as unspeci f ic mani fes ta -

t ions of t rans i tory p rocesses ( edema , neuro logica l shock , i r r i ta t ion) c o n c o m i -

tant w i th any ex tens ive b ra in surgery. T h o m p s o n and Met t le r ' s cats (73,74) d id

no t exh ib i t the deficit of Met t l e r ' s ear l ier an imals ; h o w e v e r , they still s h o w e d

" tota l and p ro longed failure to l e a rn" rather s imple tasks . P re l iminary results

in our B A c cats (81) do no t suppor t the latter f indings bu t sugges t a persevera-

t ive defect in pe r formance in l ine wi th the behav io ra l r ig id i ty mani fes ted in

the C A s . M o r e subt le defects in the cogn i t ive sphere have b e e n repeatedly

demons t ra ted in cats ( 1 6 , 1 7 , 2 1 ) , rats ( 5 7 , 6 6 , 8 8 ) , and m o n k e y s (5 ,14 ,23 ,63 ) w i th

restr icted caudatal l e s ions .

BEHAVIORAL AROUSAL AND MOTOR ACTIVITY

(INCLUDING UAC AND BFR CATS)

E n h a n c e d behav io ra l arousal and hyperac t iv i ty have long b e e n cons ide red a

result o f caudate l e s ions . A l though there are quant i t a t ive repor ts d e m o n -

strat ing hyperac t iv i ty in m o n k e y s (13 ,60) and rats (35 ,87) fo l lowing caudate

l e s ions , the repor ts of hyperac t iv i ty in the cat have b e e n b a s e d on n o n q u a n -

tified v isua l obse rva t ions (53) . T h u s , the p resen t s tudy appears to b e the first

to quant i fy the p re sence of m o t o r hyperac t iv i ty in the cat fo l lowing large

caudate l e s ions .

T h e l i terature is less c lear regard ing poss ib l e par t ic ipa t ion of l es ions

res t r ic ted to the frontal cort ical areas in p roduc ing m o t o r hyperac t iv i ty in cats .

As far as w e are aware , the on ly quan t i t a t ive s tudy is that of L a n g w o r t h y and

Rich te r (40) ; these authors repor ted a large increase in mo to r act ivi ty in cats

wi th frontal l es ions (in w h i c h s o m e damage to the s t r ia tum was also inf l ic ted) .

O t h e r s tudies dea l ing wi th frontal l e s ioned cats e i the r d id no t demons t ra te

any hyperac t iv i ty (43) or repor ted its p resence in frontal cats in w h i c h , m o s t

p robab ly , the caudate nuc le i we re also invo lved in the les ion (47 ,85) . T h u s ,

Bru tkowsk i conc ludes in h i s r ev i ew (8) that hyperac t iv i ty has no t b e e n re-

por ted in cats w i th frontal l e s ions . T h e p resen t data demons t ra te the par t ic ipa-

t ion of frontal cort ical areas in the control o f the level of m o t o r act ivi ty in the

cat. Therefore , the cats w i t h frontal l es ions appear to b e s imi lar in th i s respect

to the rhesus m o n k e y ( 3 3 , 6 0 , 6 4 ) .

S ince the inc reased m o t o r act ivi ty in our an imals w a s demons t ra t ed in the

m o n o t o n o u s e n v i r o n m e n t o f the record ing cage , i t ind ica tes that hyperac t iv i ty

exis ted i ndependen t l y of the a m o u n t of env i ronmen ta l s t imula t ion . W h e t h e r

the cats also man i fe s t ed inc reased m o t o r r e s p o n s i v e n e s s v i s -a -v is env i ron-

menta l s t imula t ion (hyperreac t iv i ty) , ha s no t b e e n demons t r a t ed quan t i t a -


t ively b y the p resen t expe r imen t s . H o w e v e r , the behav io ra l obse rva t i ons

s t rongly sugges ted that th i s w a s the case for the B A c cats only. T h e p r o b l e m of

hyper reac t iv i ty ve r sus hyperac t iv i ty in an ima l s w i t h caudate l e s ions has b e e n

prev ious ly d i scussed b y o thers (35 ,53) . K i r k b y (35) demons t r a t ed a further

increase in m o t o r act ivi ty in h i s caudate l e s ioned rats and Met t le r and Met t le r

(53) o b se rved e n h a n c e d hyperac t iv i ty in the i r caudate l e s ioned cats fo l lowing

env i ronmen ta l da rken ing for the rats or b l indfo ld ing of the ca ts , respec t ive ly .

S i n c e b o t h are noc tu rna l spec i e s , the authors in te rpre ted the change as a

hyper reac t ion to the decrease in i l lumina t ion .

T h e resul ts o f ou r s leep-wakefu lness s tud ies (49 ,80 ) , u sed as an addi t iona l

cr i ter ion to assess the level of arousal in the p resen t ca ts , fit wel l w i t h our data

on m o t o r act iv i ty . T h u s , the reduc t ion i n s leep w a s also greater dur ing the

first pos topera t ive m o n t h in the B A c cats ; it w a s less in the cats w i t h frontal

l e s ions dur ing the s a m e pe r iod , b u t in the latter an imals it b e c a m e m o r e sus-

ta ined thereafter . T h e s leep reduc t ion in the U A c cats w a s no t s tat is t ical ly s ig-

nificant.

Therefore , w h e n all three cr i ter ia to assess behav io ra l arousal and m o t o r

act ivi ty (see M e t h o d s ) are taken toge ther , our resul ts sugges t that b o t h caudate

and frontal areas (a l though the caudate nuc le i in a m o r e m a r k e d m a n n e r ) are

invo lved in the control o f the arousal -act iv i ty levels in the cat, w i t h hyper reac -

t ivi ty p r o b a b l y b e i n g ano the r factor in the acaudate an ima l s . W e have pre-

v ious ly pos tu la ted (78) a forebra in sys t em exer t ing an impor tan t cont ro l over a

powerful arousal -ac t iv i ty m e c h a n i s m loca ted p r o b a b l y at vent ra l d i encepha l i c

level. T h e p resen t e x p e r i m e n t sugges t s that the caudate nuc le i and frontal cor-

tical areas m a y b e a part of such a sys tem.

CAUDATE ABLATION IN MONKEYS

At tempt s to abla te the caudate in the m o n k e y have m e t w i t h re la t ively m o r e

success than in the cat. E v e n t hough T e u b e r et al. (72) repor ted that the i r at-

t empt to abla te the caudate bi la teral ly in the m o n k e y w a s a " d i s a s t e r " ( s ince

all the i r an ima l s d ied shor t ly after surgery) , two o ther au thors w e r e , ap-

parent ly , m o r e successful . K e n n a r d (32 ,33) l e s ioned the cauda te in a n u m b e r

of m o n k e y s , e i the r pene t ra t ing frontal areas 6 or 8 or u s ing a t ranscal losal

approach . T h e neuro log ica l and behav io ra l c h a n g e s obse rved in the m o n k e y s

wi th des t ruc t ion of areas 6 or 8 were in te rpre ted as b e i n g due to the cor t ical

les ion , w h e r e a s the a b s e n c e of s y m p t o m s in the an imals w i t h the mid l i ne

pene t ra t ion w a s cons ide red as e v i d e n c e that cauda te nuc le i ab la t ions do no t

p roduce any effects. S u c h a conc lus ion does no t s e e m fully jus t i f ied s ince in

K e n n a r d ' s m o n k e y s :

a. T h e r e w a s no t any clear s t a tement regard ing the ex tent o f the caudatal

ab la t ion , except for m e n t i o n i n g that " o n l y the head w a s l e s i o n e d in all

of t h e m " and that in o n e of t h e m the caudate l e s ions " w e r e sma l l . "


b . T h e les ions we re pe r fo rmed in two s tages .

c. N o clear in format ion w a s prov ided regarding the t ime of survival and

the m e t h o d used in s tudy ing the an imals .

d. S o m e of the m o n k e y s deve loped ep i lepsy .

Thus we th ink , the impor tan t conc lus ion quo ted above lacks exper imenta l

ev idence .

D e n n y - B r o w n (15) repor ted a r emarkab le change in b e h a v i o r in the m o n k e y

after bi la teral des t ruc t ion of the caudate nuc leus p roduced v ia a pene t ra t ion in

the prefrontal cortex. T h e m a i n effects w e r e the d isp lay of a " c o m p u l s i v e

behav iora l p r o m e n a d e " w h i c h " w a s related ent i re ly to the p resence of o t h e r s "

and of a n u m b e r of v isual c o m p u l s i o n s " o f w h i c h v isua l cha l lenge ( the leap ing

au tomat i sm) , v isua l avo id ing (facing the wal l ) , and compu l s ive v isual i nves -

t i ga t i on" were the m o s t o b v i o u s . H e conc luded that " i n s o m e m a n n e r bi la tera l

l es ion of the h e a d of the caudate nuc leus re leases v isual ly de t e rmined cort ical

a u t o m a t i s m s " and that " t h e caudate nuc leus m u s t normal ly b e c o n c e r n e d in

the in tegra t ive ba lance of cort ical r e sponses to env i ronmen ta l contact s t imul i

wi th those of v isua l con t ex t . " Unfor tunate ly , there were no t any h is to logica l

reports on the b ra ins o f these an imals . Recen t ly , W a t k i n s (83) in terpre ted

these results as ind ica t ive that the caudate " i s conce rned wi th the control of

au tomat ic mo to r pa t terns and visual ly de t e rmined r e s p o n s e s . "

SUBTOTAL CAUDATE LESIONS

N u m e r o u s o ther caudate l es ion s tudies in the cat (2 ,17 ,21 ,25 ) and in o ther

spec ies (5 ,14 ,23 ,63 ,66 ) have b e e n repor ted (see also Ref . 4 2 ) . In mos t of

these s tud ies , the l e s ion has b e e n e i ther relat ively small or purpose ly local ized

to only parts of the caudate . T h e resul ts of those s tudies , w h i c h are per t inen t

to the presen t obse rva t ions , are d i scussed be low. W e wou ld l ike to s t ress ,

howeve r , the n e e d for pe r forming ex tens ive or total l es ions in order to evalu-

ate caudate funct ion. T h i s n e e d w a s r ecogn ized early b y Met t le r and col labo-

rators after the i r obse rva t ion that l es ions des t roy ing less than 5 0 % of the

caudate and p u t a m e n failed to p roduce readi ly obse rvab le changes (51) . T h i s

is further e m p h a s i z e d b y the p resen t f indings that comple te uni la teral r emova l

of the caudate p roduces n o long- las t ing gross neuro logica l or behav io ra l

changes and that in cats w i th less than 8 0 % of the caudate r emoved , the C A s

was b o t h less marked and shor ter in dura t ion than in the an imals w i t h larger

ab la t ions .

" C o m p u l s o r y A p p r o a c h i n g " S y n d r o m e

C A s VERSUS OBSTINATE PROGRESSION

A d i scuss ion of the m a i n neurologica l behav io ra l change obse rved in B A c

cats necess i t a t es first that a clear different ia t ion b e m a d e b e t w e e n the C A s and


the obs t ina t e p rogress ion p h e n o m e n o n . Acco rd ing to the or ig ina l desc r ip t ion

b y Ba i l ey and D a v i s (3) and our o w n expe r i ence w i t h d iencepha l i c cats (78) ,

the m a i n features charac te r iz ing obs t ina te p rogress ion are

a. a pe rvas ive t e n d e n c y to wa lk incessan t ly ; i f the cat encoun te r s an o b -

stacle , he con t inues p u s h i n g aga ins t i t unt i l e i the r the obs tac le g ives

way , the an imal acc identa l ly gets started in ano the r d i rec t ion , or , i f the

p ropuls ive effort is too s t rong, p o s s i b l y infl icts se r ious damage (3 ,78)

to h i s forehead

b . a lack of awareness to cl i f f—hence the an ima l can eas i ly walk out of an o p e n cage or wa lk over the edge of a coun te r or tab le

c. a b s e n c e of any t endency to fol low the obse rve r or to a t tend to o ther

env i ronmen ta l s t imul i .

Fur the rmore , obs t ina t e p rogress ion is usual ly a t rans ien t p h e n o m e n o n , las t ing

at m o s t a few w e e k s . F ina l ly , wh i l e s o m e of the reports (40 ,47 ,53) sugges t a

t endency for the l e s ions p roduc ing it to c luster in rostral areas of the b ra in ,

f indings b y o ther inves t iga tors ind ica te that l e s ions of m a n y o ther areas

(3 ,50 ,76 ,78 ) can also p roduce the synd rome ; in fact, the m o s t marked and las t ing

obs t ina te p rog res s ion has b e e n obse rved in an imals fol lowing ex tens ive n e o -

decor t ica t ion or comple te r emova l of the cerebra l h e m i s p h e r e s (see Ref . 78) .

B e c a u s e of the a b o v e reasons w e cons ide r obs t ina te p rogres s ion to b e an

unspecif ic c o n s e q u e n c e of the remova l o f w idesp read i n h i b i t o r y te lencepha l ic

inf luences ac t ing u p o n a bas ic act iv i ty m e c h a n i s m , p r o b a b l y loca ted at a lower

d iencepha l i c level (78) .

W e did no t obse rve obs t ina te p rogress ion in our B A c cats .

In con t rad i s t inc t ion , the fo l lowing features are essen t ia l to the C A s :

a. t he d i sp l acemen t of the cat e l ic i ted b y external s t imul i and s u b s e q u e n t

m o v e m e n t b e i n g s t imulus b o u n d ; even w i t h th is qual i f icat ion,

wa lk ing is on ly o n e of the c o m p o n e n t s of the C A s

b . s te reo typed f r iendl iness ( compulsory s t imulus a t t achment , r u b b i n g ,

pur r ing , voca l iza t ion , and t reading)

c. m o n o t o n o u s , long- las t ing synd rome presen t in an imals vi r tual ly free

of o ther neuro log ica l deficits

SPECIFICITY OF THE CAUDATE ABLATION IN PRODUCING THE CAS

Severa l obse rva t i ons in the p resen t expe r imen t s as wel l as b y o thers

( 2 , 1 3 , 1 5 , 5 1 , 5 3 ) sugges t that large caudate l e s ions m a y play a centra l role in de-

t e r m i n i n g the C A s .

In the p resen t s tudy:

a. T h e C A s w a s obse rved in all the B A c cats .

b . It w a s a long- las t ing or p e r m a n e n t c h a n g e , t h e r e b y ind ica t ing that it


was not due to any nonspec i f ic , early pos topera t ive , artifact and that

the funct ional a l terat ion p roduced was no t eas i ly c o m p e n s a t e d for b y

o ther b ra in s t ructures .

c. The re we re on ly two les ions c o m m o n to all o f the cats , i . e . , the bi la t -

eral caudate remova l and the mid l ine cor t ica l -ca l losum les ion p roduced

in order to v isua l ize the caudate . T h e a b s e n c e of C A s in the sham-

opera ted cats demons t r a t ed that the latter l es ion canno t b e cons ide red

as b e i n g r e spons ib l e for the C A s ; fur thermore , reports in the l i terature

ind ica te that l e s ion ing the gyrus c ingul i r eg ion a lone t end to p roduce a

behav io ra l change jus t the oppos i t e o f the C A s (9 ,84) .

d. F inal ly , the genera l i ty of the C A s cont ras ted w i t h the fact that addi-

t ional l e s ions to o the r b ra in areas were small and d i s t r ibu ted at ran-

d o m a m o n g the cats .

Neurobehav io ra l changes w h i c h m a y b e cons ide red as f ragments of the

C A s have b e e n o b s e r v e d b y o ther authors after per forming caudate l e s ions .

A l though obs t ina te p rogress ion and o ther p h e n o m e n a ("curs ive hype rk ine -

s i a , " " l e ap ing p h e n o m e n o n , " " r u n n i n g p h e n o m e n o n " ) related to obs t ina te

p rogress ion we re consp i cuous in the f indings of Met t le r and col laborators

( 2 4 , 5 1 , 5 3 , 5 4 ) , there were b r i e f descr ip t ions of the cats " fo l lowing of s lowing

m o v i n g ob jec t s in the field of v i s i o n " and/or " fo l lowing m o v i n g o b j e c t s " (24)

w h i c h sugges t the v isua l t racking or even the actual fo l lowing obse rved in our

an imals .

In a s tudy of caudate nuc le i s t imula t ion , Aker t and A n d e r s o n (2) per formed,

as a control , e lectrolyt ic l e s ions o f b o t h caudates in two cats ( amoun t of the

damage unspec i f ied , bu t no t large) . T h e y no t i ced that " t h e formerly self-

m o v i n g cat s h o w e d n o w no spon taneous m o v e m e n t of i ts o w n . Ins tead he

could b e set in m o t i o n b y senso ry s t imula t ion l ike an au tomata" ; th is lasted

for abou t a w e e k and was a c c o m p a n i e d b y exaggera ted t read ing m o v e m e n t s .

In expe r imen t s de s igned to evaluate the inf luence of the caudate u p o n b o d y

growth , Klosovsk i i and Bo lzh ina (37) m e n t i o n e d that 2 - 3 i - m o n t h - o l d dogs

" m o v e d towards m o v i n g ob jec t s or towards wa lk ing h u m a n b e i n g s " after

bi lateral l es ions of the caudate , pe r formed th rough a mid l ine approach .

Li les and Dav i s (46) desc r ibed a the to id m o v e m e n t s in the fore l imbs of cats

wi th uni lateral electrolytic l e s ions of the anteroventra l part o f the caudate .

The i r descr ip t ion fits the features of the exaggera ted t reading or k n e a d i n g

m o v e m e n t s w h i c h we re o b s e r v e d on our B A c cats . H o w e v e r , w e prefer no t to

label these m o v e m e n t s as b e i n g a the to id b e c a u s e they:

a. lacked the wr i th ing charac ter of typical a the tos is

b . we re res t r ic ted to the musc les of the fore l imbs

c. we re marked ly rhy thmic in na ture wi th one l i m b per forming an ex ten-

s ion m o v e m e n t wh i l e the o ther w a s e n g a g e d in a flexor d isplay or v ice

versa

d. t ended to occur toge ther w i th the o ther mani fes ta t ions of the C A s


Therefore , w e in terpre t these m o v e m e n t s as b e i n g an ex t reme exaggera t ion of

the k n e a d i n g or t read ing p a w m o v e m e n t s seen in intact , f r iendly cats .

Final ly , the obse rva t i ons of D e n n y - B r o w n in m o n k e y s (see above ) are also

relevant to the p resen t f indings i n B A c cats .

THE QUESTION OF FRONTAL INVOLVEMENT IN THE CAS

T h e r e are several r easons for jus t i fy ing the poss ib i l i ty of frontal cort ical

damage par t ic ipa t ion in the genes i s o f the C A s in B A C cats . Firs t , there are

impor tan t ana tomica l c o n n e c t i o n s b e t w e e n the caudate nuc le i and frontal cor-

tical areas ( 1 1 , 1 8 , 2 9 , 3 0 ) . S e c o n d , there is good ev idence ind ica t ing that the

caudate nuc le i and the frontal cor tex are s imi lar ly invo lved in the per formance

of a n u m b e r of behav io ra l tasks , par t icular ly de layed r e sponse type of tasks

(5 ,23 ,63 ) . D ivac (16) h a s s t ressed these s imi lar i t ies to the po in t of hypo the -

s iz ing that the caudate nuc leus and frontal cor tex are phys io log ica l ly e q u i p o -

tential . T h i r d , there are reports in the l i terature sugges t ing that s o m e of the

c o m p o n e n t s of the C A s m i g h t b e p resen t in cats w i t h l e s ions invo lv ing pre-

dominan t ly frontal cort ical areas ( 2 8 , 4 7 , 6 5 , 8 5 ) .

Fea tures of the C A s obse r ve d b y s o m e authors w o r k i n g w i t h frontal l e -

s ioned cats we re the fo l lowing of the inves t iga to r b y the an ima l s and the

v isua l t racking of ob jec t s in pendula r m o t i o n . In the l i terature , exaggera ted

v isua l t racking has b e e n desc r ibed b o t h in an ima l s r ece iv ing ex tens ive frontal

cort ical ab la t ions (28 ,47 ,85 ) as wel l as in cats r ece iv ing se lec t ive frontal cort ical

l e s ions (65) . W h i l e the former expe r imen t s m a y leave s o m e doub t s (see be low)

regarding an even tua l caudate i nvo lvemen t in the ab la t ion , resul ts of

restr icted frontal l e s ions are par t icular ly c o n v i n c i n g of the fact that there is in-

dependen t frontal par t ic ipa t ion in the genes i s o f v i sua l t racking , s ince in

these expe r im e n t s (65) there w a s adequa t e h is to logica l demons t r a t ion of a b -

sence of gross caudate damage .

T h e con t r ibu t ion of damage to the frontal cor tex in the genes i s of the C A s

appears less l ikely . A l imi ta t ion in eva lua t ing the effect o f caudate versus

frontal-cort ical l e s ions u p o n behav io r s s imi lar to the C A s i s the fact that in

several papers repor t ing such behav io r s (part icularly the older o n e s ) , little or

no a t ten t ion w a s g iven e i the r to the d a m a g e w h i c h the l e s ions could have

infl icted u p o n the caudate nuc le i or to the pers i s tance of the abnormal i ty .

T h u s , W h e a t l y (85) pe r fo rmed frontal ab la t ions in five cats , two of w h i c h

exh ib i t ed v isua l fo l lowing of u n d e t e r m i n e d dura t ion . T h e s e two cats had the

les ions ex t end ing the m o r e caudal ly. T h e au thors s ta ted that "al l s t ructures an-

ter ior to the rostal e n d of the gyrus ec tosy lv ius w e r e d a m a g e d . T h e anter ior

surface of the r e m a i n i n g por t ion of the b ra in w a s necro t ic and d a m a g e d . " A

s imilar case p robab ly occur red in the M a g o u n - R a n s o n ser ies (47) in w h i c h s ix

out o f e igh t frontal cats " s h o w e d a p r o n o u n c e d d i spos i t ion to fol low the o b -

se rver . " T h e p h e n o m e n o n w a s shor t - las t ing in two cats and in the r e m a i n i n g

four an ima l s , in w h i c h it pers i s ted up to 4 w e e k s , the ab la t ion ex t ended more


caudally. I ndeed , accord ing to the au thors ' descr ip t ion of the b ra ins , b o t h in

the lat ter s ix cats and in W h e a t l e y ' s two cats , all s t ructures in front o f p lane A

12 to A 14 we re r e m o v e d . S i n c e the caudate ex tends at least to A 2 0 . 0 , it

follows that the les ion in those an imals invo lved the caudate heads . F ina l ly , in

the s tudy b y J e a n n e r o d et al. (28) in w h i c h a spatula w a s used to separa te the

frontal po les , it is expl ic i t ly s ta ted that in s o m e of the cats , the spatula w e n t

th rough the head of the caudates .

O t h e r s tudies of frontal cats have no t repor ted any behav io ra l change related

to v isual fo l lowing ( 8 , 3 9 , 6 8 , 8 2 ) . O f part icular in teres t i s a recent report b y

W a r r e n et al. (82) . In th i s s tudy, an excel lent ana tomica l control of the l e s ions

ind ica ted an a b s e n c e of caudate damage . In the latter s tudy the cats were m o r e

inc l ined to e scape ra ther than to approach ; to quo t e from the authors : " t h e s e

differences sugges t that frontal cats are m o r e indifferent toward h u m a n s than

normal c a t s . "

Severa l o ther obse rva t ions in the p resen t cats further rule out any impor tan t

con t r ibu t ion of a frontal-cort ical l e s ion in the p roduc t ion of the C A s . In the

B A c cats , the frontal-cort ical areas appeared to b e essent ia l ly intact , ana tomi -

cally and phys io logica l ly , as j u d g e d b y the fo l lowing cri ter ia:

a. E lec t rophys io log ica l expe r imen t s repor ted e l sewhere (44) have d e m o n -

strated that the spon taneous firing pa t te rns of cells o f the per ic ruc ia te

cor tex, e i ther homola te ra l or contralateral to a uni la tera l caudate re-

mova l , do not differ s ignif icant ly from each o ther or from firing pat-

terns of cells of the same areas in in tact b ra ins .

b . T h e C P R , in w h i c h the frontal cor tex plays an impor t an t role (4) , w a s

recovered in all B A c an ima l s at a t ime w h e n the C A s was still at i ts

m a x i m u m .

c. Ana tomica l damage to the in ternal capsule in B A c cats w a s s ignif icant

only in one case , b i la teral ly , and in two cases , uni lateral ly; in these

an imals , the C A s was ne i the r m o r e marked n o r longer las t ing. T h e r e -

fore, it appears that direct or ind i rec t damage to frontal-cort ical areas

was not r e spons ib l e for the C A s in our B A c cats .

Fur the rmore , our obse rva t ions in the B F r cats do no t l end any suppor t to the

no t ion that frontal damage m i g h t con t r ibu te to the behav io ra l changes of B A c

cats . In fact, e igh t out of the ten B F r an imals s tud ied d id no t exh ib i t any of the

s igns of C A s ; on the contrary , m o s t o f these an imals t ended to t imid ly run

away from the inves t iga tor . T h e r e m a i n i n g two cats exh ib i t ed a b izar re b e h a v -

ior s ince they s h o w e d e l emen t s o f b o t h aggress ion and approach . Severa l in-

ves t iga tors have o b s e r v e d aggress ion and/or rage to b e the m a i n behav io ra l af-

fect ive change consecu t ive to frontal l es ions in cats (see Ref . 8 ) . B ru tkowsk i

r ev i ewed those repor ts (8 ) , w h i c h , moreove r , are cons i s t an t w i t h h i s o w n

f indings , and conc luded that the rage or aggress ion b e h a v i o r p resen t in such


an ima l s i s p roduced b y frontal l e s ions res t r ic ted to the r eg ion o f the med ia l surface i m m e d i a t e l y rostral to the g e n u of the corpus ca l losum. T h i s last fea-ture of frontal l e s ions p roduc ing aggres s iveness sugges t s that the ab la t ion in our two cats d i scussed a b o v e m i g h t h a v e ex tended m o r e pos te r ior ly than in the r e m a i n i n g B F r cats w i th direct or indi rec t damage b e i n g infl icted u p o n the caudates .

In conc lus ion , it m a y b e s tated that a m a s s i v e bi la teral caudate l e s ion , in the a b s e n c e o f frontal d a m a g e , i s sufficient to e l ic i t the C A s . H o w e v e r , the reverse does no t appear to ho ld , i . e . , frontal damage a lone i s no t e n o u g h for i ts appearance .

ARE OTHER STRUCTURES INVOLVED IN THE CAS?

B o n d et al. (6) o b s e r v e d e l emen t s of C A s in cats w i th un i - or b i la tera l l e s ions

to pos te r ior septal , fornix, and anter ior tha lamic reg ions . In the a b s e n c e of a

clear s t a t ement regard ing the pe r s i s t ence of the v i sua l fo l lowing, o n e w o n d e r s

w h e t h e r damage to the caudate nuc le i could have occurred in h i s cats . In fact,

the l e s ions in those expe r imen t s we re per formed electrolyt ical ly w i t h ra ther

large need l e s (22 gauge) p roduc ing ex tens ive coagula t ions adjacent to the h e a d

of the cauda tes . Add i t iona l difficulty in in te rpre t ing the resul ts o f B o n d and

co-workers is the fact that over 5 0 % of h i s cats w i th bi la teral septa l -anter ior

tha lamic l es ions suffered c o m a , sudden dea th , p ro longed hype r the rmia , and

hypog lycemia , w h i c h cou ld no t b e accoun ted for b y the authors .

In our B A c cats , the an ter ior tha lamus was no t l e s ioned ; part ial uni la teral

damage to the fornix-septa l area occur red in two cases and bi la tera l d a m a g e

was p roduced in on ly o n e case . Typ ica l h i p p o c a m p a l theta act iv i ty occur red

dur ing arousal and R E M s in all s even B A c cats w i t h h i p p o c a m p a l e lec t rode

p l acemen t s verif ied h is to logica l ly inc lud ing those two w i t h the part ial fornix-

septal damage . T h i s is e lec t rophys io logica l conf i rmat ion of the in tegr i ty of the

septa l - fornix areas s ince it i s w e l l - k n o w n that l e s ions to th i s r eg ion e l imina te

the E E G theta act ivi ty (58) .

At least five o the r g roups of inves t iga tors ( 2 2 , 2 5 , 5 5 , 7 0 , 9 0 ) have per fo rmed large forn ix-pos te r ior septal area l es ions in cats w i thou t obse rv ing any of the c o m p o n e n t s o f the C A s . O n the cont rary , in o n e of these s tud ies (70) the occur rence of rage reac t ion w a s repor ted in 10 out of 17 cats . Par t icular ly per t i -nen t to th is d i scuss ion is the lack of b e h a v i o r r e s e m b l i n g the C A s in exper i -m e n t s of G y b e l s et al (25) and of Fox et al. (22) w h o pe r fo rmed fornix-septa l l e s ions as controls in caudate l es ion s tudies .

In conc lus ion , the fornix-septa l area does no t appear to b e invo lved in the g e n e s i s of the C A s .

In spi te o f the above a rgumen ta t ion , w e th ink that m o r e w o r k is n e e d e d to prove defini tely that the C A s is exc lus ive ly and ent i re ly o w i n g to the caudate nuc le i ab la t ion .


THE NATURE OF THE CAS

T w o h y p o t h e s e s , one neuro logica l , the o ther behav io ra l , m a y expla in the

C A s in B A c cats .

Neurologica l ly , the C A s could conce ivab ly b e though t of as resu l t ing from

the release of the bas i c dr ive to wa lk—prov id ing it is accep ted that the caudate

nuc leus is one of the forebra in s tructures involved in the i nh ib i t o ry control of

such a dr ive . A pure mani fes ta t ion of such a re lease is seen in obs t ina te

progress ion ; howeve r , obs t ina t e p rogress ion , as po in t ed out earl ier , is a non-

gu ided wa lk ing ac t iv i ty w h i c h can occur even in the a b s e n c e of the te lence-

pha lon [as i n the " d i e n c e p h a l i c " cat (78 ) ] , whe reas the C A s i s a s t imulus-

b o u n d event . T h e s t i m u l u s - b o u n d nature of the C A s could conce ivab ly result

from the fact that all s enso ry cort ical areas are intact in the B A c cats and there-

fore can subse rve the role o f " g u i d i n g " the re leased wa lk ing act ivi ty . T h e C A s

could thus b e under s tood as a re leased wa lk ing m e c h a n i s m " g u i d e d " b y any

of the sensory moda l i t i e s . Moreove r , one could even p ropose an e n h a n c e d ef-

fec t iveness o f s enso ry s t imul i c o n d u c i v e to hyperac t iv i ty o f the o r i en t ing reac-

t ion in B A c cats as p roposed b y Ze rn ick i (89) , for frontal cats . In th i s context ,

there are e lec t rophys io logica l data (22 ,38) l ink ing the caudate nucle i to the

p rocess ing of sensory in format ion w h i c h could p rov ide the bas i s for under-

s tand ing the inc reased effect iveness of sensory cues in caudate-abla ted cats .

T h i s poss ib i l i ty wou ld m a k e it even m o r e l ikely that a doub le re lease , that of the

wa lk ing m e c h a n i s m and o f the o r ien t ing reac t ion , could account for C A s . In

th i s context , the p re sence o f hyper reac t iv i ty in the B A c cats could also b e un-

ders tood.

T h e m a i n a rgument aga ins t the above hypo thes i s is that it does no t account

for o ther c o m p o n e n t s of the C A s , e .g . , the genera l f r iendl iness o f the B A c

cats as man i fes ted b y the i r cons tan t pur r ing , t reading , and pecul ia r m e w i n g ,

the tacti le or " b o d i l y con tac t " c o m p o n e n t , the apparent preference for the

an imals to approach an ima te versus i n a n i m a t e ob jec t s , and , finally, the pos-

s ib le , a l though no t as yet we l l -documen ted , change in sexual behav io r . T a k e n

as a w h o l e , the C A s appears to b e a great change in the b e h a v i o r of the cat.

Indeed , th is fe l ine , w h i c h is usual ly cons ide red to b e i n d e p e n d e n t and vo lub le

(45) , is sudden ly changed in to a s te reo typed , pers i s ten t fol lower, and th is

m igh t wel l b e the centra l feature of the C A s .

A n o t h e r a l ternat ive for unde r s t and ing the C A s wou ld b e to c o n c e i v e of it as

an affective c h a n g e , or at least as a c h a n g e in the approach-wi thdrawa l b e h a v -

ioral ba l ance (67) . In such a case the exaggera ted pu r r ing - t r ead ing - rubb ing , at

a m a x i m u m w h e n the an imal r eaches its target , could b e conce ived as b e i n g

part o f the " c o n s u m m a t o r y " aspec t o f the b e h a v i o r , w h e r e a s the dis t ress s igns

w h i c h the B A c cats d i sp lay w h e n a t tempts are m a d e to prevent the i r ap-

p roach ing could b e though t of as mani fes ta t ions of a s t rong " m o t i v a t i o n a l "

c o m p o n e n t . A l though m o s t o f the tes t ing to p rove or d i sprove th is h y p o t h e s i s

r e m a i n s to b e done , it is in te res t ing to no te that several papers have repor ted


i m p a i r m e n t s in avo idance and escape as a c o n s e q u e n c e of a striatal d a m a g e in rats ( 3 6 , 5 6 , 8 8 ) , dogs , r abb i t s (61) , and cats ( 2 4 , 4 1 , 7 4 ) .

Un i l a t e ra l Acauda te Ca t s

T h e ma jo r f inding in the U A c cats w a s the vir tual a b s e n c e of neuro log ica l

and behav io ra l changes . T h i s resul t is in ag reemen t w i t h obse rva t ions b y

Met t le r (51) and b y A n d e r s o n and Aker t (2) in cats w i t h large and smal l uni lat-

eral caudate l e s ions , respec t ive ly . T h i s f inding has at least two impor tan t

impl i ca t ions . T h u s , except for i ts p robab l e par t ic ipa t ion i n the contralateral

C P R , the caudate nuc le i appear no t to have any la teral ized gross n e u r o b e h a v -

ioral funct ion, and it is s t rong ev idence for a large funct ional reserve of th is

s tructure. T h i s should no t b e cons ide red as e v i d e n c e that r e m o v i n g o n e

caudate nuc leus has n o neurophys io log ica l effect wha t soeve r ; in fact, after

such ab la t ion there are changes in the firing pa t te rns of n e u r o n s in the

contralateral caudate (44) , and our o n g o i n g research (81) sugges t s s lowing of

bar p ress ing w i t h the p a w contralateral to the caudatal r emova l .

O t h e r authors have repor ted a m a r k e d and long- las t ing , compul so ry , ip-

s ivers ive c i rc l ing in an ima l s w i th uni la tera l caudate damage ( 1 2 , 5 1 , 8 6 ) . In

s o m e of these expe r imen t s (51 ,86) in w h i c h the caudate w a s approached

th rough the dorsolateral cor tex , the les ion of the latter m i g h t expla in the pos -

tural and wa lk ing asymet ry . A s imi lar m e c h a n i s m migh t expla in the ips

s ive tu rn ing found in the rat after a uni la tera l caudate l e s ion (12) s ince afa^

dant cor t ica l -subcor t ica l f ibers cross th rough the caudate in th i s spec i e s , and

these are b o u n d to b e des t royed b y any cauda te les ion .

E l ec t roencepha log ram

T h e E E G f indings in the p resen t s tudy are marked ly different from those of

K e n n a r d w h o i s , as far as w e are aware , the only o ther au thor h a v i n g e x a m -

i n e d the effects o f bi la teral caudate l e s ions upon the E E G (31) . K e n n a r d

repor ted a m a r k e d , pe rs i s t en t change in the e lec t rocor t icogram in m o n k e y s

w i th c o m b i n e d ab la t ions of the h e a d of the cauda tes and cort ical areas 4 and 6;

these cons i s t ed of in te rmi t ten t h igh ampl i tude burs t s , ma rked s h o w i n g , "un -

e v e n n e s s " and " d i m i n u t i o n of a m p l i t u d e " of the E E G pat terns . H o w e v e r , epi -

lepsy w a s a c o m m o n pos topera t ive occur rence in these an imals . M o r e o v e r , a

h is to logica l account o f the ex tent o f the l e s ions w a s not p rov ided . T h e cort ical

i nvo lvemen t plus the epi lept ic na ture of the l e s ions m a y expla in the E E G ef-

fects in K e n n a r d ' s expe r imen t s . O u r resul ts sugges t , on the cont rary , that the

caudate nuc le i are no t gross ly invo lved in the control of the E E G even t s ; it has

b e e n demons t ra t ed , h o w e v e r (10) , that E E G sp indle w a v e s can b e t r iggered b y

m e a n s of caudate s t imula t ion . In genera l , the normal i ty of poly g raphic pat-

terns in our cauda te - l e s ioned cats is further p roof of the re la t ive in tac tness of

noncauda te s t ructures .


S U M M A R Y

A m e t h o d is de sc r ibed for per forming one-s tage ab la t ion of the caudate

nucle i th rough a mid l i ne approach in cats . T e n bi lateral acaudate cats (BAc) ,

wi th an average of 8 4 % of caudate t i ssue r e m o v e d , we re s tudied for an

average of over 6 m o n t h s ( three , for over 1 year) . In addi t ion , 11 cats w i th un i -

lateral caudec tomy (UAc) , w i t h an average of 9 5 % of the nuc leus r emoved , 10

cats w i th bi lateral r emova l o f the frontal-cort ical areas (BFr) , and four s h a m -

opera ted cats ( S h O ) we re also s tud ied . M o s t cats we re implan ted wi th re-

cord ing e lect rodes in neocor t ica l areas , the h i p p o c a m p u s , the pon t ine ret icular

format ion , n e c k musc l e s , and orb i t s . T h e results o f the gross behav iora l ,

neurologica l , and po lygraphic obse rva t ions are repor ted .

1. A long- las t ing , behav io ra l change labe led " c o m p u l s o r y approach ing syn-

d r o m e " (CAs) was obse rved fo l lowing surgery in all B A c cats . T h i s s y n d r o m e

was charac ter ized b y s te reo typed , " c o m p u l s o r y , " and p ro longed approach ing

or fo l lowing of pe r sons , ca ts , or ob jec t s apparent ly a i m e d at a phys ica l contac t

w i th the approached target and pers i s t ing unde r unusua l cond i t ions . V i sua l

cues (visual t racking w a s a t ransi tory , early c o m p o n e n t of the C A s ) were mos t

effective in e l ic i t ing the C A s ; acoust ic and tacti le s t imul i we re also adequa te .

O t h e r c o m p o n e n t s of the C A s we re marked pass iv i ty , marked ly exaggera ted c ^ I m b k n e a d i n g or t read ing , m a r k e d pur r ing , and hyperreac t iv i ty . T h e

a i t u d e and dura t ion o f the C A s appeared to b e related to the ex ten t o f the

Kffllation, a l though s o m e of its bas ic features pers i s ted for over a year . T h e C A s

was never obse rved in the S h O cats .

2 . B A c cats we re r emarkab ly free of neuro logica l and gross behav io ra l defi-

ci ts . Except for s o m e shor t - las t ing deficits , p robab ly nonspec i f ic , dur ing the

early pos topera t ive pe r iod (motor w e a k n e s s , ab sence of contact p lac ing reac-

t ion , faulty awareness , defect ive ea t ing and d r ink ing , and h y p o s o m n i a ) , on ly

a modera te hyperac t iv i ty w i t h a t endency toward h y p o s o m n i a , along w i t h the

C A s , pers i s ted in the chronic s tate .

3. In all U A c ca ts , there w a s an a b s e n c e of any p e r m a n e n t gross neuro logica l

or behav iora l changes inc lud ing the C A s . T h i s f inding sugges ts that the

caudate nucle i do no t have any gross la teral ized behav io ra l or neuro log ica l

funct ion and ind ica tes an ample compensa to ry funct ional reserve .

4 . T h e B F r cats exh ib i t ed early, shor t - las t ing v isua l t racking, bu t n o n e of the

character is t ics o f the C A s were p resen t except in two an ima l s in w h i c h there

was a c o m b i n a t i o n of aggress iveness and e l emen t s of the C A s . O t h e r defects

of the frontal cats were a c o n s e q u e n c e of the ab la t ion of the senso ry -moto r

areas (pares is , hypes thes i a , and absence of contact p lac ing reac t ions) .

5. T h e cort ical and subcor t ica l E E G was normal in all cats inc lud ing the B A c

an imals .

T h e C A s i s d i scussed in te rms of the specif ici ty o f the caudate nuc le i abla-

t ion in i ts p roduc t ion . It is ten ta t ive ly exp la ined as resul t ing from a s imul-


t aneous re lease (from caudate nuclear i n h i b i t i o n ) of a bas ic wa lk ing or act ivi ty

m e c h a n i s m coupled w i t h the hyperac t iv i ty of the o r i en t ing reac t ion . O t h e r

poss ib i l i t i e s are also cons ide red .

It is conc luded that total ab la t ion of the caudate nuc le i in the cat p roduces a

character is t ic behav io ra l c h a n g e w h i c h i s bas ica l ly man i fes t ed as an exagger -

ated, s te reo typed , approach ing behav io r , the exact na ture of w h i c h r ema ins to

b e e luc ida ted . T h e caudate nuc le i do not appear to have an essen t ia l role in

the control of b a s i c me tabo l i c p rocesses , e l ementa ry s enso ry -moto r funct ions ,

e l emen ta ry cogn i t ive func t ions , and the E E G as p rev ious ly pos tu la ted .

A C K N O W L E D G M E N T S

This research was supported by USPHS Grant Nos. MH-07097, HD-05958, and HD-04612. We wish to thank Dr. C.E. Olmstead for his assistance in parts of the work and the UCLA Mental Re-tardation and Child Psychiatry Media Unit for making the 16-mm movies and video tapes.

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11 Brainstem Substrates of Sensory Information

Processing and Adaptive Behavior

JENNIFER S. BUCHWALD Mental Retardation Research Center and

Department of Physiology, School of Medicine,



U n d e r the umbre l l a of men ta l re tardat ion, the d ivers i ty of c l inical en t i t i es

clearly ind ica tes a po ten t ia l mul t i tude of causal m e c h a n i s m s . T w o s ta t is t ics ,

h o wev e r , p rov ide a po in t of depar ture for the p resen t d i scuss ion : (a) the in -

c idence of p rematu re b i r th s , w i t h anox ia as a c o m m o n per ina ta l compl i ca t ion ,

is s ignif icant ly e leva ted in menta l ly re tarded popu la t ions (13 ,29 ) ; and (b) par-

tial deafness is s ignif icant ly more c o m m o n a m o n g the menta l ly re tarded

(18 ,19) .

W h i l e p remature b i r th can potent ia l ly impl ica te any part o f the central

ne rvous sys t em ( C N S ) , several s tudies sugges t that the sensory (especia l ly

audi tory) relay nuc le i of the b r a i n s t e m are par t icular ly vu lne rab le to the anoxia

w h i c h often a c c o m p a n i e s th is b i r th compl ica t ion . In an ex t remely deta i led

quant i ta t ive s tudy e m p l o y i n g cell counts and areal m e a s u r e m e n t s , the C N S

h i s topa tho logy of 39 neona t e s ( 7 - 9 m o n t h s ges ta t iona l age) w h o d ied o f per i -

natal a sphyx ia w a s s tud ied b y Hall (14 ,15 ) , u s ing , as cont ro ls , n i n e infants w h o

suffered acc identa l dea ths w i t h o u t C N S damage . T h e a sphyx ia t ed group

s h o w e d per iod ic pos tna ta l at tacks of apnea , w h i c h failed to s tabi l ize e v e n w i t h

oxygen therapy , and dea th occur red w i t h i n a pe r iod o f 4 hour s to 22 days after

b i r th . O n the b a s i s of the deta i led h i s to r i e s w h i c h a c c o m p a n i e d these cases in -

t rauter ine anox ia and cerebra l h e m o r r h a g e we re ruled out as impor t an t pre-

natal compl i ca t ions and acute per ina ta l a sphyx ia pe r se w a s cons ide red the

mos t impor tan t e t io logy of o b s e r v e d b r a in pa tho logy . T h e b ra in s we re per-

fused intraarter ial ly w i t h i n 2 hours after dea th w i t h formal in and H e i d e n h a i n -

S o u s a so lu t ions and s u b s e q u e n t l y p repared w i t h t h i o n i n e s ta in for h i s to logica l

examina t ion . T e m p o r a l b o n e s we re addi t ional ly r e m o v e d and decalcif ied so

3 1 5

316 Jennifer S. Buchwald

that cochlear h i s to logy could also b e s tud ied . After examina t i on of the asphyx-

ia ted and control t i ssue a no rma l h is to logica l and cytological p ic ture for b o t h

groups was found in the cor tex (e .g . , audi tory cor tex) , the tha lamus (e .g . ,

med ia l genicula te b o d y ) , in the m o t o r nucle i of the b r a i n s t e m , as wel l as in the

cochlea . H o w e v e r , the a sphyx ia ted b ra in ser ies s h o w e d o the r specific loci of

pa tho logy , the mos t dramat ic of w h i c h was in the cochlear nuc leus , w h e r e a

2 0 - 4 5 % cell loss cons i s ten t ly occurred (es t imated b y a sample cell count proce-

dure) . T h e s u b d i v i s i o n of the dorsal cochlear nuc leus s h o w e d the greatest

amoun t of damage , w h i c h w a s reflected b y cell losses of 3 6 - 6 6 % . Addi t iona l

areas of cell des t ruc t ion or d a m a g e we re cons i s ten t ly p resen t in the super ior

olivary complex and infer ior col l iculus so that the b r a i n s t e m audi tory pa thway

appeared to b e part icular ly vu lne rab le to anoxic s t ress , i . e . , to have the least

safety marg in , in the neona ta l b ra in . T h i s u n i q u e suscep t ib i l i ty to anoxia m a y

b e exp la ined b y the h i g h capi l lar i ty w h i c h has b e e n quan t i t a ted for the

cochlear nuc leus (9) .

T h e suscep t ib i l i ty of the neona ta l audi tory pa thway to anoxia is also e m p h a -

s ized in the exper imen ta l s tudies of W i n d l e and h i s col leagues ( 3 0 - 3 2 ) . In

these expe r imen t s on m o n k e y s , b i r th anoxia w a s i m p o s e d for va ry ing per iods

of t ime , r ang ing from 5 to 20 m i n u t e s . T h e an imals we re s u b s e q u e n t l y ter-

mina ted at intervals of m o n t h s , w i th a m a x i m u m fol low-up t ime of 9 years ,

and b o t h behav io ra l and ana tomica l obse rva t ions were m a d e . Because the

dura t ion of the anoxic ep i sode w a s control led b y the exper imen te r , those

b ra in areas mos t suscep t ib le to th is stress could b e de t e rmined b y re la t ing o b -

served pa tho logy to degree of anoxia . In the m o n k e y the infer ior col l iculus

was cons i s ten t ly the m o s t sens i t ive b ra in reg ion to anoxic effects w i t h clearly

obse rved central r eg ions of cell des t ruct ion or damage . O t h e r b r a i n s t e m c o m -

ponen t s of the acoust ic p a t h w a y l ikewise s h o w e d marked anoxic sens i t iv i ty

wi th pa thologica l c h a n g e s in the super io r o l ivary nuc leus , nuc leus o f the lat-

eral l e m n i s c u s , and cochlear nuc leus . B r a i n s t e m nucle i of o ther sensory

sys tems , e .g. , ves t ibu la r and somat ic , also s h o w e d pa tho logy , in contrast to

the more rostral v isua l sys t em w h i c h cons i s ten t ly appeared no rma l th roughout

i ts ent i re extent . Add i t iona l loci in the basa l gangl ia also s h o w e d pa thology ,

whereas o ther b ra in areas , i nc lud ing cerebra l cor tex, appeared normal up to 10

m o n t h s . In m o n k e y s r ang ing in age from 10 m o n t h s to 9 years , h o w e v e r ,

t r ansneurona l degenera t ion b e g a n to appear as secondary c o n s e q u e n c e of the

p r imary anoxic pa tho logy (11) . T h u s , in sensory areas of the ret icular forma-

t ion , in the tha lamic relay nuc le i , and in those cort ical areas to w h i c h the

a scend ing sensory sys tems normal ly project , t r ansneurona l degenera t ion w a s

obse rved . O t h e r cort ical a reas , e .g. , the frontal cor tex, con t inued to appear

his tological ly normal .

T h e behav io ra l c o n s e q u e n c e s of such anoxic b ra in pa tho logy have not yet

b e e n ex tens ive ly inves t iga ted . Infant m o n k e y s w i t h severe ly anoxic b i r ths in i -

tially s h o w e d a var ie ty o f neuro log ica l s igns bu t , as the an imals ma tu red , the i r

11. Brainstem Substrates of Sensory Information 317

b e h a v i o r b e c a m e inc reas ing ly normal ( 2 4 , 2 5 , 3 0 - 3 2 ) ; as adul ts , these an imals

s h o w e d no gross m o t o r deficits and the i r pe r fo rmance in a v i sua l d i sc r imi -

na t ion task w a s no rma l (17) . Aud i to ry th resho lds , howeve r , w e r e found to b e

e levated w h e n quan t i t a t ed in a behav io ra l test s i tua t ion (1) . A s imi lar audi tory

f inding h a s b e e n repor ted for h u m a n sub jec t s w h o sus ta ined b i r th compl ica -

t ions of anoxia ; aud iomet r i c tes t ing s h o w e d that these ind iv idua l s h a d marked

e leva t ions of acous t ic th resho lds inc lud ing h i g h f r equency deafness (12) .

A l though soph i s t i ca ted behav io ra l s c reen ing is on ly b e g i n n i n g to b e carr ied

out on anoxic sub jec t s , s tudies of m e m o r y deficits (26) , w h i c h have b e e n

repl ica ted (17) , s h o w e d that m o n k e y s w i t h anox ic b i r ths w e r e unab l e to per-

form a 5 - second de layed r e s p o n s e task , a l though they scored at control levels

w h e n the " d e l a y " aspect of the test w a s r e m o v e d . A s no ted a b o v e , the b ra in

h i s to logy o n such an ima l s h a s s h o w n norma l frontal cor tex bu t ex tens ive

pr imary b r a i n s t e m and secondary tha lamic and cort ical pa tho logy in the

a scend ing sensory sy s t ems , w i t h the excep t ion of the v isua l pa thway . A rele-

vant c l in ical s tudy on 3-year-old chi ldren w i t h anox ic per ina ta l h i s to r ies

s h o w e d that these sub jec t s w e r e s ignif icant ly poorer than controls in tes ts o f

vocabu la ry skill and cogn i t ive abi l i ty , a l though, in m o s t o f the psycho log ica l

tests g iven , the two groups per formed equa l ly wel l (10) .

T a k e n toge ther , t hese cl inical and exper imen ta l s tud ies ind ica te a h i g h

degree of vu lnerab i l i ty of the b r a i n s t e m sensory nuc le i , par t icular ly those of

the audi tory sy s t em, to the pa thologica l effects of per ina ta l anox ia . In the

m o n k e y , control s tud ies e m p l o y i n g true anox ia (oxygen-f ree air) , ra ther than

asphyx ia (air depr iva t ion) , d id no t result in b ra in pa tho logy; thus , local in-

creases in ca rbon ic ac id ra ther than oxygen lack per se appea red to b e the

more impor tan t causal e l emen t for the o b s e r v e d pa tho logy ( 3 0 - 3 2 ) . T h e extent

to w h i c h these vu lnerab le b r a i n s t e m loci m i g h t b e se lec t ively suscep t ib le to

o ther per ina ta l s t resses has no t b e e n s tud ied , bu t the i r h i g h capi l lary dens i ty

sugges t s that any nu t r i t iona l d i s tu rbance m i g h t b e severe ly reg i s te red b y the

cells w i t h i n such r ichly vascular ized b e d s .

H i s topa tho logy of the b r a i n s t e m sensory nuc le i could s ignif icant ly d is turb

the no rma l e n c o d i n g of s t imul i in to appropr ia te neurona l firing pa t te rns ,

w h e r e b y h i g h e r centers rece ive in format ion abou t the senso ry env i ronmen t .

T h e form and degree of any resul tant behav io ra l pa tho logy w o u l d surely

depend u p o n the ex ten t of the p r imary l es ions and s u b s e q u e n t secondary

degenera t ion . Unfor tuna te ly , there are few data in the expe r imen ta l l i terature

w h i c h ex tens ive ly e x a m i n e b e h a v i o r after d i s tu rbance of sensory in format ion

p rocess ing at the b r a i n s t e m level . O n e o f the few s tudies specif ical ly d i rec ted

to th is p rob l em w a s that o f Sp rague et al. (27 ,28) w h i c h ind ica ted that dramat ic

behav io ra l aber ra t ions e m e r g e d after surgical in te r rup t ion of the somat ic and

acoust ic sensory pa thways in the i r ascent th rough the lateral b r a i n s t e m . T h e

adult cats sub jec t ed to th i s sensory in te r rupt ion p rocedure s h o w e d behav io ra l

pa tho logy that could no t b e accoun ted for solely on the b a s i s of d i m i n i s h e d


acoust ic and sensory pe rcep t ion . Fo r example , m a r k e d visual deficits were

p resen t a l though the v i sua l pa thway , inc lud ing t ec tum, was rostral to the level

of b r a i n s t e m surgery and appeared h is to logica l ly normal . T h e an imals s h o w e d

a loss o f affect, an excess ive a m o u n t o f oral ac t iv i ty w i t h a t e n d e n c y toward

hype rphag ia , s te reo typed b e h a v i o r charac ter ized b y incessan t wande r ing ,

sniff ing, and v isua l sea rch ing . W h i l e the an ima l s could learn s imple cond i -

t ioned r e sponses they s h o w e d a poor level o f re ten t ion from o n e day to the

next . Sp rague et al. (27 ,28) in te rpre ted these s y m p t o m s as a resul t o f forebra in

deafferentat ion. W i t h o u t pa t te rned inpu t to h i g h e r cen te rs v i a the specific

sensory pa thways the r e m a i n i n g por t ion of the ne rvous sys t em s e e m e d in-

capable of suppor t ing normal adapt ive behav io r . T h e b izar re nature of th is

behav io r led these authors to compare it w i th aut is t ic pe r fo rmance w h i c h they

felt shared m a n y of the same aber ran t qual i t ies .

O n the bas i s of data o b t a i n e d from sensory res t r ic t ion s tud ies , Me lzack (20)

l ikewise w a s led to compare the resul tant abno rma l b e h a v i o r w h i c h deve loped

wi th aspects of au t i sm and of ch i ldhood hyperac t iv i ty . Pupp ie s ra i sed to

adul thood unde r cond i t ions o f severe sensory res t r ic t ion s h o w e d h i g h levels

of behav io ra l arousal and hyperac t iv i ty , i m p a i r m e n t in sensory respon-

s iveness and d i sc r imina t ion , s te reo typed behav io r , and inab i l i ty to i n h i b i t

i r relevant r e sponses . T h e s e abnormal i t i e s were i r revers ib le , even after the

an imals had l ived for m o n t h s in a normal env i ronmen t . P ro longed d is rupt ive

effects of early sensory res t r ic t ion have also b e e n found in p r imates (23) ,

w h i c h con t i nue to r e spond inappropr ia te ly and apparent ly fail to d i sc r imina te

s ignif icant s t imul i for m a n y m o n t h s after b e i n g res tored to a no rma l env i ron-

men t .

W e are present ly a t t empt ing to ex tend these s tud ies w h i c h relate d i s tu rbed

sensory inf low to behav io ra l pa tho logy . In our expe r imen t s k i t t ens are b e i n g

ra ised in i so la t ion c h a m b e r s unde r cond i t ions of severe sensory res t r ic t ion, or

after lateral b r a i n s t e m t ransec t ion of the a scend ing acoust ic and somat ic

sys tems , or after med ia l b r a i n s t e m les ions of ret icular format ion areas th rough

w h i c h these moda l i t i e s a scend . In te rmi t ten t behav io ra l tests will b e carr ied

out as the an imals mature and a roster of b o t h behav io ra l and elec-

t rophys io logica l expe r imen t s will b e comple ted on the y o u n g adult an imals .

A l though perhaps mos t re levant to the p resen t conference , th is s tudy is not

yet sufficiently advanced to p rov ide any data, except for the fact that the

k i t tens survive these b r a i n s t e m l e s ion ing procedures .

To summar i ze br ief ly the thrust of m y d i scuss ion to th i s po in t , e m e r g i n g

cl inical and exper imen ta l data are b e g i n n i n g to sugges t that the b r a i n s t e m

m a y b e an impor tan t part of the funct ional mat r ix from w h i c h normal men ta l

deve lopmen t and b e h a v i o r evo lve . A l though the b r a i n s t e m has no t b e e n a

focal po in t in cons ide r ing e t io logies of men ta l re tardat ion, nor even in analy-

ses o f adapt ive b e h a v i o r , in format ion p roces s ing w i t h i n b o t h the b r a i n s t e m

sensory nucle i and the adjacent ret icular format ion m a y i n d e e d b e one of the


most cri t ical and re levant areas for such inves t iga t ions . S i n c e part ial deafness

and p remature b i r th s are r ep resen ted m o r e p r o m i n e n t l y a m o n g the menta l ly

re tarded than in the n o r m a l popula t ion , w e mus t w o n d e r w h e t h e r these sta-

t is t ics are impor tan t ly corre la ted w i th the u n i q u e suscep t ib i l i ty of the b ra in -

s t em audi tory nuc le i to per ina ta l anoxia . W e m u s t w o n d e r w h e t h e r s o m e cr i t i -

cal a m o u n t of cell d a m a g e in the in i t ia l p rocess ing s tages of s enso ry inf low has

occurred so as to garble sensory s t imul i in to r edundan t n o i s e ins tead of en-

cod ing specific pa t te rns of in format ion . S u c h garb l ing of sensory in format ion

m i g h t , i ndeed , b e m o r e deb i l i t a t ing than comple t e loss o f funct ion w i t h i n a

sensory sys tem. In the latter case , c o m p e n s a t o r y funct ions can b e a s s u m e d b y

o ther sys t ems and educa t ion can b e d i rec ted so as to m a x i m i z e these c o m p e n -

sa t ions . Par t ia l d a m a g e w i t h i n one or m o r e sensory nuc le i of o n e of m o r e sen-

sory sys t em, on the o ther h a n d , m i g h t b e difficult to de t e rmine cl inical ly and

could result in a range of unpred ic tab le behav io ra l aber ra t ions , d e p e n d i n g

u p o n the ex tent o f d is tor t ion i m p o s e d u p o n i n c o m i n g senso ry cues .

In m y labora tory w e are p resen t ly explor ing the capaci ty of the b r a i n s t e m to

encode sensory , espec ia l ly acous t ic , s t imul i and to modula te the resul tant sen-

sory in fo rmat ion so as to p roduce a " l e a r n e d " r e sponse , i . e . , a second-orde r

e n c o d i n g of the sensory s t imulus w i t h i n a greater t empora l d o m a i n . W e have

arr ived at th i s p resen t focus on the b r a i n s t e m only after a cons ide rab le a m o u n t

of w a n d e r i n g th rough the C N S look ing for a mode l sys t em s imple e n o u g h to

b e ana lyzab le , c o m p l e x e n o u g h to suppor t a var ie ty of b e h a v i o r s , and w i t h

some cl in ical re levance . In ear l ier s tud ies of no rma l adult cats behav iora l ly

t ra ined to perform a cond i t i oned leg flexion, w e we re ini t ia l ly de l igh ted b y the

m a n y b ra in loci f rom w h i c h cond i t i oned un i t act ivi ty could b e recorded (5) .

W e b e c a m e inc reas ing ly d i s enchan ted wi th th is s i tua t ion , h o w e v e r , as w e

grappled w i t h the analyt ical p r o b l e m s p o s e d b y the w e b of in te rac t ing

sys t ems , loops , and c i rcui ts w h i c h character ize the in tact b ra in . A n al ternate

prepara t ion w a s that offered b y the i so la ted sp ina l cord , in w h i c h the neura l

complex i ty is drast ical ly reduced . Unfor tuna te ly behav io ra l p las t ic i ty is l ike-

w i s e r educed and w e found that cond i t i oned (as o p p o s e d to sens i t i zed) so-

ma t i c r e sponses w e r e difficult, i f no t i m p o s s i b l e , to e s t ab l i sh in sp ina l i zed

m a m m a l s , a conc lus ion shared b y m a n y inves t iga tors (4) . W e then w o n d e r e d

wha t the m i n i m a l " b r a i n " migh t b e from w h i c h a c o n v i n c i n g behav io ra l con-

d i t ioned r e sponse could deve lop .

After r ev i ewing the l i terature , w e found that a l though cons ide rab le at-

ten t ion has b e e n d i rec ted toward sp ina l cond i t i on ing , ve ry little work has

fol lowed from the in t ense in teres t in decor t ica te cond i t i on ing w h i c h

flourished several decades ago. A s repor ted i n th i s r ev i ew (4) , s u b s e q u e n t to a

ser ies of cont rovers ia l expe r imen t s b y o ther inves t iga tors , B r o m i l y in 1948

s h o w e d that a clear , d i s c r imina t ed cond i t i oned r e sponse could b e e s t ab l i shed

in the decor t ica te p repara t ion wi th conf i rming b ra in h i s to logy , bu t on ly one

dog was used for th is s tudy; in 1958 Bard and M a c h t obse rved anecdotal ly


b o t h hab i tua t ion and cond i t i on ing in the decerebra te cat, bu t the i r exper i -

m e n t s were not focused on learn ing capaci ty and no formal t ra in ing proce-

dures were ut i l ized.

B e c a u s e the l i terature w a s unclear on the i s sue of " m i n i m a l b ra in t i s s u e "

necessa ry to suppor t cond i t i on ing , and b e c a u s e w e we re in te res ted in a

s implif ied sys t em in w h i c h w e could s tudy b o t h sensory in format ion pro-

cess ing and learn ing , w e in i t ia ted a behav io ra l s tudy of adult cats fol lowing

chronic b ra in t runca t ions of inc reas ing sever i ty (21) . Norma t ive data on the

cond i t ioned r e sponse of cho ice , the cond i t i oned eyeb l ink r e sponse , were

first col lected on a ser ies of intact adult cats . A 7 5 - 8 0 d B , 4 0 0 - m s e c tone or

CONDITIONED E Y E BL INK

CS

HABITUATION ~H •»• <M\\ ) '•WH.

£S US

FIGURE 1. Typical development of a conditioned blink response in one cat. CS duration was 400 msec and US duration was 70 msec on all traces. (Note: Bottom trace recorded on a slower time base.) CRs developed only following systematic pairing of CS and US during conditioning trials. They were characterized by a long latency (greater than 100 msec) burst of EMG activity, often superimposed on a suppression of background EMG activity early in training. Traces A through D represent changes in EMG responsiveness to the CS which typically occurred over one to two training sessions. Additional training results in the consistent appearance of sustained, relatively large amplitude CRs. CRs disappeared during extinction and did not reappear during random CS and US presentations. In the bottom trace, the arrow indicates CS onset which induces no response, although a spontaneous EMG potential just precedes the CS. (These records are from a bilaterally hemispherectomized cat but are representative of CR development in both the normal and experimental groups.) From Norman et al. (21).

EXTINCTION

RANDOMIZATION »H

CONDITIONING A M


w h i t e no i s e (CS) w a s pa i red w i th a 7 0 - m s e c shock train to the ou ter can thus of

the eye (US) w h i c h p roduced a cu taneous b l i n k reflex ( U R ) . Tria ls we re sepa-

rated b y 2 0 - to 3 0 - s e c o n d inter tr ial intervals and approx imate ly 100 trials were

run per se s s ion . E lec t romyogram re sponses w e r e recorded b ipo la r ly from the

orbicular is ocul i musc l e of the t ra ined eye , as wel l as f rom the oppos i t e eye

musc le or from musc le s e l sewhere in the face. A ser ies of p rocedures w a s rou-

t ine ly carr ied out o n all an imals w h i c h inc luded cond i t i oned r e sponse acqu i s i -

t ion , d i s c r imina t ion b e t w e e n C S + (reinforced) and CS— (nonre in forced) , ex-

t inc t ion , and r a n d o m presen ta t ions of C S and U S .

In na ive an ima l s , no r e sponse to the C S w a s ini t ia l ly presen t , a l though wi th

cons ide rab ly h i g h e r s t imulus in tens i t i e s (e .g . , 9 0 - 1 1 0 d B ) , a 1 7 - m s e c la tency ,

u n c o n d i t i o n e d acous t ic eyeb l ink reflex could b e e l ic i ted . A m o n g the no rma l

n o n l e s i o n e d cats , C S suppress ion of o n g o i n g orb icu lar i s ocul i E M G act ivi ty

typical ly occur red w i t h i n the first 200 cond i t i on ing trials a n d gradual ly gave

w a y to a re lat ively long la tency (100 msec ) cond i t i oned d ischarge w h i c h b e -

c a m e inc reas ing ly robus t w i t h c o n t i n u e d t r a in ing (Figure 1) . D u r i n g addi-

t ional t ra in ing p rocedures , the cond i t i oned E M G re sponse s h o w e d d i sc r imi -

na t ion b e t w e e n C S + and C S — , d i sc r imina t ion reversal , ex t inc t ion , and the

r e sponse d id no t deve lop dur ing r a n d o m presen ta t ions of C S and U S

(Figure 2 ) .

T h e chron ic t runca ted prepara t ions s tud ied thus far dur ing the var ious

t ra in ing procedures ou t l ined above are s u m m a r i z e d in Tab le I. Inc luded in

this ser ies have b e e n an imals wi th : l e s ions res t r ic ted to the frontal cor tex; ex-

tens ive asp i ra t ion of m o s t of the neocor tex ; b i la tera l ab la t ion of the caudate

Table I Summary of Conditioning Experiments

Treatment Name CR Ext Disc Rand Rev Shift

Normal Minuet X X X X X C-16 X X X Chastity X X X X Daffy X X X X

Decortication: El Rojo X X X X frontal cortex Freddy X X X X

Decortication: Zsa Zsa X X X X neocortex

Caudate ablation Siete X X X Bobcat X X

Bilateral Tiger X X X X X hemispherectomy Cenizas X X X X X

Puma X X X X VPM lesion Valerie X X Decerebration Tigressa X X X X X

Wrat X X X X Demian X X X X


B . NORMAL CAT (DAFFY)

C S + 2 I O O H z CS+ 3 0 0 0 Hz

------ C S - 8 0 0 Hz ------ C S - 2 0 0 Hz

1 3 1 5 10 15 20 I 4 1 6 1 3

A C Q . D I S C R I M I N A T I O N E X T . R A N D O M R E C O N D .

DAYS OF TRAINING

FIGURE 2. Summary of training procedures and typical response levels in normal animals. A. Each point represents the response average of 25 trials. The integrated EMG during the CS period (the CR response measure) is plotted on the ordinate. This animal showed CR acquisition, dis-crimination between the reinforced CS ( C S + ) , a 2100 Hz tone, and white noise, the discrimi-native, nonreinforced stimulus (CS—), extinction, a lack of reliable responding during random presentations of CS and US, and finally, reacquisition of the CR during paired CS-US trials. B . Similar data for another normal animal except that averages are for daily sessions. From Norman et al (21).

<-

EM

G

RE

SP

ON

SE

T

O

CS

I I 4 7 1 3 1 6 1 2 A C Q . D I S C R I M I N A T I O N E X T . R A N D O M R E C O N D .

DAYS OF T R A I N I N G

<_

E

MG

R

ES

PO

NS

E

TO

C

S


nuc le i ; b i la tera l h e m i s p h e r e c t o m y , i . e . , r emova l o f b o t h cerebra l h e m i s p h e r e s ,

inc lud ing l i m b i c nuc le i , basa l gangl ia , and in ternal capsule , l eav ing the thal-

amus as the m o s t rostral ex tens ion of the C N S ; and an imals w i t h m i d -

coll icular t r ansec t ion of the b r a i n s t e m , i . e . , comple te dece rebra t ion . All these

an imals have s h o w n the capaci ty to acqu i re a d i sc r imina ted cond i t i oned

A

FIGURE 3. The diencephalic brain. A. The brain from one of the chronic diencephalic animals (Tiger) is shown; the hemispheres were removed 4 months prior to termination. The remaining diencephalic stump is devoid of all telencephalic structures. (The probe is lifting a nonfunctional remnant of the fornix.) B . Histology from this animal shows marked degeneration throughout the thalamus. Note the enlarged ventricles and reduced thalamic mass. From Norman et al. (21).

B


r e sponse of 1 0 0 - m s e c la tency, to ex t ingu i sh th is r e sponse and not to develop

the r e sponse dur ing r a n d o m C S and U S p resen ta t ions (Table I ) . A l though

n o n e of the an imals s h o w e d na ive r e sponses to the C S , at h i g h e r s t imulus in-

tens i t i es (e .g . , 9 0 - 1 0 0 d B ) , all o f the t runcated prepara t ions s h o w e d the short

17-msec la tency, u n c o n d i t i o n e d acoust ic reflex. Data from the m o s t ex tens ive ly

t runcated p repara t ions , the h e m i s p h e r e c t o m i z e d and decerebra te an imals , are

summar i zed in F igures 3 , 4 , and 5. T h e h e m i s p h e r e c t o m i z e d cats b e g a n to

s h o w cond i t i on ing w i t h i n the first 2 w e e k s pos topera t ive ly and once acquis i -

t ion of the cond i t i oned r e sponse was wel l e s t ab l i shed , s u b s e q u e n t behav io r

appropr ia te to the addi t ional t ra in ing procedures deve loped at a no rmal rate

(Figure 4 ) .

In the decerebra te cats a longer pos topera t ive recovery per iod w a s necessary

before cond i t i oned r e sponse acqu i s i t i on b e g a n to appear . D u r i n g th is 4 - to

6 -week per iod a fasc ina t ing degree of funct ional recovery occurred ; for the

sake of b rev i ty I shall dwell on ly u p o n those aspects w h i c h relate to acoust ic

r e spons ivenes s . For several days fo l lowing b r a i n s t e m t ransec t ion there w a s a

d i sappearance of acoust ic reflex act ivi ty . In contrast , reflexes i n d u c e d b y cu-

taneous s t imula t ion , inc lud ing the b l ink reflex, never d i sappeared a l though

thresholds were t rans ien t ly e levated immed ia t e ly after decerebra t ion . Gradu-

ally, b y the s econd to th i rd pos topera t ive w e e k , f ragmented acoust ic startle

r e sponses b e g a n to reappear b u t th resholds we re h igh and ex t remely loud

sounds were necessa ry to el ici t any re sponse . B y the end o f the first m o n t h ,

the o r i en t ing r e sponse had b e g u n to re turn w i t h inc reas ing ly wel l - in tegra ted

neck , head , and p i n n a m o v e m e n t s . D u r i n g the second m o n t h acous t ic reflex

th resholds , inc lud ing that for the 17 -msec la tency , u n c o n d i t i o n e d acoust ic

b l ink reflex, b e g a n to s tab i l ize at levels s imi lar to p reopera t ive va lues and

100 -msec la tency c o n d i t i o n e d r e sponses b e g a n to emerge .

W h i l e the cond i t i oned e y e b l i n k r e sponses in t he decerebra te an ima l were

less s table than those of the no rma l cat , they deve loped wi th a s imi lar ini t ia l

suppress ion of E M G act ivi ty dur ing early t ra in ing fol lowed b y the appearance

of an E M G discharge 100 m s e c or m o r e after C S onse t . In the three chronic

decerebra te an imals s tud ied thus far ex t inc t ion , d i sc r imina t ion b e t w e e n C S +

and C S - , and r a n d o m C S and U S presen ta t ion procedures have b e e n carr ied

out. T h e cond i t i oned r e sponse per formance dur ing acqu i s i t i on reached levels

of 80 to 9 0 % bu t did no t cons i s ten t ly r ema in at h igh levels from one day to the

next , a l though there appeared to b e s o m e savings across days. D i s c r i m i n a t i o n

b e t w e e n w h i t e no i s e C S + and 1000 H z C S - (or 1000 H z C S + and wh i t e no i se

CS—) w a s e s t ab l i shed , a l though per formance w a s poorer than normal . Dur ing

ex t inc t ion the r e sponse d i m i n i s h e d ; dur ing r a n d o m C S and U S presen ta t ions

the C S re sponse ini t ia l ly reappeared , bu t aga in , it gradually d i m i n i s h e d over

s u b s e q u e n t trials (Figure 5 ) .

T h u s , the chron ic decerebra te prepara t ion appears capab le o f suppor t ing a

d i sc r imina ted c o n d i t i o n e d r e sponse , a l though the cond i t i on ing is no t as clear

or robus t as in the o ther less severe ly l e s ioned an imals that w e have s tudied .


BILATERALLY HEMISPHERECTOMIZED CAT (TIGER)

CS+ 2IOOHz CS+ 1000 Hz

A C Q . D ISCRIMINATION E X T . DISCRIM. R A N D O M

DAYS OF TRAINING

BILATERALLY HEMISPHERECTOMIZED CAT (CENIZAS)

2.0-

— cs+

----- c s -

100Hz

1000Hz

p

CS+

— c s -

1000 Hz

100 Hz

1.5-

7

Q

o

\\ ?t — q

\ Q jA 0.5- 6

B nteg. Volt.

1 2 3 4 5 6

D I S C R I M I N A T I O N

7 8 1 2

R A N D O M

1 2

DISCRIM

3 4

R E V E R S A L

DAYS OF TRAINING

FIGURE 4. Summary of conditioning in two diencephalic cats. Acquisition of conditioned blink response was similar to that of the normal animals. These cats showed the parametric character-istics of conditioning, including discrimination and discrimination reversal, in the absence of all telencephalic structures. From Norman et al. (21).

H o w e v e r , the re la t ive s impl i c i ty o f the i so la ted b r a i n s t e m w i t h on ly o n e asso-

c ia t ion area ( the re t icular fo rmat ion) , w i t h access to two sensory moda l i t i e s

(acoust ic and t r igemina l ) , and w i t h an eas i ly quant i f iab le m o t o r ou tpu t ( the

eyeb l ink) m a k e it an in t r igu ing sys t em in w h i c h such behav io ra l p h e n o m e n a

as cond i t i on ing , hab i tua t i on , and funct ional recovery m a y b e m o r e suscep -

t ib le to analys is than in the in tac t b ra in .

In re levant e lec t rophys io log ica l s tud ies our da ta thus far are p r imar i ly from

EM

G

RE

SP

ON

SE

T

O C

S

EM

G

RE

SP

ON

SE

T

O C

S


ACQ. EXT. DISCRIMINATION EXT. DISCRIM. 100..

DAILY SESSIONS (days post-op) FIGURE 5. This figure expresses percent CR per day during the conditioning training of one

decerebrate cat. At the end of the first postoperative month the conditioned response began to

look consistent so the response to white noise was extinguished and a discrimination procedure

was used. Retraining to the C S + (1 kHz) was very rapid and was maintained at high levels. The

CS— was introduced using a "fading" procedure in which the intensity was initially set below

that of the C S + but was increased gradually over training so that by the end of training the two

stimuli were of equal intensity. The stimulus previously used as the CS— was subsequently rein-

forced and conditioning could be demonstrated. As in other cats, random shock did not pro-

vide responses to the CS. From Buchwald et al. (8).

the acoust ic relay nucle i . W e have found that in the acute decerebra te cond i -

t ion uni t record ings of o n g o i n g act ivi ty and acous t ic r e spons ivenes s from the

cochlear nuc leus and infer ior col l iculus are very s imi la r to the record ings o b -

ta ined in the imp lan ted , in tact cat ( 6 ,7 ,22 ) . T h u s , the acute loss of acoust ic

reflex r e s p o n s i v e n e s s in the decerebra te is no t due to a depress ion of the

p r imary acoust ic pa thway . O n the o ther h a n d , the recovery of acoust ic reflex

funct ion ind ica tes that the necessa ry c i rcui t ry is p resen t in the isola ted b ra in -

s tem. A s in o the r reflex sys t ems , th is local c i rcui t ry apparent ly b e c o m e s

increas ing ly suppressed as h i g h e r tha lamic and cort ical centers mature and

thei r de scend ing inf luences b e c o m e inc reas ing ly potent . Re lease , b y decere-

bra t ion , of the b r a i n s t e m from these de scend ing inf luences al lows the p r imi -

t ive , or la tent , funct ions of the b r a i n s t e m to r eemerge w i t h a capaci ty suf-

ficient to suppor t no t on ly acous t ic startle and o r i en t ing reflexes bu t an

acoust ic cond i t i oned r e sponse as wel l . T h e s e data po ignan t ly por t ray the re-

%

CO

ND

ITIO

NE

D

RE

SP

ON

SE

MESENCEPHALIC CAT (TIGRESSA)


t icular format ion as a s t ructure so capt iva ted dur ing matu ra t ion b y the

descend ing in f luences of h i g h e r cen te rs that its i n d e p e n d e n t assoc ia t ive and

reflex func t ions , pe rhaps h igh ly s ignif icant in s h a p i n g neona ta l b e h a v i o r ,

b e c o m e largely m a s k e d .

W h i l e it ha s long b e e n k n o w n that acous t ic r e sponses can b e recorded from

the re t icular fo rmat ion , these have genera l ly b e e n classif ied as " n o n s p e c i f i c "

r e sponses reg i s te r ing nove l s t imul i t h rough the arousal s y s t e m ra ther than

subse rv ing any specific acous t ic in fo rmat ion transfer . O n the o the r h a n d , in

the chron ic decerebra te , cond i t i oned d i sc r imina t ion b e t w e e n an acous t ic C S +

and acoust ic C S - sugges t s that acous t ic in fo rmat ion is hand led ve ry specifi-

cally b y the neurop i l suppor t ing the p roduc t ion or the w i thho ld ing of the

cond i t i oned r e sponse—neurop i l w h i c h w e presen t ly p r e s u m e to b e w i t h i n the

reticular format ion . Suppor t ive data for the concep t of s enso ry r e sponse spec i -

ficity in the re t icular fo rmat ion have c o m e from other e x p e r i m e n t s , the in i t ia l

purpose of w h i c h w a s to explore acous t ic r e sponse specif ic i ty in the l imb ic

sys tem (2 ,3 ) . T h e un i t r e sponse pat terns in h i p p o c a m p u s and amygda la we re

found to different iate t ones of different f r equency or i npu t lateral i ty (2) .

S u b s e q u e n t l y , the source of acoust ic inpu t to these s i tes w a s inves t iga ted b y

p lac ing l e s ions e i ther in the b rach i a of the infer ior coll iculi to in ter rupt the

p r imary acous t ic pa thway , or in the ret icular fo rmat ion of the med ia l b ra in -

s t em to in ter rupt acoust ic p ro jec t ions a scend ing th rough th is sy s t em (3) .

A l though an in i t ia l , re la t ively small c o m p o n e n t of the r e sponses d i sappea red

fol lowing the p r imary acoust ic p a t h w a y sec t ions , the qua l i ty o f r e sponse

specif ici ty w a s p rese rved (Figure 6 ) . In contras t , after the re t icular l e s i o n s , the

h i p p o c a m p a l a n d amygda la acous t ic r e sponses largely d i sappea red (Figure 7 ) .

T h u s , specific in fo rmat ion c o n c e r n i n g the na ture of the acoust ic s t imulus w a s

pro jec ted to these l i m b i c s i tes largely th rough the ret icular format ion . T a k e n

toge ther , these obse rva t i ons suppor t a concep tua l i za t ion of the re t icular for-

ma t ion as a s t ructure w h i c h can rece ive and t r ansmi t acoust ic in fo rmat ion

wi th re ten t ion of specif ic s t imulus cod ing , ra ther than s imply a degrada t ion of

the s t imulus in to a nonspec i f ic s ignal o f arousal . W e are p resen t ly pu r su ing

th is p r o b l e m in a m a p p i n g s tudy of acous t ic r e sponse specif ic i ty in the

chron ic decerebra te p repara t ion .

In order to deve lop a t empora l frame of reference for the b r a i n s t e m struc-

tures potent ia l ly invo lved in sensory in format ion p rocess ing and lea rn ing

p h e n o m e n a , w e have also b e g u n to es tab l i sh w h a t w e refer to as " t i m e con-

stants of r e sponse p la s t i c i ty" at different levels of the b r a i n s t e m sys tem. For

example , after an acous t ic s t imulus has b e e n p re sen ted , over w h a t pe r iod of

t ime does that s t imulus c o n t i n u e to exert an in f luence ; h o w different is the

dura t ion of th is in f luence in the acous t ic ne rve than in the cochlear nuc leus ,

the infer ior col l iculus , or the re t icular fo rmat ion? A l t h o u g h a great deal o f

w o r k has b e e n d o n e in the area of audi tory hab i tua t ion , it has b e e n on ly

w i t h i n the pas t decade that the n u m e r o u s va r iab les l ead ing to artifactual data


L A T E R A L SECTIONS

A. R HIPP

ipsi

Pre nTTTflrrTTTfTTTTrrn

Post

B. R M G B

contra

Pre

Post rrnrlTTlTnTrTTT^

C. L M G B contra

Pre rnrrfTTTrnrTTTTTTTi

Post

FIGURE 6. Persistence of limbic (hippocampal, HIPP) acoustic response after bilateral damage to primary auditory pathways. Extents of pathway sections are shown in coronal diagrams at slightly different A-P levels on each side (R and L). MGB = medial geniculate body, SC = superior colliculus. A. Multiple-unit discharge frequency histograms from ventral hippocampal site are averaged over five trials. Top trace: Presection excitatory response to ipsilateral 80 dB white noise stimulus (dark bar) is not eliminated after auditory pathway damage (bottom trace). B and C. Five-trial averaged multiple-unit discharge frequency histograms from right and left MGB showing typical presection excitatory responses to contralateral white noise stimulation (top traces) and sharp reduction of postsection (bottom traces) response magnitudes. From Brown and Buchwald (3).

have b e e n r ecogn ized and control led (7) . S u b s e q u e n t expe r imen t s have b e e n more carefully carr ied out bu t s t imulus pa ramete rs and r e sponse measu re s have con t inued to b e so var ied that for any sol id in terpre ta t ion of the hab i tua -t ion process to e m e r g e a cohes ive s tudy of the re levant subs t ra tes m u s t b e made . In our approach to th is p r o b l e m , a ra ther w i d e range of s t imulus duty cycles ( i . e . , s t imulus durat ion/ inter tr ial interval) have b e e n u t i l ized , w i t h a ser ies of 25 success ive acoust ic s t imula t ion trials at each duty cycle. S ing le and mul t ip le uni t r e sponses we re recorded from decerebra ted cats m a i n t a i n e d unde r Flaxedi l and artificially resp i red and the percen t of r e sponse dec remen t w h i c h deve loped across the 25 trials at each duty cycle w a s de te rmined . T h e range of duty cycles ex t ended from very l ight , i . e . , 1-second tones every 10 seconds , to very heavy , e .g. , 8 .5 - second tones every 10 seconds ; in tens i ty w a s he ld cons tan t at 75 or 80 dB S P L (re 0 .0002 dyn /cm 2 ) .


MEDIAL SECTION

A. L HIPP ipsi

Pre flMTTflrM

Post (TTHwfTHTTThftTfhrTmnnwmTTTTL

B. L HIPP contra

Pre l l f l l l l t l ^ ^

Post " - mfTTTTmmmmT WFFFRRFL

C. L MGB D. R MGB contra fTrnrnm^i

Pre r n J | | | | ^ Pre fTmnJllIM

Post nrnrnnl^^ P o s t f l t l J l ^ ^ 2SO MSEC

FIGURE 7. Reduction of limbic (hippocampal, HIPP) acoustic responses after medial section of

brainstem at geniculate level (A 6.0). Extent of section shown on coronal diagram. MGB = medial

geniculate body. A and B. Top traces show five-trial averaged multiple-unit discharge frequency

histograms from same ventral hippocampal site before section. Presection excitatory responses

(top traces) to ipsilateral (A) and contralateral (B) 80 dB white noise (dark bar) are markedly

diminished in postsection (bottom traces). C and D. Five-trial averaged multiple-unit discharge

frequency histograms from left and right MGB show typical excitatory responses (top traces) to

contralateral 80 dB white noise which are not markedly reduced in postsection (bottom traces).

From Brown and Buchwald (3).

T h e cochlear ha i r cell m i c r o p h o n i c potent ia l recorded from the round

w i n d o w s h o w e d no change as a funct ion of any of these duty cycles (7 ,16) .

O n l y dur ing te rmina l levels of barb i tu ra te did the m i c r o p h o n i c po ten t ia l b e g i n

to d i m i n i s h and w i t h E K G cessa t ion it d i sappeared . U n i t r e sponse s recorded

from the acoust ic ne rve also s h o w e d m a r k e d s tabi l i ty dur ing inc reas ing ly

heavy duty cyc les , w i t h essent ia l ly no adapta t ion as long as 100 m s e c or m o r e

in t e rvened b e t w e e n success ive s t imul i (Figure 8 ) . D u r i n g con t inuous tone

s t imula t ion , h o w e v e r , s o m e degree of r e sponse dec remen t appeared bu t

recovery occurred a lmost i m m e d i a t e l y after s t imulus cessa t ion . At the central

level of the cochlear nuc leus there w a s a m a r k e d increase in the pe r s i s t ence of

the acoust ic r e sponse trace as ind ica ted b y the inter tr ial in terval across w h i c h

r e sponse dec remen t s deve loped , i . e . , at re la t ively l ight duty cycles (Figure 9 ) .

T h u s , in add i t ion to the r e sponse pat tern e n c o d e d b y the cochlear nuc leus ,

w h i c h funct ional ly d i s t i ngu i shes it from the acoust ic ne rve , pe r s i s t ence of the

acoust ic r e sponse trace occurs to a qual i ta t ive ly greater degree in the cochlear

nuc leus than in the ne rve . T h i s difference in r e sponse trace pe r s i s t ence can

b e defined in t e rms of the duty cycle w h i c h p roduces r e sponse changes

over success ive trials. At the level of the infer ior col l iculus , greater complex i ty

is e n c o d e d into the acous t ic r e sponses and a m o r e dramat ic degree of r e sponse

pers i s tence occurs (22) . For example , a s t imulus duty cycle of 1-second tone/5-

A 6 . 0


CH

AN

GE

IN

R

ES

PO

NS

E

DU

E

TO

2

5 /

T

RIA

LS

Cat a

ACOUSTIC NERVE

Cat b

A. T O N E DURATION E F F E C T

Cat b Cat a

B. ITI E F F E C T

FIGURE 8. For cat a, multiple unit responses at the acoustic nerve did not show any decrement at any tone duration, including continuous tone stimulation. For cat b , small response decrements were observed but were sharply in contrast with the data from the cochlear nucleus shown in Figure 9. From Huang and Buchwald (16).

s econd inter tr ial interval p roduced a r e sponse dec remen t of 80 to 9 0 % in the

col l iculus, in contras t to the 10 to 2 0 % dec remen t p roduced b y the s a m e duty

cycle in the cochlear nuc leus . Bo th loci are clearly inf luenced b y l ighter duty

cycles than those neces sa ry to i nduce changes at the m o r e per iphera l audi tory

s ta t ions .

COCHLEAR NUCLEUS

| A. TONE DURATION E F F E C T B. ITI E F F E C T s

FIGURE 9. A. For a fixed intertrial interval (ITI) of 1.5 seconds, increased tone (80 dB, 3000 Hz)

duration enhanced response decrement in cochlear nucleus multiple unit activity during a block

of 25 trials. Increasing the tone duration beyond 8-10 seconds had little effect on the percent

change. B. Percent change of the cochlear nucleus multiple unit activity became less significant

with increasing ITI while tone (90 dB, 3000 Hz) duration is fixed at 1.5 seconds. For ITI beyond

8-10 seconds, the habituation procedure had essentially no effect. From Huang and Buchwald (16).

J -i 1 . r , , , , , H \—r 4 , , , , , , , , , r

£ 0 1 2 3 4 5 6 7 8 9 3 0 1 2 3 4 5 6 7 8 9 10

s e c TONE DURATION M , n S e c IT!

-10-

-20-

-30-

-40-

-50-

-70-

%

CH

AN

GE

IN

R

ES

PO

NS

E

DU

E

TO

2

5

/

TONE DURATION ITI


T h e s e data m a p the t empora l d o m a i n of exc i tab i l i ty and recovery p rocesses ,

i . e . , the pe r s i s t ence o f the acous t ic r e sponse t race , for the acous t i c centers

tes ted thus far. B y deve lop ing s imi lar data for the r e m a i n i n g b r a i n s t e m

acoust ic nuc le i and for the var ious areas o f the ret icular fo rmat ion w h i c h s h o w

acoust ic r e s p o n s i v e n e s s , we h o p e to es tab l i sh a h ie ra rchy of " t i m e cons tan t s

of r e sponse p las t i c i ty" w i t h i n the b r a i n s t e m . S u c h a h ie ra rchy w o u l d provide

a t empora l frame of reference for b r a i n s t e m e l emen t s , b a s e d on the i r exc i tab i l -

i ty and recovery p rocesses , and should sugges t those r eg ions w i th lowes t

modu la t ion thresholds . T h o s e reg ions part icular ly sens i t ive to the repea ted

s t imula t ion of an hab i tua t ion pa rad igm m i g h t also b e part icular ly suscep t ib le

to the success ive convergen t sensory inf low of a cond i t i on ing pa rad igm.

In s u m m a r y , w h i l e the va r ious expe r imen ta l s tud ies I h a v e d e s c r i b e d are far

from comple t ion , w e h o p e that they wil l expand our p resen t unde r s t and ing of

sensory in fo rmat ion p rocess ing , the modu la t ion of th is p roces s ing , and the

deve lopmen t of adapt ive behav io r . A s ou r in teres ts have focused inc reas ing ly

on the acous t ic and ret icular b r a i n s t e m sys t ems , the c l inical and exper imen ta l

data ind ica t ing the u n i q u e suscept ib i l i ty of the b r a i n s t e m sensory sys t ems to

per ina ta l anox ia have s e e m e d inc reas ing ly re levant , as has the subpopu la t ion

of men ta l ly re ta rded ind iv idua l s w h o are part ial ly deaf w i t h a h is tory of

p remature b i r th .

S U M M A R Y

E m e r g i n g cl inical and exper imen ta l data sugges t that the b r a i n s t e m m a y b e

an impor tan t part o f the funct ional ma t r ix from w h i c h no rma l men ta l deve lop-

m e n t and b e h a v i o r evolve . A l though the b r a i n s t e m has no t b e e n a focal po in t

in cons ide r ing e t io log ies of men ta l re tardat ion no r even in ana lyses o f adap-

tive behav io r , in format ion p rocess ing w i t h i n b o t h the b r a i n s t e m sensory

nuc le i a n d adjacent re t icular format ion m a y i n d e e d b e o n e o f the m o s t cr i t ical

and re levant areas for such inves t iga t ions . Da ta are s u m m a r i z e d from several

o n g o i n g expe r imen t s w h i c h are d e s i g n e d to explore the capac i ty of the b ra in -

s t em to e n c o d e senso ry , especia l ly acous t ic , s t imul i and to modu la t e the resul-

tant sensory in fo rmat ion so as to p roduce a " l e a r n e d " response . S u c h data in-

dicate that in the a b s e n c e of forebra in or even m i d b r a i n s t ructures , a s imple

cond i t i oned r e sponse can b e suppor ted b y the b r a in s t em. T h e ret icular

neurop i l is cons ide red par t icular ly impor tan t to th i s r e sponse acqu i s i t i on and

is concep tua l i zed as a s t ructure w h i c h can rece ive and t ransmi t acoust ic infor-

ma t ion w i t h re ten t ion of specific s t imulus cod ing . It is further sugges ted that

i n d e p e n d e n t assoc ia t ive and reflex funct ions of the ret icular fo rmat ion m a y b e

h igh ly s ignif icant in neona ta l b e h a v i o r bu t , w i t h the deve lopmen t of de scend -

ing inf luences f rom h i g h e r centers dur ing matura t ion , these func t ions m a y

b e c o m e largely m a s k e d . In order to deve lop a t empora l f rame of re ference for


b r a i n s t e m structures potent ia l ly invo lved in senso ry in format ion p rocess ing

and learn ing p h e n o m e n a " t i m e cons tan t s of r e sponse p las t i c i ty" are b e i n g es -

t ab l i shed for different levels of the b r a i n s t e m sys tem.


This work has been supported by USPHS Grant Nos. NS-05437, HD-04612, HD-05958, and NIH

Special Resources Grant No. RR-3.

R E F E R E N C E S

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8. Buchwald, J . S., Norman, R. J . , Villablanca, J . R., Brown, K. A., and Schwafel, J . A. Classical conditioning of the eyeblink response in chronic decerebrate cats. Proc. 26th Int. Physiol. Cong., 1974, 11: 216.

9. Craigie, E. H. The comparative anatomy and embryology of the capillary bed of the central nervous system. Res. Publ. Assoc. Res. Nerv. Ment. Dis., 1938, 18: 3-28.

10. Ernhart, C , Graham, F . , and Thurston, D. Relationship of neonatal apnea to development at three years. Arch. Neurol, 1960, 2: 504-510.

11. Faro, M. D. , and Windle, W. F. Transneuronal degeneration in brains of monkeys asphyxi-ated at birth. Exp. Neurol, 1969, 24: 38-53.

12. Fisch, L. The aetiology of congenital deafness and audiometric patterns. / . Laryngol. Otol, 1955, 69: 479-193.

13. Gottfried, A. W. Intellectual consequences of perinatal anoxia. Psychol. Bull, 1973, 80: 231-242.

14. Hall, J . G. On the neuropathological changes in the central nervous system following neonatal asphyxia. With special reference to the auditory system. Acta Otolaryngol, Suppl, 1963, 188: 331-338.

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16. Huang, C.-M., and Buchwald, J. S. Multiple unit responses in cochlear nucleus and acoustic nerve during acoustic habituation procedures. Anat. Rec, 197A, 178: 379-380.

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18. Kodman, F. The incidence of hearing loss in mentally retarded children. Am. J. Ment. Defic, 1957, 62: 675-678.

19. Lloyd, L. L. , and Reid, M. J . The incidence of hearing impairment in an institutionalized mentally retarded population. Am. J . Ment. Defic, 1967, 7 1 : 746-763.

20. Melzack, R. Effects of early experience on behavior: Experimental and conceptual consider-ations. In: Psycho-pathology of Perception. (P. H. Hoche and J. Zubin, Eds.). Grune & Stratton, New York, 1965: pp.271-299.

21. Norman, R. J . , Villablanca, J . R., Brown, K. A., Schwafel, J . A., and Buchwald, J . S. Classical eyeblink conditioning in the bilaterally hemispherectomized cat. Exp. Neurol., 1974, 44: 363-380.

22. Regan, D. , and Buchwald, J . S. Single unit response decrements in the inferior colliculus of decerebrate cats during repeated acoustic stimulation. Proc. Soc. Neurosci., 1974, 4: 389.

23. Riesen, A. H. Plasticity of behavior: Psychological aspects. In: Biological and Biochemical Bases of Behavior. (H. F. Harlow and C. N. Woolsey, Eds). Univ. of Wisconsin Press, Madison, 1958: pp. 425-450.

24. Saxon, S. V. Effects of asphyxia neonatorum on behavior in the rhesus monkey. / . Genet. Psychol, 1961, 99: 277-282.

25. Saxon, S. V. Differences in reactivity between asphyxial and normal rhesus monkeys. / . Genet. Psychol, 1961, 99: 283-287.

26. Sechzer, J . A. Memory deficits in monkeys brain damaged by asphyxia neonatorum. Exp. Neurol, 1969, 24: 497-507.

27. Sprague, J . M. , Chambers, W. W. , and Stellar, E. Attentive, affective and adaptive behavior in the cat. Science, 1961, 133: 165-173.

28. Sprague, J . M. , Levitt, M. , Robson, K., Liu, C. N. , Stellar, E. , and Chambers, W. W. A neuroanatomical and behavioral analysis of syndromes resulting from midbrain lemniscal and reticular lesions in the cat. Arch. Ital. Biol, 1965, 1 0 1 : 225-295.

29. Towbin, A. Mental retardation due to germinal matrix infarction. Science, 1969, 164: 156-161. 30. Windle, W. F. Neuropathology of certain forms of mental retardation. Experiments on

monkeys illustrate probable mechanisms of brain damage in human infants. Science, 1963, 140: 1186-1189.

31. Windle, W. F. Brain damage at birth. JAMA, 1968, 206: 1967-1972. 32. Windle, W. F . , Jacobsen, H. N., de Arellano, R., and Combs, C. M. Structural and functional

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HA Discussion: Neurophysiological Substrates of

Behavioral Habituation and Conditioning

C H A R L E S D . W O O D Y




T h e obse rva t i ons repor ted b y Dr . B u c h w a l d are of cons ide rab le s ign i f icance .

T h e abi l i ty to p roduce audi tory cued cond i t i oned r e sponses in a m e s e n -

cepha l ic p repara t ion sheds l ight on p r o b l e m s that h a v e b e e n p rev ious ly ra i sed

b y Bard and M a c h t (2) and b y M e r i n g o f the Sov ie t U n i o n (18) . Before pro-

ceed ing to further specific c o m m e n t s , h o w e v e r , I should l ike to address a few

remarks to var ious d u b i o u s l y re la ted, bu t re levant aspects of the m o r e genera l

ques t ion of neu rophys io log ica l subs t ra tes of cond i t i on ing .

CONDITIONING IS DEFINITIONALLY OVERSIMPLIFIED, THE UNDERLYING PROCESSES ARE COMPLEX

O u r task as neu rob io log i s t s is to s implify. Bu t our na ture , it s e e m s , is to

overs impl i fy . A l so , sc ience t ends ini t ia l ly to b e desc r ip t ive , and descr ip t ive

sc i ence , i tself, can b e overs impl i s t i c . W e m a y b e j u s t app roach ing the po in t

w h e r e our unde r s t and ing c o n c e r n i n g c o n d i t i o n e d b e h a v i o r pe rmi t s an aware-

ness of s o m e overs impl i f ica t ions in th i s field.

Let us e x a m i n e detai ls of two par t icular ly mer i to r ious a t tempts to define as-

pects of classical cond i t i on ing . O n e s ta tes , r ea sonab ly e n o u g h , that an essent ia l

feature o f c lass ical c o n d i t i o n i n g invo lves the re l iab le evoca t ion of a m e a s u r a b l e

u n c o n d i t i o n e d r e sponse b y an u n c o n d i t i o n e d s t imulus (13) . T h i s descr ip t ive

charac ter iza t ion m u s t n o w b e qual i f ied in l ight of s tud ies in w h i c h cond i -

t i on ing has b e e n a t t empted in p repara t ions in w h i c h the per iphera l efferent

ne rves have b e e n c rushed . In such p repara t ions , the u n c o n d i t i o n e d per iphera l

335

336 Charles D. Woody

m o t o r r e sponse does no t occur dur ing t ra in ing , ye t the an imals learn a cond i -

t ioned r e sponse w h i c h is i nd i s t i ngu i shab l e from the usual type of c o n d i t i o n e d

b e h a v i o r (3 ,7 ) .

A n o t h e r def ini t ional a t tempt is appropr ia te ly conce rned wi th the p re sence

or a b s e n c e of a r e sponse to the cond i t i oned s t imulus (CS) pr ior to t r a in ing

(16) . T h e p r o b l e m here is that the phys io log ica l level of the r e sponse in q u e s -

t ion m a y i tself r equ i re s tr icter def ini t ion. Tha t i s , o n e m a y n e e d to d i s t ingu i sh

b e t w e e n r e sponses at the level o f pos t synap t ic po ten t ia l s , ne rve ac t ion po ten-

tials, musc le ac t ion poten t ia l s , and finally gross m o v e m e n t . Pos t synap t ic po-

tent ials are ve ry l ikely genera ted in m o t o r n e u r o n s b y m a n y types of C S s

del ivered in naive an ima l s (35) . Un i t a ry act ion potent ia ls genera ted b y or in a

s ingle neu ron in the m o t o r cor tex can lead to a measu rab l e pe r iphera l m y o -

graphic r e sponse (34) . S ince ne i t he r o f the a b o v e n e e d b e assoc ia ted w i t h any

obse rved gross m o v e m e n t , one w o n d e r s i f the general ly accep ted usage of " o b -

servable behav io ra l r e s p o n s e " in the context o f cond i t i on ing theory requ i res

s o m e modif ica t ion . In addi t ion , specific a t tempts to d i s t ingu i sh b e t w e e n

alpha cond i t i on ing and classical cond i t i on ing on the bas i s of the p r e sence or a b -

sence of obse r ved r e s p o n s e s p r io r to t ra in ing should also no te Pavlov ' s obse r -

va t ions of sal ivary r e sponses to m e t r o n o m e C S s in s o m e of h i s an imals pr ior

to t ra in ing (20) .

P r o b a b l y the complex na ture of p rocesses under ly ing cond i t i on ing also con-

t r ibutes to def ini t ional confus ion . A n ana logous p r o b l e m has long b e e n obv i -

ous in research on " s c h i z o p h r e n i a " and the same m a y undoub ted ly b e sa id for

" m e n t a l r e ta rda t ion . " E v e n the appl ica t ion of s o m e os t ens ib ly s imp le , e lec-

t rophys io logica l inves t iga t ive t e c h n i q u e s m a y b e more complex (11 ,29) than

some m i g h t r ecogn ize .

PARTITIONING COMPLEX PROCESSES ACCORDING TO SUBSETS OF UNDERLYING, CONSEQUENTIAL

OPERATIONS MAY HELP THEIR ANALYSIS

W h a t pos s ib l e so lu t ions m a y b e sought to these p r o b l e m s ? M o s t useful

approaches (cf. 16 ,20) r equ i re r ecogn i t ion of consequen t i a l r e la t ionsh ips

b e t w e e n the under ly ing process - subs t ra tes and the o b s e r v e d behav io r s . T h u s ,

pe rhaps , the p resen t s e s s ion m i g h t have b e e n ent i t led "Neurophys io log i ca l

Subs t ra tes Are Behav io ra l Hab i tua t i on and C o n d i t i o n i n g . "

It wou ld appear useful to par t i t ion " l e a r n i n g " or " c o n d i t i o n i n g " into

subse t s of measu rab l e " b e h a v i o r a l " opera t ions de r iv ing consequen t i a l ly from

co r re spond ing sets o f unde r ly ing p rocesses o r subs t ra tes m e d i a t e d th rough

se lec ted popula t ions of neu rons . To take a specific example , the fo l lowing par-

11 A. Neurophysiological Substrates 337

t i t ion ing m a y he lp to charac ter ize cond i t i oned b l i nk reflexes resul t ing from pa i r ing c l i ck -CS w i t h glabel la tap U S . *

a. Cer ta in specific s t imul i such as the CS evoke the c o n d i t i o n e d r e sponse

after t r a in ing w h i l e o the r s t imul i do not (9) .

b . T h e m o t o r r e sponse evoked after cond i t i on ing is a specific o n e invo lv ing

measu rab ly specif ic pa t te rns o f musc l e act ivi ty (37, cf. F igure 5 ) .

c. S o m e changes m u s t occur w i t h i n the C N S such that the s ame phys ica l

s t imulus w h i c h fails to p roduce a specif ic r e s p o n s e pr ior to t r a in ing does so

afterward (33 ,36) . t

DESCRIPTIONS OF SOME POTENTIALLY RELEVANT PROCESSES AND OPERATIONS

W h a t p rocesses and subs t ra tes m i g h t relate s ignif icant ly to the above opera-

t ions? A part ia l l i s t ing of some w h i c h m i g h t profi tably b e cons ide red at

present fol lows:

Temporal Contiguity

Processes that p r o m o t e and under l i e the deve lopmen t o f pers i s ten t changes

in affected n e u r o n s are poor ly under s tood at present . H o w e v e r , " t empora l

con t i gu i t y , " i . e . , the specific t empora l assoc ia t ion b e t w e e n pa i red s t imul i , ap-

pears to inf luence t h e m signif icant ly (see also 8 ) . Tempora l con t igu i t i e s of

2 0 0 - 4 0 0 m s e c b e t w e e n assoc ia ted s t imul i appear to b e near the o p t i m u m

delays for efficient deve lopmen t o f s o m e key changes subse rv ing c o n d i t i o n i n g

(23). No te , h o w e v e r , that longer tempora l con t igu i t i e s are op t imal for the

deve lopmen t of o ther types of cond i t i on ing , such as those s tud ied b y Dr .

Garc ia (12) . Dif ferences in still o ther aspects o f t empora l con t igu i ty , for ex-

ample , the difference b e t w e e n trace and de layed cond i t i on ing , m a y p roduce

o ther s t r ik ing differences in the opera t iona l c o n s e q u e n c e s of s t imulus assoc ia-

t ion (14) .

Anatomical Representation

A n o t h e r aspect of p rocesses under ly ing cond i t i oned b e h a v i o r conce rns thei r

ana tomica l represen ta t ion . O s t e n s i b l y and superficial ly s imi la r cond i t i oned

behav io r s can d e p e n d , ana tomica l ly , on qui te different parts of the bra in .

^Specific, related physiological operations are described in detail elsewhere (9 ,32-40) . tSome responses may occur initially, but they are of questionable motor specificity and do not persist with repeated stimulus presentation in contrast with the persistent, specific responses observed after training.


Smal l differences in s t imulus charac ter and tempora l con t igu i ty can , for ex-

ample , lead to s t r ik ingly different opera t iona l c o n s e q u e n c e s for the deve lop-

m e n t o f cond i t i oned b l i nk ing , inc lud ing differences in r e sponse la tency and

ana tomica l represen ta t ion (cf. 4 - 6 , 4 0 ) . S o m e types of cond i t i oned b l i n k i n g

clearly d e p e n d upon p rocesses subse rved b y n e u r o n s of rostal cor tex (32 ,36) .

In contras t , b l i nk c o n d i t i o n i n g of the type repor ted b y Dr . B u c h w a l d appears

to d e p e n d u p o n p rocesses subse rved b y neu rons at b r a i n s t e m levels . T h o s e

in te res ted in cort ical funct ion m a y find the former type of cond i t i on ing of par-

t icular re levance , w h i l e those in te res ted in per formance subse rved b y isola ted

b r a in s t em wil l prefer to inves t iga te the latter. It i s , parenthet ica l ly , paradoxical

that the cond i t i oned r e sponse in the " s i m p l e r " b r a i n s t e m prepara t ion , w h i c h

migh t b e expec ted to e m p l o y s imple r c i rcui t ry , is of five to ten t imes longer

reflex la tency than is that in the in tact prepara t ion . T h e ques t ion of the

m i n i m a l reflex c i rcui t ry (6) useful for phys io log ica l inves t iga t ion is p rob lem-

atic. R e s p o n s e la tenc ies as shor t as 1 2 - 2 0 m s e c (35 ,39) are m u c h longer (even

after subt rac t ing k n o w n conduc t ion delays) than the 0 . 3 - 0 . 6 m s e c requ i red for

each synaptic delay a long the r e sponse p a t h w a y and h e n c e still too long for

some useful approaches to c i rcui t analys is (21) .

"Physiological Locus": Presynaptic Changes, Postsynaptic Changes, and Growth of New Connections

A third po in t of re levance to b io log ica l p rocesses subse rv ing cond i t i on ing is

the "phys io log ica l l o c u s " of the p rocesses . W e m i g h t w i s h to d i s t ingu i sh

b e t w e e n var ious types of process- loc i . O n e wou ld involve pre- or in-

t rasynapt ic c h a n g e s w h i c h could affect synapt ic po tency . W i t h poss ib l e excep-

t ions (e .g . , " l o n g - l a s t i n g " hab i tua t ion p h e n o m e n a in Aplysia) such p rocesses

appear to b e conce rned pr imar i ly w i t h shor t - las t ing changes . T h e s e inc lude

pos t te tanic po ten t ia t ion , p resynap t ic i n h i b i t i o n , and c o m m o n he te rosynapt ic

faci l i tat ion (30) . Ra the r ana logous m e c h a n i s m s m a y subse rve hab i tua t ion

( 1 0 , 2 6 - 2 8 ) .

A n o t h e r p rocess - locus invo lves pos t synap t ic m e c h a n i s m s w h i c h could affect

cellular exci tabi l i ty . A l though the w o r k is still i n its p re l iminary s tages (34 ,36 ) ,

there is s o m e ind ica t ion that such m e c h a n i s m s m a y b e invo lved in es tab l i sh-

m e n t of the m o t o r specif ic i ty o f s o m e types of cond i t i oned b l i nk ing .

Still ano the r class of p rocesses conce rns those to do w i t h n e w g rowth and dif-

ferent ia t ion of cells and the i r connec t i ons as desc r ibed in earl ier chapters i n th i s

vo lume . T h e role of such p rocesses in adult l ea rn ing r ema ins to b e d e m o n -

trated. In complex compu te r au tomata , s o m e t h i n g m u s t b e he ld cons tan t once

adapt ive , complex , l ea rn ing- l ike p rocesses are b e g u n ( 1 , 1 9 , 2 2 , 2 4 ) . M y presen t

b i a s as to wha t is he ld relat ively cons tan t in the adult h u m a n favors neuro -

ana tomica l connec t i ons w h i c h wou ld , as set forth genet ica l ly and deve lop-

menta l ly , p rov ide appropr ia te b a s e s for later in te rac t ions . T h e p r o b l e m of

11A. Neurophysiological Substrates 339

h o w then w o u l d " s w i t c h i n g func t ions" b e a c c o m p l i s h e d in in format iona l

t ransac t ions w i t h i n the adult b r a in i s , as wil l b e s een , eas i ly reso lved .

Functional Ensembles of Neurons

A fourth po in t charac te r iz ing p rocesses subse rv ing c o n d i t i o n i n g conce rns

the s tochas t ic in te rac t ion of funct ional ly related popu la t ions of neu rons . It ap-

pears inc reas ing ly l ikely that cod ing and t r ansmis s ion of in fo rmat ion re levant

to the pe r fo rmance of ve ry s imple c o n d i t i o n e d reflexes are subse rved v ia

changes in synapt ic po t ency or cellular exc i tab i l i ty over funct ional ly related

e n s e m b l e s or sets of un i t s (32) . Unfor tuna te ly , it m a y b e difficult to m e a s u r e

precise ly c h a n g e s in the p rocesses o f a s ing le un i t re levant to complex con-

sequen t ia l behav io ra l ope ra t ions b e c a u s e the m a g n i t u d e of such c h a n g e s ap-

pears to b e qu i t e smal l (34) . Ident i f icat ion and sampl ing of m a n y such e le-

m e n t s w i t h i n funct ional ly related neurona l popu la t ions m a y b e neces sa ry if

one is to demons t ra t e such c h a n g e s . Ident i f ica t ion and sampl ing of such func-

t ional e n s e m b l e s appear to b e afforded in cat m o t o r cor tex b y e s t ab l i sh ing the

moto r p ro jec t ion of the n e u r o n s and in sensory cor tex b y e s t ab l i sh ing the i r

sensory recept iv i ty ( 9 , 3 3 , 3 7 , 3 8 ) .

Tempora l - spa t i a l s u m m a t i o n or e n s e m b l e averag ing over funct ional ly re-

lated neurona l popu la t ions is o n e m e d i u m th rough w h i c h the opera t iona l

c o n s e q u e n c e s of al tered neurona l p rocess - subs t ra tes is mani fes t . N o t i o n s tha t

learn ing and m e m o r y m i g h t b e subse rved b y diffuse p rocesses have b e e n

a round for s o m e t ime ( 1 5 , 1 7 , 2 0 ) . Ideas conce rn ing cod ing (and " s w i t c h i n g " ) of

in format ion v ia the stochastic interactions of neura l e n s e m b l e s are also no t n e w

(25 ,31) . O n e w o n d e r s if comparab l e p rocesses exis t to b e ident i f ied b y those

w o r k i n g in the area of cell g rowth and different ia t ion or in o the r areas w i t h i n

the field of research on men ta l re tardat ion.

Substances and Mechanisms

A final cons ide ra t ion conce rn ing p rocesses under ly ing b e h a v i o r conce rns

subs t ra tes under ly ing the p rocesses t hemse lves . A l t h o u g h in the last decade

and a ha l f p rogress has b e e n m a d e toward unde r s t and ing s o m e elec-

t rophys io logica l p rocesses potent ia l ly invo lved w i t h l ea rn ing and plas t ic i ty ,

n o n e of t hese p rocesses is as yet exp la ined b iochemica l ly . For p rocess there

is m e c h a n i s m , and unde r s t and ing of m e c h a n i s m surely m u s t d e p e n d u p o n

knowledge of invo lved b i o c h e m i c a l subs t ra tes and the i r in te rac t ions . It is j u s t

as easy for th is to b e over looked in the con ten t of p sychophys io log ica l i nves -

t iga t ion as it is for those w h o w o u l d define the subs t ra tes to over look the

p roper p sychophys io log ica l context .

In conc lus ion , r ecogn i t ion of the complex i ty of p rocesses and subs t ra tes that

under l ie c o n d i t i o n i n g is neces sa ry i f w e are to formulate and pose the ques -


t ions that mus t b e asked , inves t iga t ive ly , to advance our under s t and ing of

cond i t i oned behav io r . The re is also n e e d to secure adequacy of m e a s u r e m e n t

in a t t empt ing inves t iga t ive so lu t ions to any p o s e d ques t ions . M o d e l or s impl i -

fied sys t ems and par t i t ion ing of complex p rocesses accord ing to the i r con-

sequen t i a l opera t ions m a y b e useful in th is regard , bu t one mus t none the l e s s

b e w a r e of overs impl i f ica t ion .

R E F E R E N C E S

1. Arbib, M. A. Brains, Machines, and Mathematics. McGraw-Hill, New York, 1964: A1-A9.

2. Bard, P., and Macht, M. B . The behavior of chronically decerebrate cats. In Ciba Foundation Symposium on the Neurological Basis of Behavior. (G. E. W. Wolstenholme and C. M. O'Connor, Eds.). Little, Brown, Boston, Massachusetts, 1958: 55-75.

3. Beck, E. C , and Doty, R. W. Conditioned flexion reflexes acquired during combined catalepsy and de-efferentation. / . Comp. Physiol. Psychol, 1957, 50: 211-216.

4. Black-Cleworth, P. Conditioned blink acquired by pairing click and electrical stimulation of facial nerve. In: Cellular Mechanisms Subserving Changes in Neuronal Activity. (C. D. Woody et al, Eds.). Brain Information Service/Brain Research Inst., UCLA, Los Angeles, 1974: 111-118.

5. Buchwald, J . S. Classical conditioning of the eyeblink response in the simplified mammalian central nervous system. In: Cellular Mechanisms Subserving Changes in Neuronal Activity. (C. D. Woody et al, Eds.). Brain Information Service/Brain Research Inst., UCLA, Los Angeles 1974: 143-151 .

6. Buchwald, J . S. Neurophysiological substrates of behavioral habituation and conditioning. Chapter 11, this volume.

7. Crow, T. J . , and Woody, C. D. Acquisition of a conditioned eyeblink response during revers-ible denervation of orbicularis oculi muscles in the cat. Brain Res., 1973, 64: 414-418.

8. Doty, R. W. Discussion. In: Brain Mechanisms and Learning. (A. Fessard et al, Eds.). Blackwell, Oxford, 1961: 659.

9. Engel, J . , J r . , and Woody, C D . Effects of character and significance of stimulus on unit activ-ity at coronal-pericruciate cortex of cat during performance of conditioned motor response. / . Neurophysiol, 1972, 35: 220-229.

10. Farel, P. Habituation and persistent PTP of a monosynaptic response. In: Cellular Mechanisms Subserving Changes in Neuronal Activity. (C. D. Woody et al, Eds.). Brain Information Service/ Brain Research Inst., UCLA, Los Angeles 1974: 4 5 - 5 6 .

11. Freeman, J . A. , and Stone, J . A technique for current density analysis of field potentials and its application to the frog cerebellum. In: Neurobiology of Cerebellar Evolution and Development. R. Llinas, Ed.). Amer. Med. Ass. Educ. & Res. Found., Chicago, Illinois, 1969: 4 2 1 ^ 3 0 .

12. Garcia, J . , and Ervin, F. Gustatory-visceral and telereceptor-cutaneous conditioning: Adapta-tion in internal and external milieus. Commun. Behav. Biol, 1968, 1: 389^415.

13. Gormezano, I. Classical conditioning. In: Experimental Methods and Instrumentation in Psychol-ogy. (J. B . Sidowski, Ed.). McGraw-Hill, New York, 1966: 385-420.

14. Gormezano, I. The interstimulus interval and mechanisms for CS-CR functions in classical conditioning. In: Cellular Mechanisms Subserving Changes in Neuronal Activity. (C. D. Woody et al, Eds.). Brain Information Service/Brain Research Inst., UCLA, Los Angeles 1974: 97-110 .

15. Hebb, D. O. The Organization of Behavior. Wiley (Interscience), New York, 1949. 16. Kandel, E. R., and Spencer, W. A. Cellular neurophysiological approaches in the study of

learning: Physiol Rev., 1968, 48: 65-134. 17. Lashley, K. S. See: The Neuropsychology ofLashley. (F. A. Beach et al, Eds.). McGraw-Hill, New

York, 1960.

11 A. Neurophysiological Substrates 341

18. Mering, T. A. Aspects of Conditioned Reflex Closure Involving Auditory Analyzers. Med. Publ. House, Moscow, 1967: 1-155.

19. Minsky, M. , and Papert, S. Perceptrons. An Introduction to Computational Geometry. MIT Press, Cambridge, Massachusetts, 1969.

20. Pavlov, I. P. Conditioned Reflexes. (G. V. Anrep, Transl. and Ed.). Oxford Univ. Press, London and New York, 1927.

21 . Renshaw, B . Activity in the simplest spinal reflex pathways. /. Neurophysiol, 1940*, 3: 373-387. 22. Rosenblatt, F. The Perceptron. Cornell Aeronaut. Lab. , Cornell University, Ithaca, New York,

1958: Rep. UG-1196-G-1. 23. Schneiderman, N. , and Gormezano, I. Conditioning of the nictitating membrane of the rabbit

as a function of CS-US interval. / . Comp. Physiol Psychol, 1964, 57: 188-195. 24. Selfridge, O. G. Pandemonium: A Paradigm for Learning. Armed Services Technical Information

Agency (ASTIA) 1960: AD 236251; also JA-1140 (1958).

25. Siebert, W. M., and Communications Biophysics Group. Processing Neuroelectric Data. MIT Press, Cambridge, Massachusetts, 1959.

26. Spencer, W. A. , Thompson, R. F . , and Neilson, D. R., Jr. Response decrement of the flexion reflex in the acute spinal cat and transient restoration by strong stimuli. / . Neurophysiol, 1966, 29: 221-239.

27. Spencer, W. A., and Thompson, R. F . , and Neilson, D. R., Jr. Alterations in responsiveness of ascending and reflex pathways activated by iterated cutaneous afferent volleys. / . Neuro-physiol, 1966, 29: 240-252.

28. Spencer, W. A. , Thompson, R. F . , and Neilson, D. R., Jr. Decrement of ventral root electro-tonus and intracellularly recorded PSPs produced by iterated cutaneous afferent volleys. / . Neurophysiol, 1966, 29 : 253-274.

29. Stevens, C. F. Neurophysiology: A Primer. Wiley, New York, 1966: 161-173. 30. Tauc, L. Transmission in invertebrate and vertebrate ganglia. Physiol. Rev., 1967, 47: 521-593. 31. Woody, C. D. Some Aspects of Information Processing in the CNS. Honor's Thesis, Harvard

Medical School, Cambridge, Massachusetts, 1962.

32. Woody, C D . Aspects of the electrophysiology of cortical processes related to the develop-ment and performance of learned motor responses. Physiologist, 197A, 17: 49-69 .

33. Woody, C. D. Mechanisms underlying blink conditioning in the cat. In: Cellular Mechanisms Subserving Changes in Neuronal Activity. (C. D. Woody et al, Eds.). Brain Information Service/ Brain Research Inst., UCLA, Los Angeles 1974: 5 -12 .

34. Woody, C D . , and Black-Cleworth, P. Differences in excitability of cortical neurons as a func-tion of motor projection in conditioned cats. / . Neurophysiol, 1973, 36: 1104-1116.

35. Woody, C. D. , and Brozek, G. Changes in evoked responses from facial nucleus of cat with conditioning and extinction of an eye blink. / . Neurophysiol, 1969, 32: 717-726.

36. Woody, C. D. , Carpenter, D. , Knispel, J . D . , Crow, T. , and Black-Cleworth, P. Prolonged increases in resistance of neurons in cats motor cortex following extracellular iontophoretic application of acetylcholine (ACh) and intracellular current injection. Fed. Proc, 1974, 33: 399.

37. Woody, C. D. , and Engel, J . , Jr. Changes in unit activity and thresholds to electrical micro-stimulation at coronal-pericruciate cortex of cat with classical conditioning of different facial movements. / . Neurophysiol, 1972, 35: 230-241.

38. Woody C D . , Vassilevsky, N. N. , and Engel, J . , Jr. Conditioned eye blink: Unit activity at coronal-precruciate cortex of the cat. / . Neurophysiol, 1970, 33: 851-864.

39. Woody, C. D. , and Yarowsky, P. J . Conditioned eye blink using electrical stimulation of coronal-pericruciate cortex as conditional stimulus. / . Neurophysiol, 1972, 35: 242-252.

40. Woody, C , Yarowsky, P., Owens, J . , Black-Cleworth, P., and Crow, T. Effect of lesions of cortical motor areas on acquisition of conditioned eye blink in the cat. / . Neurophysiol, 1974, 37: 385-394.

12 Unit Recordings during Pavlovian

Conditioning

JAMES OLDS Division of Biology, California Institute of Technology,

Pasadena, California

T h e s c i ence of l ea rn ing and m e m o r y w o u l d b e advanced b y a be t t e r under -

s tand ing of the swi t ches that o p e n and close connec t i ons b e t w e e n neu rons .

T w o k i n d s of s w i t c h i n g m e c h a n i s m s are k n o w n . T h e s imples t is a n e u r o n

w h i c h s tands b e t w e e n two others . Unt i l i ts th resho ld is c rossed , it bars the

path. O n l y w h e n i ts th resho ld i s c rossed i s the pa th open . S l igh t ly m o r e

compl ica ted are p resynap t ic i nh ib i to r s (4) . T h e s e are n e u r o n s w h i c h modi fy

m e s s a g e flow b e t w e e n two o the r n e u r o n s w i t h o u t s t and ing b e t w e e n t hem. In-

s tead, these del iver the i r m e s s a g e on to the j u n c t i o n b e t w e e n ano the r two . B y a

burs t of act ivi ty they can b lock the m e s s a g e p a t h w a y al together . T h e s e two

k inds of swi t ches have the d i sadvantage that w h e n a t empora ry reverbera t ion

ceases , the swi tch flops b a c k to its s teady s tate , open or c losed as the case m a y

b e . T h u s t hey lack the at tract ive feature of the magne t i c core m e m o r i e s in a

compute r . Core swi t ches ho ld the i r se t t ings and thus p rov ide for long- run

m e m o r i e s of words and p rograms . Bra in swi t ches that ho ld the i r se t t ings s e e m

on the verge of b e i n g d i scovered . Compl i ca t ed neurona l c o n n e c t i o n s w i th a

great var ie ty of shapes and m e c h a n i s m s are s tud ied phys io log ica l ly and o b -

served w i t h the e lect ron mic roscope . A n a t o m i s t s s h o w that c o n n e c t i o n s

change in n u m b e r and s ize d e p e n d i n g on h o w they are used (5) . N e u r o p h y s -

io logis ts s h o w that u n u s e d inpu t s are funct ional ly d isp laced b y u sed ones

(17) . Mic roe lec t rodes pene t ra te into the compl ica ted dendr i te sy s t ems that

form w h a t looks l ike a s w i t c h i n g n e t w o r k on the inpu t s ide of the larger

n e u r o n s of the b ra in (8 ) . There fore I expec t several m e c h a n i s m s for s tab le

swi t ch ing ac t ion wi l l soon b e d i scovered , and these wil l b e the bas i c l ea rn ing

and m e m o r y m e c h a n i s m s for record ing in fo rmat ion and for p r o g r a m m i n g the

bra in .

343

344 James Olds

T h i s p rob lem wil l rece ive con t r ibu t ions from m a n y d i rec t ions . M y s tudies

are to put tools of neu ropsycho logy to work on it. M o r e specifically they are to

locate and character ize s o m e of the swi tches invo lved in a cond i t i on ing exper-

iment . The re are usual ly four s teps : (a) trace out m e s s a g e pa ths pr ior to

swi tch ing ; (b) cause swi t ch ing to occur b y s o m e opera t ion ; (c) find w h e r e the

message b r a n c h e d into n e w pa ths ; (d) find w h a t k i n d of swi t ch ing act ion

caused the b r a n c h i n g ( i . e . , the dynamic k i n d d e p e n d i n g o n reverbera t ions or

the more structural and s table k ind) .

T h e expe r imen t s are m a d e w i t h a v i e w to lay ing traps o r snares to he lp us

find and character ize these k inds . I wil l try to s h o w h o w the snares work , and

prove that they can work , ra ther than display the catch. T h i s is because the

p rogram has no t p rogressed fast e n o u g h to give a good catch yet.

The re are five me thodo log ica l b a s e s that n e e d to b e m a d e explici t . T h e first

bas i s is a stat is t ical s ampl ing a s sumpt ion pa t te rned after that under ly ing

publ ic o p i n i o n pol ls . I pose a set of ques t ions and ask a fair sample of neu rons

in each part of the b ra in to g ive answers . M y p remi se is that a sample n u m -

be r ing in the thousands can g ive a fair r epresen ta t ion even i f the popula t ion is

indef ini te ly large.

T h e second is a specia l un i t - record ing m e t h o d . M o n i t o r i n g e lec t rodes are

b rough t next to small famil ies of s ingle ne rve cells so that cells can " a n s w e r

the q u e s t i o n s " wi th the i r firing pat terns . It is a s t range k ind of e lec t rode, not

smal l e n o u g h to b e mic ro , no t large e n o u g h to b e macro . It t ouches abou t 26

large neu rons . It " s e e s " on ly the sp ikes of these . I coun t a s ized ba tch of the

larger ones ; these c o m e from abou t ten neu rons . Each sp ike that gets coun ted

is a s ingle ac t ion poten t ia l , a s ingle m e s s a g e uni t , from a s ingle neu ron . Bu t

the total count a t t r ibuted to a " u n i t " is actually the s u m of the sp ikes con t r ib -

u ted b y abou t t en different o n e s . T h i s has d rawbacks and v i r tues . A d rawback

is that neu rons m a y b e w o r k i n g aga ins t one ano ther , and good p h e n o m e n a

m a y b e mi t iga ted or h idden . A v i r tue is that i f the p robe sl ips to any degree

the n e w ba tch is m o r e l ike the old o n e than if th is were really a s ingle uni t .

T h e third bas i s o f these expe r imen t s is averaging data ep i sodes to e l imina te

no i se and unre la ted even ts . Neurona l act ivi ty unre la ted to the s t imul i are

" e l i m i n a t e d " b y averag ing data ep i sodes and synch ron i z ing or l in ing these up

on the bas i s of the onse t of the s t imul i . W h e n th is is done , i r relevant excesses

are supposed to b e m a t c h e d b y deficits , and thus to cancel . In the end , all of

the inf lect ion po in t s are therefore p r e s u m e d to represen t neurona l (or b e h a v -

ioral) ac t iv i ty caused b y the s ignal . If there are e n o u g h data ep i sodes in the

average th is p r e sumpt ion is val id to a point . There fore , the data are averaged ,

and a t tent ion is pa id ma in ly to inf lect ion po in t s .

T h e fourth me thodo log ica l u n d e r p i n n i n g is to use success ive r e sponse la-

tenc ies at success ive po in t s in the b ra in , eventua l ly h o o k i n g up wi th b e h a v -

ioral r e sponse la tencies to es tab l i sh p re sumpt ive causal pa thways . In isola ted

cha ins of neu rons w h e r e the a n a t o m y is clear th is wou ld not b e necessa ry

12. Unit Recordings during Pavlovian Conditioning 345

(unless mag ic or ac t ion at a d i s tance were invoked) . Bu t to follow a s ignal in

the b ra in k n o w i n g the ana tomica l pa thways is no t e n o u g h b e c a u s e there is

such a superf lui ty of pa ths . La t ency m a p s are to find w h i c h p a t h w a y the

s igna l actual ly t akes (before and after cond i t i on ing ) .

T o m a k e these , a s ignal w i t h a sharp onse t is appl ied and the arrival o f i ts

m e s s a g e is t racked in success ive t i m e frames th rough the va r ious s ta t ions of

the b ra in . T h e behav io ra l r e sponses of the an ima l can b e put in to the s ame

pic ture . A n d it is to a l imi ted degree pos s ib l e to j u m p over gaps i n our ana-

tomica l k n o w l e d g e . B y this m e t h o d m e s s a g e m a p s por t ray ing the course of the

s ignal t h rough the b ra in in t ime and space are genera ted . B y over lay ing suc-

cess ive m a p s m a d e at the different s tages of cond i t i on ing it shou ld b e poss ib l e

to trace out a family of changes that succeed o n e ano ther dur ing the course of

t ra in ing.

Ave rag ing and la tency m a p p i n g b e t w e e n t h e m cons t i tu te the m a i n at tack on

the p r o b l e m of m o v e m e n t artifact and on the neura l correlates o f m o v e m e n t

w h i c h w o u l d b e a lmost as d i s rup t ing as artifacts. T h i s is b e c a u s e I do not

wan t the neu rona l correlates fed b a c k from m o v e m e n t s to b e coun ted as

learned b r a in r e sponses . Ave rag ing e l imina tes the effects of m o v e m e n t s w h i c h

are not caused b y the s ignal . T h e n res t r ic t ing s tudies to shor t la tency

r e sponses e l imina te s the effects of m o v e m e n t s w h i c h are caused b y the s ignal .

Res t r i c t ing a t ten t ion to the b ra in r e sponses of shor t l a tency has d r awbacks as

wel l as advan tages . T h i s is b e c a u s e in s o m e cases on ly relat ively un in te re s t ing

lea rned even t s occur in th i s first b r i e f in terval . T h e s e are the l ea rned o r ien t ing

r e sponses that p recede the dec i s ion p rocess . W e m a y lose the dec i s ion process

a l together . It i s a lmos t l ike t h rowing the b a b y out w i t h the b a t h water . Bu t

ge t t ing at s o m e va l ida ted swi t ch ing po in t s m a y b e wor th the h i g h pr ice .

T h e fifth bas i s o f these expe r imen t s is a cond i t i on ing procedure . A " n o n -

s e n s e " s ignal is appl ied first to find the p a t h w a y it takes wh i l e it has m i n i m a l

m e a n i n g , t hen it is conver ted in to a mean ingfu l s ignal b y assoc ia t ion wi th

food, and the sw i t ch ing p rocesses that med ia t e the change are obse rved .

T h e m a i n p r o b l e m that en ters an expe r imen t o f this k ind has to do w i t h

s tab i l i z ing the an ima l so that the only difference b e t w e e n the " b e f o r e " and

"a f t e r " cond i t i ons wi l l b e in the m e a n i n g of the part icular s igna l unde r s tudy.

The re shou ld b e no change in the genera l cond i t ion of the an imal w h i c h

wou ld account for an al tered r e spons ivenes s and thus for al tered m e s s a g e

pa thways . It is c lear that a ve ry hung ry an ima l in an inhosp i t ab l e e n v i r o n m e n t

has a different genera l cond i t i on from a hung ry an ima l w h i c h is b e i n g per iod-

ically fed. T h e per iod ic feed ing p rovokes a state o f r ead ines s and it also

modif ies the an ima l ' s genera l a t t i tude toward the tes t ing cage . O u r expe r i ence

ind ica tes that an audi tory s ignal p r e sen t ed dur ing pe r iod ic feed ing ( in an

expe r imen t w h i c h w e call p s e u d o c o n d i t i o n i n g ) often has different effects from

the s a m e s igna l p re sen ted a lone . B e c a u s e of th is it is r equ i r ed that feed ing go

on dur ing the p re l imina ry control pe r iod ; and therefore w e have u s e d p seudo-

346 James Olds

cond i t i on ing as the p re l imina ry ba se l i ne cond i t ion for m a k i n g the first m e s -

sage m a p .

T h e expe r imen t i s run and t i m e d b y a compute r . The re are three s ignals :

two rela t ively pure tones of 1 and 10 k H z , and the m a g a z i n e w h i c h m a k e s a

no i se and g ives a pellet . T h e r e is an average of 1 m i n u t e b e t w e e n s t imul i and

an average o f 3 m i n u t e s b e t w e e n re in fo rcements . T h e in te r s t imulus intervals

range from 10 seconds to 2 m i n u t e s , and the in te r re in forcement intervals from

10 seconds to 10 minu t e s . Dur ing p seudocond i t i on ing , the different s ignals

are r a n d o m i z e d (really p s e u d o r a n d o m i z e d ) , bu t are separa ted from one an-

other b y at least 10 seconds . D u r i n g cond i t i on ing the food is regularly pre-

sented 1 second after o n e of the tones . T h i s n o w b e c o m e s the C S + and the

o ther tone therefore b e c o m e s the CS—. One-k i loHer t z and 10-kHz tones are

al ternated as C S + from sub jec t to subjec t .

It has b e e n po in t ed ou t that th is k i n d o f p s e u d o c o n d i t i o n i n g schedule m a y

cause the acqu i s i t ion of act ive i nh ib i t o ry proper t ies b y the C S + and the C S —.

A n d therefore these are no t true " n o n s e n s e " s ignals dur ing the control pe r iod .

The re is ev idence that th i s occurs to a m i ld degree ; b u t cer ta in ly a g e n u i n e

swi t ch ing opera t ion occurs dur ing the conve r s ion of these neutra l or mi ld ly

i nh ib i to ry cues to act ive exci ta tory cond i t i oned s t imul i .

Rescor la (13) conc luded that there is little ev idence that s imply nonre in -

forcing a C S in i so la t ion is sufficient to p roduce any act ive proper t ies ( i nh ib i -

tory or exci ta tory) . I f the C S is nonre in fo rced w h e n in c o m b i n a t i o n wi th o ther

cues it wi l l b e c o m e inh ib i to ry . In our case there is no c o m b i n a t i o n . Bu t unde r

some c i rcumstances in w h i c h re in forcement is regular ly g iven in the a b s e n c e

of a nonre in fo rced C S , it i s pos s ib l e that the s i tua t ional cues wil l acqu i re ex-

ci tatory value and this m a y lead to the acqu i s i t ion of i nh ib i to ry proper t ies

b y the C S . T h e cond i t ions for the effect, name ly , that the re in forcement b e

regularly appl ied and b e d i sp laced b y a regular t ime after each p resen ta t ion of

the C S , we re not me t in our s tudy. T h e in te r re inforcement in terval var ied

from 10 seconds to 10 m i n u t e s (mean = 3 m i n u t e s ) and the interval b e t w e e n

C S and re in forcement also va r ied from 10 seconds to 10 m i n u t e s (mean = 1 . 5

minu t e s ) . T h u s the non re in fo rcemen t in terval guaran teed b y the C S (10 sec-

onds ) w a s m i n i m a l c o m p a r e d w i t h the actual t i m e b e t w e e n re in forcements .

Sti l l there w a s ev idence in s o m e cases of a mi ld i nh ib i t o ry effect.

Psycho log i s t s d iv ide cond i t i on ing in to two types called Pav lov ian and

operant , d e p e n d i n g on w h e t h e r the expe r imen te r fully de t e rmines the t ime of

re inforcement b y direct appl ica t ion of the u n c o n d i t i o n e d s t imulus , or w h e t h e r

the an imal b y s o m e voluntary- l ike r e sponse par t ic ipates in d e t e r m i n i n g the

t ime of appl ica t ion of the re in forcement . W h e n the expe r imen te r does all the

s t imula t ing it is cal led Pav lov ian ; w h e n the an imal he lps b y s o m e do-i t -your-

self b e h a v i o r it is called operant . Usua l ly in operan t cond i t i on ing there i s n o

real C S at all, on ly a pedal and a reward . T h i s could not b e used b e c a u s e there

wou ld b e no s t imulus to synchron ize the record ings . O p e r a n t cond i t ion ing


w i th a s igna l (in w h i c h a r e sponse is re inforced on ly dur ing or after a s ignal)

is really a mix tu re o f the two . Because there is qu i cke r l ea rn ing in th i s case

than in pure Pav lov ian cond i t i on ing , w e used th is ve r s ion . T h e expe r imen te r

(who w a s a c o m p u t e r i n our case) d i scharges a food m a g a z i n e and offers the

an imal a food pellet ; bu t it is up to the an ima l w h e t h e r it wil l eat the food or

not . B y the t ime p re t ra in ing is d o n e — a n d th is occurs dur ing a 5-hour pe r iod

jus t before the b e g i n n i n g of the expe r imen t—the s igna l o f the food m a g a z i n e

is b y i tself re inforc ing; and moreove r the an imal regular ly takes the pellet . T h e

an imals are at abou t 7 5 - 8 0 % of no rma l b o d y w e i g h t and the i r pel let retr ieval

b e h a v i o r b e c o m e s exper t qu ick ly .

M E T H O D S

(a) T h e sub jec t s w e r e rats, (b) Severa l p r o b e s we re imp lan ted in each rat so

that r ecord ings were m a d e s imul taneous ly from several different b r a in areas,

(c) T h e exper imenta l ca lendar was as fol lows: day 1 = surgery; day 5 =

p seudocond i t i on ing ; day 6 = cond i t i on ing ; day 7 = h is to logy , (d) T h e sched-

ule on days 5 and 6 w a s as fol lows: 240 cond i t i on ing or p s e u d o c o n d i t i o n i n g

trials in 13 hours . T h i s is abou t one in 3 m i n u t e s . T h e s e were d iv ided in to

four g roups of 60 trials each for data analys is . O n day 5 all of these were

p seudocond i t i on ing . O n day 6 the first b lock of 60 w a s p s e u d o c o n d i t i o n i n g

and the rest (180 trials) were cond i t i on ing . O n b o t h days there were also 240

presen ta t ions of the CS— and 240 presen ta t ions of the m a g a z i n e and food.

O n day 5 the 240 C S + trials were separa ted from the m a g a z i n e presenta-

t ions (as de sc r ibed prev ious ly) ; dur ing the c o n d i t i o n i n g tr ials o n day 6 they

were correlated, (e) T h e trials we re d iv ided in to 200 t ime b i n s (1.5 to 10 m s e c

in different expe r imen t s ) . T h e first 100 b i n s were pr ior to C S onse t . T h e sec-

ond 100 b i n s we re dur ing the C S . T h e C S las ted for 1.5 seconds a n d over-

lapped the U S dur ing the last i s econd (in c o n d i t i o n i n g tr ials) .

T h e day 5 and day 6 expe r imen t s started at 5 PM one day and f in ished at 6

AM the next . B e c a u s e of the diurnal cycle and hunge r , ac t iv i ty w a s h ighes t

dur ing the first 3 g roups of 60 trials and s lacked off dur ing the last 60 tr ials.

Therefore p e a k c o n d i t i o n i n g per formance regularly occurred dur ing the next

to the last 60- t r ia l g roup .

O n e o ther detai l in the presen t m e t h o d is no tab le . W h e n lea rned b ra in

r e sponse s b e g a n to b e o b s e r v e d i n the lower parts o f the audi tory s y s t e m the

mos t r easonab le in terpre ta t ion at first w a s that the an imal had learned s o m e

n e w w a y to pos i t i on h i m s e l f so that he could be t t e r hea r the s ignals . T h i s was

no t exact ly t he k i n d of e n g r a m w e h a d set ou t to s tudy and w e h a v e sought

ways to assure ourse lves that th is w a s not h a p p e n i n g . O n e w a y has b e e n to

record from the cochlear nuc leus and the e igh th nerve in h o p e s of p rov ing that

the n e w r e sponse s w o u l d no t occur in these lowes t centers . T h e o the r was to

348 James Olds

FIGURE 1. Rat with cable and plastic auditory horn. In the background is the food chute and, on the right of that, the water tube.

create a sort of " s t ab i l i zed audi tory i m a g e " so that the an imal wou ld stay in

the same relat ion to the sound source even after m o v i n g i ts head . To do th is

we used a plast ic ho rn and a t tached it to the headp iece (see F igure 1) . Th i s

w a s r igidly affixed and all s ignals in recent expe r imen t s were sent down a

sound tube and del ivered th rough this dev ice . T h e y also have b e e n of very

small in tens i ty , b e i n g abou t 20 dB at 1 inch from the ho rn (which is abou t the

d is tance of the ear) .

T h e s e m e t h o d s and closely related ones have b e e n desc r ibed in a ser ies of

pub l ica t ions b y inves t iga tors of our group ( 1 - 3 , 6 , 7 , 9 - 1 2 , 1 4 - 1 6 ) . I wil l b o r r o w

freely from those pub l i ca t ions and from u n p u b l i s h e d obse rva t ions b y these

inves t iga tors in the fo l lowing d i scuss ion . T o keep the record straight , J o h n

Dis terhof t (current ly at Nor thwes te rn ) is r e spons ib l e for m u c h of the data on

the infer ior col l iculus , the med ia l genicula te , the pos te r ior nuc leus , and the

audi tory cortex. M e n a h e m Sega l (current ly at N I M H ) is r e spons ib l e for m u c h

of the data on the h i p p o c a m p u s . M a r i a n n e Olds (Cal tech) is r e spons ib le for

m u c h of the data on the hypo tha l amus . Carol Kornb l i th (Univers i ty of Nor th


Carol ina) and J a c q u e s Mon tp l a i s i r (McGil l ) are r e spons ib l e for the data on the

ret icular ac t iva t ing sys tem. M a r y A n n L i n s e m a n (Toronto) is r e spons ib l e for

data on the basa l gangl ia and n e a r b y s tructures .

P H A S E S O F L E A R N I N G

For the m e t h o d to work , o n e r e q u i r e m e n t i s that it separa te the different

l ea rn ing even t s in a neurona l cha in b y s h o w i n g t h e m to occur at different

trials or p h a s e s o f l ea rn ing . T h i s r e q u i r e m e n t can b e demons t r a t ed b y a s sum-

ing s o m e cha in of n e u r o n s arrayed in a success ion : A - B - C . If all three had

thei r r e sponses c h a n g e d b y cond i t i on ing , th is w o u l d at first on ly locate the

change at the i npu t s ide o f A . A n y change in the A - B c o n n e c t i o n or the B - C

connec t ion w o u l d go unde tec ted . B u t i f the c h a n g e in C s r e sponses occurred

at a different po in t in the trial s e q u e n c e than the change in the A and B

re sponses (e i ther ear l ier or la ter) , this w o u l d separa te the l inks , and s h o w the

B - C connec t i on to b e plastic or swi tchab le . Therefore the l ea rn ing of different

b r a in centers at different rates w o u l d b e no t on ly in te res t ing in itself, bu t

me thodo log ica l ly it w o u l d provide an addi t ional ba s i s to separa te different

l inks in the cha in to s h o w w h e r e plast ic coup l ing po in t s lay.

Actual ly , different b ra in areas d id appear to " l e a r n " at different t imes as

thei r different funct ions b e c a m e appropr ia te . Behav iora l l ea rn ing occurred in a

s equence of phase s (see F igure 2 ) . At first, the an ima l con t i nued to ignore the

hab i tua ted s ignal , bu t r e n e w e d in teres t was g rowing . T h e n the an imal for a

b r i e f n u m b e r of trials paused w h e n the s t imulus w a s appl ied and looked

toward it. After th i s , the s ignal b e g a n to cause over t b e h a v i o r d i rec ted in a

s low and a w k w a r d w a y toward the food tray. Later th i s was fast and wel l -

d i rec ted . F ina l ly , after o b v i o u s b e h a v i o r i m p r o v e m e n t w a s comple t ed , the

b e h a v i o r still b e c a m e m o r e ing ra ined so it w a s harder to un t ra in later.

T h e data correlat ing different b ra in sys tems w i t h these different phases

have a f law, bu t I p re sen t t h e m a n y w a y b e c a u s e the n e w data to replace t h e m are

not r eady yet . T h e flaw is that they are b a s e d on the w h o l e 1-second in terval

b e t w e e n the C S and the U S and therefore cou ld b e in f luenced b y feedback

from behav io r , and p r o b a b l y are. I a m no t sure h o w m a n y of the p h e n o m e n a

wil l s tand u p w h e n w e look on ly at the first 20 m s e c after s t imulus onse t .

Hopeful ly several of the in te res t ing ones wil l . Tha t the b ra in " l ea rn ing c u r v e s "

do not jus t fol low the b e h a v i o r l ea rn ing curves i s s o m e cause for h o p e (see

F igures 3 and 4 ) . If they did so they wou ld no t differ f rom each o the r as they

do.

W h i l e r e n e w e d in teres t w a s a t taching i t se l f to the s ignal , n e w bra in r e sponses appeared in severa l areas a long the b a s e of the b ra in , inc lud ing areas in the pons , the m i d b r a i n , the hypo tha l amus , and the t e l encepha lon (see Tab le I ) . All of these areas were potent ia l ly related to the dr ive a n d reward

Behavior learning

1—i—i—i—i—i—i— r

m m 2

Response = rate during I sec ofter CS onset

Background rate (between trials)

Conditioning trials

FIGURE 2 . Behavioral learning curve showing average behavior rates during the 1 second after

stimulation (O) and average behavior rates during the 1 second before stimulation ( • ) at different

stages of conditioning. The "movement detections' 7 constitute an arbitrary measure derived by

counting the voltage deflections in a noisy cable attached to the head. The Roman numerals

denote the phases of learning: I = prior to behavioral learning; II = orientation response

learning; III = purposive behavior; IV = skilled behavior; V = further behavior improvement.

Posterior pons (Kornblith) 7 units

Central midbrain (Kornblith) 17 units

Caudate (Linseman) 8 units

m m 2 i

T " 1 1 Rate of r e s p o n s e ^ ^ j

Background rate

i n m m e -

~T

/ I '

I I 1 S I

20 40 60 20 40 60 0 20 40 60 Conditioning trials

FIGURE 3 . Unit learning curves showing average spike rates during the 1 second after stimulation

(O) and average spike rates during the 1 second before stimulation ( # ) at different stages of condi-

tioning. The posterior pons group was in a region where brain reward was induced by stimu-

lating; the central midbrain group was in an area where arousal thresholds were low. The caudate

was a part of the extrapyramidal motor system. The Roman numerals stand for the same phases as

in Figure 2 . The data are from Kornblith and Olds ( 6 ) and from Linseman and Olds ( 7 ) .

350

Mov

emen

t d

ete

ctio

ns

per s

ec

o

o

o

o

0 I I I i i i I i i 0 20 40 60

Sp

ike

s p

er

seco

nd


Two kinds of learning

Medial geniculate (Disterhoft) 9 units

Auditory cortex (Disterhott) 12 units

i—r—i—i i—i i i

Conditioning trials

FIGURE 4. Unit learning curves for medial geniculate and auditory cortex. Details as in Figure 3.

centers of the hypo tha l amus . C h a n g e s in these centers c a m e before there were

any s igns of c h a n g e d behav io r . D u r i n g the p r e b e h a v i o r l ea rn ing per iod ,

c h a n g e s d id no t occur e l sewhere .

T h e n w h e n the an ima l s b e g a n to look toward the source of the s ignal , there

we re n e w r e s p o n s e s for the first t ime at several s ta t ions o f the ret icular

sys t em, no tab ly in the m i d b r a i n and the tha lamus (see T a b l e II) . At th is t ime ,

a lso, there we re n e w r e sponses in the ventral nuc leus of the tha lamus , the in-

ternal capsule , and the denta te gyrus ( w h i c h is one of the m a i n inpu t sys t ems

for the h i p p o c a m p u s ) . W h e t h e r these we re ma in ly related to m o v e m e n t is

open to ques t ion . In any event , the b ra in r e sponses c a m e first and the m o v e -

m e n t r e sponses s econd in the la tency cha in . The re we re also at th is s tage s o m e

learned r e sponses in assoc ia t ion cen te rs o f the cor tex and tha lamus .

At phase th ree , w h e n the b e h a v i o r b e c a m e purpos ive , there w e r e the first

s igns of l ea rn ing in the ex t rapyramida l m o t o r sy s t ems (see T a b l e III) . T h i s w a s

true for b o t h the caudate nuc leus and the g lobus pal l idus . T h e r e were also

n e w re sponses in the preopt ic area. A n d at th i s t ime the d u b i o u s s igns of

Table I Parts of the Brain That Evidenced Learning at Stage P

Brain Part

1. Hypothalamus

2. Posterior pons

17 7 9

10 5 8

3. Posterior midbrain

4. Central pons 5. Pyriform-amygdala

6. Preoptic (inhibitory)

u Trials 10 -20 , prior to the appearance of behavior signs of learning. Stage I = learning within 20 trials. 6 N = number of cases tested.

Sp

ike

s p

er

seco

nd

352 James Olds

Table II Parts of the Brain That Evidenced Learning at Stage I P

Brain Part Nb

1. Nonspecific thalamus 28 2. Posterior diencephalon 11 3. Central midbrain 17 4. Ventral nucleus 22 5. Dentate gyrus 35 6. Internal capsule 40

Questionable Evidence at this Stage 1. Middle cortex 28 2. Posterior nucleus 11

" At the same time as behavioral signs of learning appeared. Stage II = learning at 20-30 trials. h N = number of cases tested.

Table III Parts of the Brain That Evidenced Learning at Stage III of Behavior Learning"

Brain Part Nb

1. Posterior nucleus (or stage II) 11 2. Preoptic (excitatory) 11 3. Anterior midbrain 4 4. Caudate 8 5. Globus pallidus 16

Questionable Evidence at this Stage 1. Lateral nucleus 13 2. Posterior cortex 13

" Stage III = learning at 30 -40 trials. b N = number of cases tested.

Table IV Parts of the Brain That Did Not Evidence Learning until Stage IV

of Behavior Learning 0

Brain Part Nb

1. Posterior cortex (or stage III) 12 2. Medial geniculate 9 3. Lateral nucleus (or stage III) 13 4. Middle cortex (or stage II) 28 5. Anterior pons (to cerebellum) 14 6. CA 3 (hippocampus) 38

a Stage IV = learning at 40 -50 trials. b N = number of cases tested.


Table V Parts of the Brain That Did Not Evidence Learning until Stage V of Behavior Learning**

Brain Part Nb

1. Frontal cortex 22 2. Dorsomedial nucleus 17 3. CA 1 (hippocampus) 34

4. Subiculum (hippocampus) 40

a Stage V = learning at 50 -70 trials. '' N = number of cases tested.

l earned r e s p o n s e s in the assoc ia t ion cen te r s of the tha lamus lost the i r du-

b i o u s n e s s and b e c a m e p r o n o u n c e d .

At p h a s e four, w h e n the b e h a v i o r b e c a m e s m o o t h and fast, n e w r e sponses

appeared for the first t i m e in the audi tory cor tex (see T a b l e IV) . T h e r e were

also impor tan t upsurges in r e sponses recorded from the med ia l genicu la te ,

from the assoc ia t ion cor tex , from the p o n t i n e reg ion w h i c h forms a l ink

b e t w e e n cor tex and ce rebe l lum, and also from the m a i n field of the h ip -

p o c a m p u s , that i s , the C A 3 field.

Dur ing the fifth p h a s e as behav io ra l changes b e c a m e comple t e , there we re

still three parts of the b ra in w h i c h s e e m e d to go on lea rn ing (see T a b l e V ) . T h e

first of t hese w a s the frontal cor tex. T h e s e c o n d was the do r somed ia l nuc leus

of the tha lamus w h i c h is corre la ted w i t h the frontal cor tex . T h e th i rd w a s the

m a i n output field of the h i p p o c a m p a l cor tex , that i s , the C A 1 field. B e c a u s e

mo to r cor tex and frontal a s soc ia t ion cor tex are over lapped in the rat, it is no t

clear w h e t h e r th i s late l ea rn ing (after b e h a v i o r l ea rn ing w a s comple te ) w a s

correlated w i t h these m o t o r ou tput funct ions or w i t h assoc ia t ion func t ions .

H o w e v e r , it is t e m p t i n g to suppose that it w a s assoc ia t ion centers that were

invo lved and that th is w a s s o m e k i n d of conso l ida t ion p rocess .

W h i l e these data va l ida ted s o m e older ideas abou t the different funct ions of

different a reas , the i r m a i n impor t ance w a s to s h o w that a m a i n r e q u i r e m e n t of

the m e t h o d wou ld work . T h e m e t h o d n e e d e d different b r a in areas to / T e a r n , ,

at different ra tes . T h e y did so and the differences s e e m e d to b e large o n e s .

A U D I T O R Y T U N I N G

T r a i n i n g caused the s igna l to b r a n c h off in to n e w p a t h w a y s at a lmost every

audi tory s ta t ion. It also caused p reex i s t ing r e sponses in the audi tory s ta t ions

to b e modi f ied , usual ly to b e ampli f ied o r e n h a n c e d . B e s i d e s b o t h of these ,

there w a s also a subs tan t ia l c h a n g e in the b a c k g r o u n d firing rate , the so-cal led

spon taneous d i scharge rate of neu rons in s o m e of the audi tory s ta t ions .

354 James Olds

T h e Lower Aud i to ry Cen te r s

R e s p o n s e s recorded from the e igh th ne rve , the coch lear nuc leus , and the

super ior o l ive all b e g a n w i t h i n 3 m s e c of the arrival o f the audi tory s ignal at

the ear (see F igures 5 and 6 ) . T h e average data taken from 19 h igh ly r e spons ive

p robes in the cochlear nuc leus and the e igh th ne rve d id no t s h o w any impor -

tant effects of t ra in ing . T h e s ame w a s true for ten r e spons ive p robes implan ted

in the super io r o l ive . H o w e v e r , there we re ind iv idua l cases w h e r e improve -

ment , even i n the first 1 -3 -msec part o f the cochlear nuc leus r e sponse , d id

occur (see F igure 7 ) . T h e p r o b e s were r e spons ive in the first p lace , h igh ly re-

spons ive . T h e i m p r o v e m e n t a m o u n t e d to abou t 3 0 % . B e c a u s e these were small

p ropor t iona te ly , and b e c a u s e they did no t s h o w up in the overall average, I

wou ld have s o m e inc l ina t ion to doub t w h e t h e r the real effects of cond i t i on ing

reached d o w n th is far. H o w e v e r , b e c a u s e of data from W e i n b e r g e r ' s labora-

tory (Norman M . W e i n b e r g e r , Un ive r s i ty of Cal i fornia , I rv ine , pr ivate c o m -

munica t ion ) that evoked potent ia l changes occurred in the cochlear nuc leus in

the curar ized cat, I take t h e m ser ious ly and wil l explore these centers further.

10 \ - c s +

Cochlear Nucleus + 3ZHL-nerve (19 cases)

h c s - y r - Day 2 (cond 'g )

' 6 0 ' 120 I80|

Tr ia ls

• J L / - D a y I (pseudo)

*^ ^ ^ ^ ^ ^ts» S$*J 1 6 0 1

120 180

Tr ia ls

- 2 3 to - 3

1 6 0 1

120 I80 |

Tr ia ls

6 0 1

120 I 80 |

T r ia ls

I 6 0 1

120 180

T r i a l s

1 60 120' 180

T r i a l s

6 0 1

120 180

Tr ia ls

1 6 0 1

120 I 8 0 |

Tr ia ls

1 6 0 ' 120 I 80 |

Tr ia ls

1 6 0 1

120 I 80 |

T r ia ls

6 0 1

120 180

T r i a l s

1 6 0 1

120 I 8 0 |

T r ia ls

120 180]

T r ia ls

- 3 t o O O t o 3 3 t o 6 6 t o 9 9 to 12 12 to 15 15 to 18 l 8 t o 2 l 2 l t o 2 4 2 4 to 27 to 3 0 to 2 7 3 0 3 3

Milliseconds from stimulus onset

FIGURE 5. Average cochlear nucleus and eigth nerve response during pseudoconditioning

( ) and conditioning ( ). Each group of four points stands for the neuron firing rate in a

particular latency interval. Each of the points is the score for a particular 60-trial block. For the

solid curves, the first point is the 60 trials before conditioning, and the other three are the succes-

sive blocks of conditioning.

Ra

te

in

mu

ltip

les

of

ba

ckg

rou

nd

I I

I I

I I

I I

I I

I I

I I

hi

I I

I I

I I

I


10 r - CS +

\ - c s -

" 7 ^ Superior Olive

(10 cases)

- Day I (pseudo)

• Day 2 (cond'g)

o 1 1 1 o 1 1 1 o ' l l o ' l l o ' l l o ' l l o ' l l o'1 I OIL 60 1 60 1 60 1 60 1 60 1 60 1 60' 60 1 60 1 60 1 60 1 60' 60" 1201 1201 1201 1201 1201 1201 1201 1201 1201 1201 1201 1201 1201

180 180 180 180 180 180 180 180 180 180 180 180 180

Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials

-23 to -3 to 0 0 to 3 3 to 6 6 to 9 9 to 12 12 to 15 !5tol8 !8to2l 21 to24 24 to 27 to 30 to -3 27 30 33

Mi l l iseconds f rom st imulus onset

FIGURE 6. Average superior olive response during pseudoconditioning (-

tioning ( ). The details are the same as in Figure 5 .

-) and condi-

The Inferior Colliculus

At the level of the infer ior col l iculus , the r e sponse to the audi tory s ignal

b e g a n dur ing the s econd 3 -msec interval after arrival o f the s ignal at the ear ,

bu t it r e ached i t s p e a k 3 to 6 m s e c after that . T h e average t aken from 47 re-

spons ive p r o b e s imp lan ted in the infer ior col l iculus s h o w e d an augmen ta t i on

b y c o n d i t i o n i n g of the average p e a k r e sponse (see F igure 8 ) . T h i s occur red in

spi te of the fact that in a full th i rd of the cases , there w a s a subs tan t i a l t rend in

the oppos i t e d i rec t ion . W h e n averages w e r e taken from a specia l ly se lec ted 2 1 -

m e m b e r s u b g r o u p from w h i c h all cases w i t h o u t the m a i n effect w e r e e l imi -

na ted , several m a i n t rends we re o b v i o u s (see F igure 9 ) . Firs t , the average

r e sponse of these n e u r o n s was abou t doub led dur ing the s e c o n d 60 r e sponse s

of cond i t i on ing . S e c o n d , i nc reased r e s p o n s i v e n e s s appea red e v e n in the

3 - 6 - m s e c la tency t ime pe r iod ; thus the i m p r o v e m e n t could no t have b e e n fed

b a c k after the m e s s a g e h a d advanced to h i g h e r cen te rs . T h i r d , there w a s a ten-

dency at the end of the day, at a t i m e w h e n mot iva t ion w a s lower , for the

r e sponses i n the infer ior col l iculus to fall off. Four th , there w a s n o apprec iab le

t rend of any k i n d o n day 1 dur ing p s e u d o c o n d i t i o n i n g . Fif th , a l though there

was no t rend of change in the course of p s e u d o c o n d i t i o n i n g , there w a s a sub-

Ra

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ba

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gro

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I I

I I

I I

I I

I I

I I

I I

I I

A I

I

I I

I I

356 James Olds

I I

5 0

5 0

5 0

150

100

5 0

2 0 0

150

100

5 0

Learning curves for different "latency" intervals ( 5 probes in one animal - 2 4 6 2 )

Overt behavior

Anterior medial geniculate(2) ^ 9

Posterior medial geniculate (3) mjr* (left) * S

Posterior medial (right) -

geniculate (6)

Inferior coll iculus (4)

5 - 9 msec

Cochlear nucleus (5)

' 6 0 ' 120

I80| T r ia ls

- 2 0 3 to - 3

1-5 msec

120 180

Tr ia ls

120 120 120 120 180 180 180 180

Tr ia ls Tr ia ls Tr ia ls Tr ia ls

120 I80|

T r ia ls

- 3 to +1 I to 5 5 to 9 9 to 13 13 to 17 17 to 21

M i l l i s e c o n d s f r o m s t imulus onset

52J 120

I80| T r ia ls

2 0 0 to 2 8 0

FIGURE 7. Behavioral response and five individual brain probe responses for the same rat dur-ing conditioning. The details are as in Figure 5.

stant ial i m p r o v e m e n t b e t w e e n p s e u d o c o n d i t i o n i n g on the first day and pseu-docond i t i on ing on the s econd day. T h i s was no t a chance occur rence . It was not res t r ic ted to jus t these 21 cases , as y o u wil l see later. W h a t caused th is subs tan t ia l change?

S o m e t h i n g w h i c h occur red on the first day m u s t have had an effect even

Un

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sp

ike

s/s

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p i t 1 1 I I I 1

- 4 0 0 40 80 120 160 200

Tr ia ls

FIGURE 8. Average responses for the inferior colliculus, cochlear nucleus, and eighth nerve

during conditioning. Each curve is for just one latency interval (as marked); this was the one that

showed the greatest improvement during conditioning. The trials before and after the start of con-

ditioning are marked on the baseline.

Inferior Colliculus • Group (21 cases)

CS +

4**

\ - c s -

Day 2 (cond'g)

Day I (pseudo)

U-t* »»«4

60' I201, \8C\

-23 to -3

60' I201, 180

o ' l l o ' l l i ! 11 o ' l l o ' l l o ' l l 911 1 o i l 1 o ' l l o' 1

60 1 60' 60 1 60 1 60 1 60' 60 1 60 1 60' 60' I201 I201 I201 I201 120' I201 I201 I201 I201 I201

180 180 180 180 180 180 180 180 180 180 Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials

1 60 1

I20 1, I80|

Trials

-3toO Oto3 3 to 6 6 to 9 9 to 12 12 to 15 15 to 18 18 to 21 21 to 24 24 to 27

27 to 30

30 to 33

FIGURE 9. Average response of 21

tioning ( ) and conditioning ( -


'augmented" inferior colliculus probes during pseudocondi-

). The details are the same as in Figure 5.

Inferior coll iculus i U (N = 4 7 ) 9-12 msec

i> i \

Re

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iple

s o

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ack

gro

un

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Rat

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mu

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les

of

back

grou

nd

358 James Olds

t hough it s tayed h i d d e n or latent . T h e effect is surpr i s ing and in te res t ing for

several r easons . T h e first is that n o h in t that it was h a p p e n i n g could b e o b -

served in the data o f day 1. It ha s b e e n sugges ted that there m i g h t b e two effects

cance l ing one ano the r on the first day; bu t m y expe r i ence is that the cance l ing

could no t b e so perfect as to g ive the flat curves that appeared . Therefore it

looks to m e as if it w e r e some effect p lanted b y the first day 's t ra in ing w h i c h

then conso l ida ted in s o m e w a y dur ing the 11-hour wa i t b e t w e e n exper imen t s .

T h e second in te res t ing po in t is that i t is no t b y any m e a n s a genera l effect in

the b ra in . It w a s res t r ic ted to the infer ior col l iculus and to very c losely l inked

parts of the audi tory sys t em. It was far m o r e c o m m o n in the coll iculus and

m a y b e in the med ia l gen icu la te than in any of the l inked n e i g h b o r s . It d id no t

occur in the ret icular fo rmat ion or in the hypo tha l amus , o r in any of the parts

of the b ra in w h e r e o n e w o u l d expec t mot iva t iona l arousal effects. It s eems to

m e that i f it we re an effect of a pos i t ive assoc ia t ion b e t w e e n th is s ignal and

food, or of a nega t ive assoc ia t ion b e t w e e n th is s ignal and food (bo th of w h i c h

could b e conce ived as go ing on dur ing the first day of p s e u d o c o n d i t i o n i n g ) , it

\ - cs+

Inferior Colliculus { group (18 cases)

-Day 2 (cond'g)

-Day I (pseudo)

120 I80|

Trials

120 i ' l l o ' l l i ' l l i ' l l o ' l l i ' l l i ' l l i i l l i ' l l oil 60 1 60 1 60 1 60 1 60 1 60 1 60 1 60 1 60 1 60 1

1201 120' I201 I201 I201 I201 I201 I201 I201 1201

180 180 180 180 180 180 180 180 180 180 Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials

60' I201,

\eo\ -23 to -3 toO Oto3 3 to 6 6 to 9 9 tol2 12 Tol5l5tol8 18 fo2l 2lfo24 24 to 27to 30 to

-3 I 27 30 33


FIGURE 10. Average response of 18 "diminished" inferior colliculus probes during pseudocondi-

tioning ( ) and conditioning ( ). The details are the same as in Figure 5.

Rat

e in

mu

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of

back

grou

nd


should have b e e n ref lected o n e w a y or ano the r in arousa l or mot iva t iona l

r eg ions . O u r o the r data g ive good ev idence that mot iva t iona l and arousal

e m a n a t i n g from a s igna l do get reflected in h y p o t h a l a m u s and i n the re t icular

format ion . There fore I a m inc l ined to reject t hese and to be l i eve that th i s effect

in the audi tory sys t em has s o m e t h i n g to do w i t h fami l ia r iza t ion; the an imal

b e c o m e s famil iar w i t h th is s t imulus and p o s s i b l y " l e a r n s " to pe rce ive it. T h e

seeds are p lan ted on the first day. Bu t the effect has a de layed onse t . T h i s is a

specula t ion that wil l n e e d to b e tes ted b y e l imina t ing the o the r p o s s i b l e m o -

t iva t ional in f luences .

T h e averages from 18 col l iculus cases w h o s e r e sponse s we re d i m i n i s h e d b y

c o n d i t i o n i n g gave a p ic ture that w a s inverse in several respec ts (see F igure

10) . The re w a s a 3 0 - 5 0 % d i m i n u t i o n in the peak r e sponse . T h a t the d i m i n u -

t ion w a s caused b y c o n d i t i o n i n g w a s a t tes ted b y i ts a b s e n c e on day 1. L ike the

augmen ta t i on it occur red even in the 3 - 6 - m s e c la tency in terval , and it w a s at-

t enua ted dur ing the last 60 trials o f the day. In o n e respec t , h o w e v e r , these op-

posed cases w e r e no t the inverse . T h e r e w a s the s ame subs tan t ia l r i se in

ampl i tude b e t w e e n the day-1 p s e u d o c o n d i t i o n i n g tests and day-2 p s e u d o c o n -

d i t ion ing tes ts . T h e day 1 — day 2 difference w a s thus the mos t d e p e n d a b l e

f inding w e have c o m e u p o n so far (wha tever it m a y m e a n ) .

T h e N u c l e u s o f the T r a p e z o i d B o d y

W h a t e v e r else i t m a y m e a n , it sugges ts that s o m e t h i n g h a p p e n e d on day 1

w h i c h w a s no t e x h i b i t e d in the r e sponses of n e u r o n s in the infer ior col l iculus .

W e r e there any n e u r o n s that s h o w e d subs tan t ia l r e s p o n s e changes on day 1?

Y e s . T h e m o s t m a r k e d of these occurred in an audi tory s ta t ion b e l o w the

col l iculus in the ana tomica l cha in bu t c o m i n g after the col l iculus in the la tency

cha in (see F igure 11) . It could b e a s ta t ion that r ece ives m e s s a g e s after they

have c i rcu i ted th rough the uppe r b ra in a n d then re tu rned , a l though it looks to

b e in a p lace to r ece ive collaterals from n e u r o n s of the coch lear nuc leus (and

m a y b e e v e n from the e igh th ne rve ) . T h i s is the nuc leus of the t rapezoid b o d y .

It i s on the b o u n d a r y of the decussa t ing ne rve fibers on the i r w a y from the

cochlear nuc leus to the super io r ol ive . It i s adjacent to the nuc leus g igan-

tocellularis of the medul la ry re t icular fo rmat ion , and the la tency of its audi tory

r e sponses ( 6 - 1 2 m s e c ) and the i r p las t ic i ty s e e m to labe l it as a part of the

ret icular fo rmat ion . It i s t e m p t i n g to suppose it r ece ives collaterals f rom the

t rapezoid audi tory f ibers b u t that i ts plast ic synapses s o m e h o w s low d o w n the

message . Tha t ' s really a roman t i c no t ion wi th little l ike l ihood , bu t it i s attrac-

t ive all the s a m e . A group of 13 cases c h o s e n m o r e or less ana tomica l ly we re

averaged . T h e r e w a s a shade of a r e sponse he re at 3 - 6 m s e c . A be t t e r bu t still

m i n i m a l r e sponse at 6 - 9 m s e c , and then a defini te r e sponse at 9 - 1 2 m s e c .

E v e n the 9 - 1 2 m s e c r e sponse w a s pre t ty m u c h absen t at the b e g i n n i n g of

p s e u d o c o n d i t i o n i n g . It g r ew over the course of the day. T h e overal l r e sponse

360 James Olds

£ 5 h -

cs+

The Nucleus of the Trapezoid Body (13 cases)

c s -

Day I (pseudo)

Day 2 (cond'g)

60 120 , 180

-23 to -3

'60 120 , 180

4JJ 4JJ °L\\ AMI o 'M AMI r 0

60 1 60' 60 1 601 60 1 60 1 60 1 60 1 60 1 60 1

I 1201 1201 1201 1201 1201 1201 I201 I201 1201 1201

180 180 180 180 180 180 180 180 180 180 Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials T

60' 120, 180

• 3 t o O O t o 3 3 t o 6 6 t o 9 9tol2 12 to 15 15 to 18 18 to 21 21 to 24 24 to 27

27 to 30

30 to 33


FIGURE 11. Average trapezoid nucleus response during pseudoconditioning ( ) and condi-

tioning ( ). The details are the same as in Figure 5.

ga ined so m u c h in s ize as to sugges t an order o f m a g n i t u d e change dur ing the

p s e u d o c o n d i t i o n i n g procedure . T h e i m p r o v e m e n t that accrued dur ing the

course of the p s e u d o c o n d i t i o n i n g trials then d i sappea red comple te ly dur ing

the day 1-day 2 interval bu t reasser ted i tself on the s econd day. T h e improve -

m e n t dur ing pseudo t ra in ing and the loss dur ing the t ime b e t w e e n ses s ions

m a d e th is the inverse of the infer ior col l iculus . Bu t b o t h areas po in t ed to a

h a p p e n i n g on the first day.

T h e T h a l a m u s

T h e neu rons of the pos te r io r tha lamic nuc leus we re far less r e spons ive to

these mi ld tones (20 dB) w i th " s t ab i l i zed audi tory i m a g e s " than they were to

the in t ense tones ( 7 0 - 8 0 dB) from a wall l oudspeaker w h i c h had b e e n used in

p rev ious expe r imen t s (2) . T h e average r e s p o n s e at the he igh t of cond i t i on ing

was bare ly doub le the b a c k g r o u n d rate (see F igure 12) . In th is case , h o w e v e r ,

there w a s n o s ign of the r e sponse in the averages from day 1. T h e la tency of

these r e sponses , h o w e v e r , fell b e t w e e n 15 and 18 m s e c . T h i s put t h e m sub -

stantial ly b e h i n d n e w r e sponses in o ther par ts of the b ra in and pe rmi t t ed the

in terpre ta t ion that cond i t i oned r e sponses here have a secondary status.

In ind iv idua l cases w i t h p r o b e s on the b o u n d a r y b e t w e e n the med ia l gen ic -

ulate and the pos te r io r nuc leus there were m u c h ear l ier l ea rned r e sponses

(wi th 9 - 1 3 - m s e c la tency) w h i c h also arose de novo f rom prev ious ly unre-

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M ^ t M-*>« » v * > r*f$ *CV» r » 3 ^

Posterior Nucleus (15 cases)

C S -

- Day I (pseudo)

/-Day 2 (cond'g)

60' 120', I80|

Trials

-23 to -3

120 180

°!l 1 Sf f t i l l ° ' l l o' l l oU| i l l ol l l 5 f f f

60 ' 60 1 60 1 60 1 60 1 60 1 60 1 60 1 60 1 60 1

120' I201 I201 120 120' 120' I201 120 120' 120 180 180 180 180 180 180 180 180 180 180

Trials Trials Trials Trials Trials Trials Trials Trials Trials Trials

' 6 0 1

120 , I80|

Trials

•3 toO 0 to 3 3to6 6 to9 9tol2 l2tol5 15 to 18 l8to2l 2lto24 24 to 27to 30 to 27 30 33


FIGURE 12. Average posterior nucleus response during pseudoconditioning ( - -

tioning ( ).

- ) and condi-

spons ive p r o b e s dur ing the course of c o n d i t i o n i n g (see F igure 7 ) . I a m not yet

in a pos i t ion to g ive a genera l p ic ture o f the data for the med ia l gen icu la te and

the audi tory cor tex. T h e data are col lected bu t the p robes still n e e d to b e

located, ca tegor ized b y r e sponse , and averaged .

T h e Re t i cu l a r S y s t e m

Data from a s imi la r , bu t no t iden t ica l , e x p e r i m e n t of Mon tp l a i s i r (9) s h o w e d

that n e u r o n s of the pon t i ne ret icular format ion had learned r e s p o n s e s w h o s e

la tenc ies also fell in the 9 - 1 0 - m s e c range . In th i s case , p s e u d o c o n d i t i o n i n g and

cond i t i on ing tests were m a d e success ive ly on the same day (120 trials of p seu -

docond i t i on ing fo l lowed b y 120 trials of cond i t i on ing ) and there w e r e i n t ense

audi tory s ignals from loudspeakers m o u n t e d abou t 1 foot from the head . T h e

peak r e sponse of these n e u r o n s c a m e 6 - 9 m s e c after the onse t of the s ignal

( 5 - 8 m s e c after i ts arrival at the ear) . T h i s r e sponse w a s a l ready subs tan t ia l

even before cond i t i on ing ( amoun t ing to abou t 7 t imes the b a c k g r o u n d firing

level) . It was no t c h a n g e d apprec iab ly b y hab i tua t i on . B u t it w a s doub l ed b y

cond i t i on ing (see F igu re 13) . T h e effect was large and the data we re conv inc ing

because they w e r e all ga the red on one day. D i d these account for the changes

in the audi tory centers? It i s un l ike ly that t hey could accoun t for the changes

in the infer ior col l iculus . T h e latter appea red in the 3 - 6 - m s e c in terval even

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362 James Olds

ROSTRAL MIDBRAIN (26)

CENTRAL MIDBRAIN (40)

msec msec

FIGURE 13. Averaged responses for different groups of reticular formation probes during pseudo-conditioning and conditioning. On the baseline are marked the end times of the latency intervals. There were 120 trials before conditioning and 120 trials during conditioning. A = first 60 trials before. B ( • ) = second 60 before. C (A) = first 60 of conditioning. D ( • ) = final 60.

with a very mi ld tone; and these appeared in a 5 - 8 - m s e c interval w i th a very

in t ense tone . T h u s they d id no t l ikely s tand b e h i n d the coll iculus changes .

D Y N A M I C T R A C E S

W h a t do y o u suppose d id s tand b e h i n d the changes in the infer ior coll ic-

ulus , and those in o ther lower audi tory cen te rs? M y first guess wou ld b e that

there w a s s o m e specia l b o m b a r d m e n t e m a n a t i n g from the cor tex and that the

k ind of swi tches invo lved we re dynamic ones that wou ld flop b a c k after the

cessa t ion of the cort ical p rocess .

T h e message from the cor tex migh t b e e i ther an exci ta tory or an inh ib i to ry

one . A n d it m i g h t b e appl ied e i ther l ike presynapt ic i n h i b i t i o n , or pos t synap-

tically as e i ther a faci l i tatory or i n h i b i t o r y input . If t ra in ing c h a n g e d the

background firing level of cort ical un i t s and if these inne rva ted the audi tory

inpu t channe l s , th is wou ld suffice to expla in s o m e of the changes obse rved .

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M y first gues s w o u l d b e tha t each c h a n n e l from the ear to the cor tex (whe the r

it b e a f r equency c h a n n e l o r a channe l b a s e d on s o m e o the r audi tory feature)

m i g h t h a v e , no t on ly i ts pr iva te l ine to the cor tex , bu t the spec ia l i zed cort ical

cells at the e n d of the l ine m i g h t b e in a pos i t i on b o t h to r e s p o n d to and to

gate the s igna ls in the channe l . T h i s is g u e s s e d m a i n l y on the b a s i s of ana tom-

ical ev idence tha t s e e m s to s h o w th i s k i n d of o rgan iza t ion . If a smal l set of cor-

t ical cells w e r e invo lved , they m i g h t b e ha rd to find.

T o a n s w e r the ques t i on of w h e t h e r any th ing l ike th is h a p p e n s , I have to

refer b a c k to the older data w h i c h , as I sa id p rev ious ly , we re f lawed. T h e s e are

the l ea rn ing curves for the different b ra in areas w h i c h s u m all neu rona l act iv-

i ty dur ing the C S - U S in terval and thus are suscep t ib le to con t amina t i on b y

m o v e m e n t s . A s prev ious ly , I jus t i fy use o f t hese data b y the fact that data from

all b ra in areas shou ld b e equa l ly c o n t a m i n a t e d b y m o v e m e n t s , and ye t the dif-

ferent b r a in areas gave qu i t e different c h a n g e s dur ing learn ing .

D i d the cor tex s h o w any c h a n g e s in b a c k g r o u n d act ivi ty dur ing lea rn ing?

W a s it different in th i s regard from o ther parts o f the b r a i n ? T h e answer on

b o t h scores is " y e s / ' Le t u s look at c h a n g e s i n b a c k g r o u n d ra tes dur ing cond i -

t ion ing . T h e b a c k g r o u n d level of m o v e m e n t (see F igure 2) i s s een to dec l ine

dur ing the pe r iod o f c o n d i t i o n i n g ( this is sad , b e c a u s e it puts a pall over the

cor tex data) . A l t h o u g h m o v e m e n t scores e x h i b i t e d th is change in b a c k g r o u n d

level , i t w a s n o t ref lected in m a n y o f the b r a in cen te rs . In the mot iva t ion

cen te rs , arousal cen te r s , and ex t rapyramida l m o t o r cen te rs , t r a in ing caused

only b r i e f c h a n g e s in b a c k g r o u n d firing rate a n d left t h e m wel l w i t h i n cont ro l

ranges (see F igure 3 ) . T h e m a i n effect of t ra in ing in these cases w a s to cause a

w i d e n i n g gap b e t w e e n the firing rate caused direct ly b y s t imula t ion and the

b a c k g r o u n d firing rate . O n l y in the cor tex w a s there a s table and sus ta ined

change i n the b a c k g r o u n d firing rate i t se l f (see F igure 4 ) . D u r i n g the first two

s tages of t r a in ing , th i s w a s the on ly c h a n g e o b s e r v e d in the cor tex . T h e r e w a s

abou t a 2 5 % dec l ine in the average b a c k g r o u n d sp ik ing rate of these audi tory

cor tex n e u r o n s . At first, the act iv i ty rate dur ing the C S - U S in terval fell a long

w i t h the b a c k g r o u n d . T h e y s tayed parallel for the first, s econd , and th i rd

s tages (dur ing the pe r iod w h e n in teres t , a t ten t ion , and t hen pu rpose were

a l ready b e g i n n i n g to charac te r ize the lea rned behav io r ) . T h i s leaves the

i m p r e s s i o n that i f cor tex par t i c ipa ted at all dur ing th is pe r iod , it d id so b y its

changes in b a c k g r o u n d fir ing, no t b y its c h a n g e d r e sponses . T h e n , dur ing the

fourth a n d later s t ages , the r e sponses to the audi tory s igna l b e g a n to r i se , e v e n

t hough the b a c k g r o u n d ra tes r e m a i n e d depressed . It w a s appea l ing to suppose

therefore that t hese n e u r o n s in audi tory cor tex first r educed the i r b a c k g r o u n d

rate (or pe rhaps the i r c o n n e c t i o n to o ther i r re levant no i se s ) and then inc reased

thei r connec t i on to the spec ia l audi tory s ignal that had jus t acqu i r ed a n e w

m e a n i n g . W h i l e it is o f course pos s ib l e that s lowing of t hese cor tex n e u r o n s

w a s ju s t a ref lect ion of the behav io ra l c h a n g e , ano the r poss ib i l i t y m u s t also b e

taken ser ious ly . T h e dec reased s p o n t a n e o u s d i scharge rate m i g h t b e part of an

364 James Olds

early and t empora ry m e m o r y process invo lved in caus ing c h a n g e d neu ron

r e sponses in o ther parts o f the b ra in . It m i g h t b e that a large n u m b e r of the

audi tory cor tex n e u r o n s had the i r ac t iv i ty suppressed wh i l e a small specia l set

w i th a priori t ies to the C S + b e c a m e m o r e act ive. In th is case , even though the

average r ep resen t ing the major i ty wou ld dec l ine , the specia l d y n a m i c eng ram

wou ld b e an exci ta tory p rocess and it wou ld facil i tate the l ower m e s s a g e

pa thways . A n equa l ly at t ract ive a l ternat ive is that the audi tory s ignal is dif-

fusely represen ted b y a d y n a m i c process in cor tex; and that th is process is

act ive if the s ignal is hab i tua ted . T h e dynamic p rocess in th is case would b e

the gate that bars the path. S u c h a process migh t b e set in m o t i o n b y ha-

b i tua t ion procedures and a t tenuated b y re inforcement . T h i s hypo thes i s sug-

gests several expe r imen t s . O n e is that a n e w s igna l added to the envi ron-

m e n t and then hab i tua t ed should raise the b a c k g r o u n d in the correlated

cortex; and every t ime such a s ignal b e c a m e meaningfu l there wou ld b e a re-

duct ion. W i t h m a n y n o i s e s go ing , the b a c k g r o u n d act ivi ty o f the b i g filter

wou ld b e so h i g h y o u could hard ly hea r yourse l f th ink.

H o w e v e r these expe r imen t s turn out , it is clear that i f there are dynamic

eng rams o p e n i n g and c los ing swi t ches b y descend ing (or a scend ing or local)

b o m b a r d m e n t s , these can b e de tec ted b y look ing for sys temat ic changes in the

b a c k g r o u n d firing rates . " L e a r n i n g c u r v e s " in the p res t imulus firing rate

wou ld b e the key . T h e r e is n o reason w h y these changes shou ld no t b e as easy

to detect as the lea rn ing curves in the r e sponses . Therefore , i f t hey exis t w e

are qu i te l ikely to find t hem. T h i s g ives us one m e t h o d to look for dynamic

swi tches . Is there any s imi lar ly effective m e t h o d for de tec t ing the o ther k i n d

of swi tch or for separa t ing d y n a m i c from structural o n e s ?

T I M E - O U T E X P E R I M E N T S

The re are two answers . O n e that c o m e s from a priori cons ide ra t ions is that

dynamic eng rams should d i sappear dur ing a t ime-ou t pe r iod and wou ld n e e d

re ins ta tement . T h e c h a n g e s that we re i nduced in the nuc leus of the t rapezoid

b o d y dur ing p s e u d o c o n d i t i o n i n g d id th i s . T h e y d i sappeared dur ing the

11-hour delay b e t w e e n the two expe r imen t s . T h u s if the dynamic eng ram

were not too rapidly rep laced the next day, it should b e poss ib l e to d e m o n -

strate the t empora r iness . For th is reason a lone , tes t ing after a t ime-ou t pe r iod

m i g h t b e a m e t h o d of separa t ing d y n a m i c from structural t races.

S o m e unexpec t ed obse rva t ions that have b e e n m a d e several t imes sugges t

that it m a y wel l b e a still s t ronger m e t h o d to ident i fy at least some of the act iv-

i ty de r iv ing from structural t races . T h i s is b e c a u s e of the poss ib i l i ty that struc-

tural t races m a y no t get fo rmed rapidly or i m m e d i a t e l y dur ing t ra in ing bu t

m i g h t ins tead n e e d t ime to mature . If the g rowth or matura t ion con t inued

after t ra in ing w a s comple ted , then these connec t i ons m i g h t s h o w themse lves

b y i m p r o v e m e n t dur ing relat ively long in te rexper imenta l pe r iods .


T h e r e w e r e actual ly m a n y cases of i m p r o v e d r e s p o n d i n g after the t ime-ou t

pe r iod . A s I have a l ready po in t ed out , s o m e of th i s is a sc r ibab le to the fact

that mo t iva t i on (and therefore r e spond ing ) dec l ined toward the e n d of the

1 2 - 1 4 - h o u r expe r imen ta l se r ies and was then r e n e w e d dur ing the t ime-out .

B e c a u s e of the mot iva t iona l p r o b l e m , expe r imen t s to demons t ra t e the k i n d of

g rowth that conce rns us wil l have to b e repea ted and done the s econd t ime a

specia l way . H o w e v e r , the data that w e have are p r o m i s i n g , b e c a u s e there

were some r e sponse s after the t ime-ou t that l ooked far ou t o f l ine w i t h any-

th ing that appea red be fo rehand , and b e c a u s e there w e r e subs tan t ia l dif-

ferences in the i m p r o v e m e n t o b s e r v e d i n different par ts of the b ra in . T o i l lus-

trate, I wil l g ive three examples .

Firs t were the i m p r o v e d r e sponses of the neu rons in the infer ior col l iculus

w h i c h I m e n t i o n e d earl ier (see F igures 6 and 7 ) . It s e e m s unl ike ly that these

were due to the mot iva t ion change because there w a s no s ign of these

r e sponses at the he igh t of mo t iva t ion on the first day. P o s s i b l y two wel l -

ba lanced p rocesses cance led o n e ano the r all the w a y , bu t I do no t be l i eve th is

is so . If t h i s effect we re no t o w i n g to mot iva t iona l c h a n g e s , i t m a y h a v e b e e n

due e i ther to s o m e nega t ive or pos i t ive cond i t i on ing that occurred o n the first

day, or to s o m e " l ea rn ing to p e r c e i v e . " In any of these cases , the ove rn igh t

i m p r o v e m e n t w o u l d a m o u n t to s o m e sort o f a conso l ida t ion w i thou t further

t ra in ing. A n d it s e e m s to m e that th i s k i n d of i m p r o v e m e n t m i g h t b e the hall-

mark of s low-g rowing structural connec t i ons .

S e c o n d , there we re s imi la r cases w h i c h we re not so surpr i s ing in exper i -

m e n t s w h i c h h a d two success ive days of cond i t i on ing . In these expe r imen t s

the first half of the first day w a s devo ted to p s e u d o c o n d i t i o n i n g . T h e second

half to cond i t i on ing . O n the s econd day, the first ha l f w a s devo ted to cond i -

t ion ing and the s econd ha l f to p s e u d o c o n d i t i o n i n g aga in (wh ich w a s n o w

called ex t inc t ion) . T h e expe r imen t s of Sega l (14) on h i p p o c a m p u s w e r e of th i s

type (see F igure 14) . In these expe r imen t s there was a very large i m p r o v e m e n t

in behav io ra l pe r fo rmance after the long t ime-ou t interval . T h i s w a s exp la ined

to s o m e degree b y the spec ia l expe r imen ta l a r r angement . T h e first day ' s

t ra in ing occur red late in the day , dur ing the s econd half of the day ' s feeding .

B e c a u s e o f th i s , the t ra in ing h a d on ly a hal fway effect o n the first day. T h e r e

was " l e a r n i n g " for the first 50 trials o f cond i t i on ing ; b u t there w a s a subs t an -

tial backs l i d ing i n the further tr ials. A t the b e g i n n i n g of the nex t day, h o w -

ever , pe r fo rmance w a s at a level far a b o v e any th ing that occur red on the first

day. S o m e of the i m p r o v e m e n t w a s cer ta in ly due to the n e w h i g h mo t ive

level , ene rg i z ing la ten t - lea rn ing w h i c h occurred on day 1. Pe r fo rmance no t

only s tar ted h i g h , b u t shot up still further dur ing the first 10 or 20 trials.

Because s o m e behav io ra l i m p r o v e m e n t w a s apparen t at the start and s o m e

w a s added qu ick ly , it w o u l d b e poss ib l e to conc lude that b o t h structural and

dynamic t races could have b e e n invo lved i n m e d i a t i n g the m a x i m u m perform-

ance pat tern. I canno t appor t ion the i m p r o v e m e n t w i t h accuracy to these two

sources b e c a u s e the data we re accumula ted in 10-tr ial b locks a n d I canno t tell

366 James Olds

D A Y I D A Y 2 D A Y I D A Y 2

100 2 0 0 3 0 0 100 2 0 0 3 0 0 TR IALS 100 2 0 0 3 0 0 100 2 0 0 3 0 0 TR IALS

IcoNomoNiNol C 0 N D I T I 0 N I N G | [CONDITIONING] EXTINCTION I | C O N D I E T I O N I N G | c o n d i t , o n , n 8 I |CONDITIONING| EXTINCTION I

FIGURE 1 4 . Unit learning curves for sectors of hippocampus and related areas; and behavior learning curves from the same experiments. The break in the curves indicates an 11 -hour delay between days. Pseudoconditioning occurred on the first half of day 1 ; conditioning occurred on the second half. Conditioning occurred on the first half of day 2 ; pseudoconditioning (extinction) occurred on the second half. The vertical lines indicate the beginning of conditioning on day 1 . The data are from Segal ( 1 4 ) .

h o w m u c h i m p r o v e m e n t occur red dur ing the first 10 trials. A n d of course

m u c h of the i m p r o v e m e n t w a s accoun ted for b y the improved m o t i v e state

caused b y a per iod o f s tarvat ion. Never the le s s , it is in te res t ing that some

b ra in areas improved (like behav io r ) and s o m e did not .

In the septal area there w a s very little in te rexper imenta l i m p r o v e m e n t . T h e

same w a s true for neu rons recorded from the ventra l h i p p o c a m p u s . Bu t in the

fields of the h i p p o c a m p u s (denta te , C A 3 , C A 1, and sub icu lum) there w a s

subs tan t ia l i n t e rexpe r imen t i m p r o v e m e n t . T h i s w a s largest propor t ional ly in

the denta te gyrus w h i c h looked ve ry l ike b e h a v i o r itself. Bu t even in the o ther

h ippocampa l fields the r e sponse w a s m o r e than doub led b y the t ime-out . In

C A 3 and C A 1 there w a s also a subs tan t ia l w a r m - u p effect at the b e g i n n i n g

of day 2 (as there was in behav io r ) . T h i s w a s to s o m e degree absen t in the

subicu lar cor tex and the denta te gyrus . T w o afferent sources of m e s s a g e s to

the h i p p o c a m p u s s tood in contras t to the h i p p o c a m p u s itself. In the pos te r ior

c ingula te cor tex there w a s a subs tan t ia l i m p r o v e m e n t in the 11 -hour in terex-

pe r imen t interval ; bu t there w a s no wa rm-up . O n e m i g h t guess from th is that

the c ingula te changes we re all b a s e d on structural eng rams . In the en torh ina l

cor tex, the r e sponse caused b y the audi tory s ignals was a dece lera t ion ra ther

than an exci ta t ion . C o n d i t i o n i n g caused the s lowing to b e exaggera ted . T h i s

SP

IKE

S

PE

R

300

MS

EC


r e sponse comple te ly d i sappea red overn igh t and d id no t r eappear unt i l after

abou t 50 trials on the s econd day.

It is clear that there are m a n y different k inds of change m e d i a t e d b y the in-

t e rexper iment interval . T h e data are p romis ing b e c a u s e they s h o w that b y

i m p r o v i n g the m e t h o d to c i r cumven t the mot iva t ion p r o b l e m , it m a y wel l b e

poss ib l e to locate structural b r a in c h a n g e s , i . e . , the ones that invo lve s o m e

k ind of g rowth p rocess and therefore improve dur ing t ime-ou t pe r iods .

O n e m e t h o d to i m p r o v e the a n s w e r is o b v i o u s . If cond i t i on ing w e r e con-

t inued for several days w i th repea ted i n t e r expe r imen t in tervals , the mot iva -

t ion c h a n g e m i g h t b e qu i t e s table from day to day. T h e c h a n g e o w i n g to s o m e

connec t ive g rowth p rocess wou ld b e a m o r e " o n e - t i m e " affair. E v e n t hough it

m igh t take several days , it w o u l d no t go on indef ini te ly .

A thi rd example of the k i n d of c h a n g e s m e d i a t e d b y t ime a lone c a m e from a

special set of expe r imen t s on s t imulus reversal . In th is case there were 2 days

of p re t ra in ing w i t h the C S + and the CS— backward . O n the first day after

th i s , i . e . , the first day of reversal t ra in ing , there w a s a gradual dec r emen t in

behav io ra l r e s p o n s i v e n e s s to the CS— and a gradual inc rease to the C S + (see

F igure 15) . T h e first day ' s e x p e r i m e n t was hal ted after abou t 150 reversal trials.

At th i s po in t the exci ta tory value of the two s ignals w a s at an i n - b e t w e e n

level , abou t equa l . T h e n , after an 11-hour rest , there was a subs tan t ia l

i m p r o v e m e n t in the behav io ra l r e s p o n s e to the C S + and a total loss of exci ta-

tory r e sponse to the C S —. B e c a u s e the t ime-ou t caused b o t h a loss of r e spon-

1 I 1 1 1 I | Behavior

1—^ '

- | V ^ , ^ ^ i 8 hr

1 * ~ ^ - o \ 1

1 JQ^

1 rest

1

Wr~^! 1 I i I — i ->

40 80 _ y ^ T r i a l s of reversa l training

• o

t CS+

New CS+

Old CS+

FIGURE 1 5 . Behavior and cortex unit responses before and after reversal of C S + and CS - . Open

symbols were the ones reinforced. Circles were reinforced originally; squares after the reversal. The

data are from an as yet unpublished work by J . Disterhoft.

Rat

e in

cre

me

nts

ca

use

d b

y s

tim

uli

un

its/s

ec

be

h/s

ec

D —

ro

O

J

cn

OO

OO

OO

368 James Olds

s iveness to the CS— and a ga in to the C S + , the events could not b e asc r ibed

ent i re ly to the mo t ive change .

W h a t part of the b ra in m e d i a t e d th is behav io ra l i m p r o v e m e n t ? T h e answer

is that no part of the b ra in t racked the behav io ra l changes very well . Tes t s

were m a d e wi th p r o b e s in the cor tex, in the h i p p o c a m p u s , and in the non-

specific centers of the tha lamus . O n l y in the cor tex we re there day 1 to day 2

i m p r o v e m e n t s in r e s p o n s i v e n e s s to the C S + ; bu t in th is case there were also

lesser i m p r o v e m e n t s in the r e sponse to the CS— (see F igure 15) . T h e latter

wou ld b e looked on as " s p o n t a n e o u s r ecove ry" of an ex t ingu i shed response if

it appeared in behav io r . H o w e v e r , it d id not appear in the behav io r , and

taken toge ther w i t h the i m p r o v e m e n t in the cor tex r e sponse to the C S + it

looked l ike a specia l p roper ty o f cort ical connec t i ons m i g h t b e to g row (or to

g row back—like w e e d s ) . In any even t , the cor tex y ie lded correlates of the

b e h a v i o r to the C S + bu t no t of that to the C S —. W e r e there any correlates of

b e h a v i o r loss to the C S —? Y e s . T h e s e occur red in b o t h h i p p o c a m p u s and in

the tha lamic record ings (see F igure 16) . In h i p p o c a m p u s , the r e sponse to the

C S + s tayed in place dur ing the t ime-ou t per iod . Bu t the r e sponse to the CS—

disappeared . In tha lamus there w a s a specia l change on day 1 that deserves

c o m m e n t . T h e r e spons ivenes s to b o t h the C S + and the CS— improved w h e n

• New C S +

O • O l d CS +

t t cs+ c s -

FIGURE 16. Hippocampus and thalamus unit responses before and after reversal of CS + and

C S - . The details are the same as in Figure 15. The data are from as yet unpublished work by

J . Disterhoft and M. Segal.

Ra

te

incre

me

nts

ca

use

d

by

sti

mu

li (u

nit

s/s

ec)


the reversal e x p e r i m e n t b e g a n . T h i s sugges ted that t h ings b e c a m e especia l ly

in te res t ing to th i s part of the b ra in w h e n there w a s assoc ia t ive sw i t ch ing

go ing on. In any event , t ha lamus , l ike h i p p o c a m p u s , y ie lded s o m e loss of re-

s p o n s i v e n e s s to the CS— dur ing the t ime-ou t , bu t no ga in to the C S + .

T h e s e three obse rva t i ons of changes dur ing t ime-ou t were no t a i m e d at th i s

p rob lem. T h e y n e e d repe t i t ion , therefore, w i t h careful cont ro ls . Jus t obse rv ing

such c h a n g e s does no t po in t direct ly to a g rowth or conso l ida t ion of c o n n e c -

t ions . Bu t the fact that they take such different shapes and d i rec t ions in dif-

ferent parts of the b ra in (even in very closely c o n n e c t e d parts of the b ra in as in

the parts of the h i p p o c a m p a l circui t) m a k e s t h e m ve ry p romis ing . If there are

some c o n n e c t i o n s that are p lanted b y lea rn ing and w h i c h then g row as a func-

t ion of t ime , it shou ld b e easy to d i scover t hem. A n d if there are ent i re ly tem-

porary c h a n g e s that d i sappear as a funct ion of t ime it m a y even b e poss ib l e to

ident i fy t hem.

S U M M A R Y

T h e a im is to find s o m e swi t ches in the b ra in and to charac ter ize t h e m i f

poss ib l e as e i the r t empora ry and r equ i r ing s o m e reverbera tory p rocess o r as

more s table and structural .

The re w e r e five a s s u m p t i o n s involved: (a) that the ve ry large popu la t ion of

neu rons in the b ra in could b e effectively sampled in a l imi ted set of exper i -

m e n t s ; (b) that a midd le k ind of e lec t rodes could di rect t hemse lve s to a suf-

ficiently s table s ingle un i t popula t ion to pe rmi t ex t ended cond i t i on ing exper i -

men t s ; (c) that averag ing could b e used to e l imina te the effects of all u n w a n t e d

var iab les that we re no t corre la ted w i th the m a i n audi tory s igna ls ; (d) that la-

t ency m a p p i n g w o u l d pe rmi t the t rac ing of a s ignal even th rough the mul -

t ip le -channe led pa thways of the b ra in (and wou ld he lp us to ident i fy

swi t ch ing po in t s ) ; (e) that a midd le k ind of c o n d i t i o n i n g (not Pav lov ian en-

t irely no r S k i n n e r i a n ent i re ly) could b e used to get us the rap id l ea rn ing of a

S k i n n e r expe r imen t w i t h the clear C S of a Pav lov ian one .

To pe rmi t the separa t ion of the l inks of a m e s s a g e cha in to he lp us find a

larger n u m b e r of the plast ic coup l ing po in t s it w a s h o p e d that different parts

of the b ra in wou ld " l e a r n " at different rates . T h i s d id h a p p e n to s o m e degree ,

different funct ional sys t ems in the b ra in s h o w i n g the i r changes at t imes w h e n

the i r funct ions were m o s t re levant ; hypo tha l amus c h a n g e d w h e n the s ignal

was r eacqu i r ing in teres t . T h e arousal sys t em c h a n g e d w h e n the s igna l caused

o r i en t ing r e sponses . T h e ex t rapyramida l sy s t em c h a n g e d w h e n the s ignal

caused purpos ive behav io r .

After th i s , the search for the shor tes t l a tency n e w r e sponses led surpr is -

ingly , or no t ( depend ing on you r po in t o f v i e w ) , to the ve ry ear ly s ta t ions of

the audi tory sys t em. Clear l ea rned r e sponses w e r e no t p roved for the shor tes t

370 James Olds

la tency c o m p o n e n t s o f the r e sponses in the first and second s ta t ions ( the

cochlear nuc leus and the super io r o l ive) , bu t t hey we re no t exc luded e i ther .

Lea rn ing did charac ter ize the first ( 3 - 6 msec ) part of s o m e re sponses in the

infer ior col l iculus. In th i s case it w a s ma in ly an ampli f icat ion of a p reex is t ing

response a l ready of^subs tan t ia l m a g n i t u d e before t ra in ing . In the med ia l

genicula te , learned b ra in r e sponses wi th a la tency of 9 - 1 2 m s e c arose de novo.

In the nuc leus of the t rapezoid b o d y and the re t icular format ion there were

learned r e sponses of s imi la r l a tenc ies .

In te res t ing r e sponse c h a n g e s occurred dur ing p s e u d o c o n d i t i o n i n g in the

infer ior coll iculus and the t rapezoid nuc leus . T h e col l iculus had s table

r e sponses on the first day, b u t then a m a r k e d i m p r o v e m e n t demons t ra ted

after an 11-hour rest pe r iod . T h i s sugges ted s o m e latent change created or

p lanted on the first day w h i c h then was deve loped or conso l ida ted dur ing the

inter test interval . T h e t rapezo id b o d y r e sponses s h o w e d learn ing scores that

were a lmost as good on the first day dur ing p s e u d o c o n d i t i o n i n g as on the sec-

ond day dur ing cond i t i on ing . Bu t in th is case the i m p r o v e m e n t w a s lost

dur ing the long in ter tes t interval .

A search for dynamic p rocesses , i . e . , changes in b a c k g r o u n d firing rates ,

that could account for the changes in lower audi tory centers po in ted in pre l im-

inary tes ts to the audi tory cor tex w h e r e c o n d i t i o n i n g w a s a c c o m p a n i e d b y a

drop in the genera l b a c k g r o u n d firing rate.

Final ly , changes that occur red dur ing an 11-hour in ter tes t interval sugges ted

that the t ime-ou t m e t h o d m a y d i s t ingu i sh dynamic from structural t races: the

dynamic ones migh t d i sappear w i t h t ime ; the structural ones m a y wel l g row

or b e c o m e conso l ida ted . T h e expe r imen t s demons t r a t ing such effects so far are

con tamina ted to s o m e degree b e c a u s e there w a s a major change in the mo t ive

state dur ing the t ime-ou t per iods . Bu t p l anned expe r imen t s a i m e d expl ic i t ly at

this should pay good d iv idends .

R E F E R E N C E S

1. Disterhoft, J . F. Unit response in the auditory system and posterior thalamus of rat during classical conditioning and extinction. Proc. 2nd Annu. Meet. Soc. Neurosci., 1972: 84.

2. Disterhoft, J . F . , and Olds, J . Differential development of conditioned unit changes in thal-amus and cortex of rat. /. Neurophysiol., 1973, 35: 665-679.

3. Disterhoft, J . F . , Stuart, D. K., and Olds, J. Emergence of changed unit responses in the audi-tory system of the alert rat during classical conditioning. Fed. Proc, 1973, 32: 383.

4. Frank, K., and Fuortes, M. G. F. Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. Proc, 1957, 16: 39-40.

5. Globus, A. , and Scheibel, A. B . The effect of visual deprivation on cortical neurons: A Golgi study. Exp. Neurol, 1967, 19: 331-345.

6. Kornblith, C., and Olds, J . Unit activity in brain stem reticular formation of the rat during learning. J . Neurophysiol, 1973, 36 : 489-501.

7. Linesman, M. A., and Olds, J. Activity changes in rat hypothalamus, preoptic area, and striatum associated with Pavlovian conditioning. /. Neurophysiol, 1973, 36: 1038-1050.


8. Llinas, R. , and Nicholson, C. Electrophysiological properties of dendrites and somata in alligator Purkinje cells. / . Neurophysiol., 1971, 34: 532-551.

9. Montplaisir, J . , and Olds, J . Conditioned responses in the reticular formation. Proc. 3rd Annu. Meet. Soc. Neurosci., 1973: 115.

10. Olds, J. Brain mechanisms of reinforcement learning. In: Pleasure, Reward and Preference. (D. E. Berlyne and K. B . Madsen, Eds.). Academic Press, New York, 1973: 35-63 .

11. Olds, J . , Disterhoft, J . F . , Segal, M. , Kornblith, C , and Hirsh, R. Learning centers of the rat brain mapped by measuring the latencies of conditioned unit responses. / . Neurophysiol., 1972, 35: 209-219.

12. Olds, M. E. Short-term changes in the firing pattern of hypothalamic neurons during Pavlo-vian conditioning. Brain Res., 1973, 58: 95-116.

13. Rescorla, A. Pavlovian conditioned inhibition. Psychol. Bull, 1969, 72: 77-94. 14. Segal, M. Flow of conditioned responses in the limbic telencephalic system of the rat.

/. Neurophysiol, 1973, 36: 840-854. 15. Segal, M. , Disterhoft, J . F . , and Olds, J . Hippocampal unit activity during classical aversive

and appetitive conditioning. Science, 1972, 175: 792-794. 16. Segal, M. , and Olds, J . Behavior of units in hippocampal circuit of the rat during learning.

/. Neurophysiol, 1972, 35: 680-690. 17. Wiesel, T. N., and Hubel, D. H. Comparison of the effects of unilateral and bilateral eye

closure on cortical unit responses in kittens. / . Neurophysiol, 1965, 28: 1029-1040.

12A Discussion: "Operant Sensitization" and Some

Remarks on Unit Recording in Conscious Animals

S. STEFAN SOLTYSIK Mental Retardation Research Center,



I admire Dr . O l d s ' w o r k and a m espec ia l ly fond of h i s "o rgan i z ing i d e a s . "

T h e y m a y no t represen t the on ly logical ou t come of h i s obse rva t ions , b u t they

do, i ndeed , emerge as a ve ry e legant and h igh ly insp i r ing product of b o t h h i s

data and h i s persona l tas te and b i a se s . A n d I share t h e m wholehea r t ed ly , the

more so that m a n y o£ t h e m fit so neat ly w i t h m y o w n pre jud ices w h i c h I

acqui red wh i l e expe r imen t ing in the field of classical a n d ins t rumenta l cond i -

t ion ing . For lack of t i m e , I shall m e n t i o n only o n e idea that was genera ted b y

behav io ra l expe r imen t s carr ied out in W a r s a w and for w h i c h I have found in

Dr . O l d s ' data a conve rg ing of ev idence c o m i n g from the e lec t rophys io log ica l

expe r imen t s . T h e idea of c h a n g i n g th resho ld for a s t imulus r ewarded w i t h

food has b e e n deve loped dur ing the ins t rumenta l cond i t i on ing o f " i nvo lun -

ta ry" r e sponses , and in th i s d i scuss ion , I i n t end to couple it w i t h Dr . O l d s '

demons t ra t ion of w h a t he calls " r ecep to r tun ing . "* Fo l lowing early expe r imen t s

of Fedorov (1) , Yakov l eva (9) , and Kupa lov (5) , J a n k o w s k a , and I (4) o b s e r v e d

that w h e n a tacti le s t imulus (e l ic i t ing a scra tch reflex) is r ewarded w i t h food, a

hype res thes i a deve lops in the loca t ion of the s t imula t ion . It m a y occas iona l ly

develop to such an ex ten t that the an imals ignore food for a wh i l e and con t inue

* Earlier studies by Hernandez-Peon et al. (3), Galambos (2), and others who used evoked po-tential techniques were clouded by the controversy over uncontrolled variations of the audi-tory input due to the animal's changing position of the head in relation to the source of sound (6). Dr. Olds copes with this problem by delivering the sound through a divided tube attached to the head of the animal.

373

374 S. Stefan Soltysik

s t r iv ing to r emove an imag ina ry ob jec t from the skin . T h i s lower ing of th resh-

old for reflexes from a recept ive field, the s t imula t ion of w h i c h w a s pa i red

wi th a reward , is res t r ic ted to the exper imenta l s i tua t ion and is not carr ied

over to the h o m e cage . It i s , howeve r , unde r the control of a dr ive , and the

an imal mus t b e h u n g r y for th i s p h e n o m e n o n to occur re l iably. T h e s e features

p rompt m e to sugges t a n a m e for the p h e n o m e n o n , name ly , "ope ran t sens i -

t i za t ion . "

Dr . Old ' s data s h o w i n g an e n h a n c e m e n t of un i t r e sponse to an audi tory

s t imulus in the cochlear nuc leus , i f no t in the cochlear ne rve itself, after th is

s t imulus w a s cons i s ten t ly pa i red w i t h food, g ives s t rong suppor t to one of

three poss ib le m e c h a n i s m s . Fedorov and Yakov leva faithfully recalled Pavlov 's

theory of ins t rumenta l cond i t i on ing and located the l ea rn ing change in the

connec t ion b e t w e e n cort ical taste and k ines the t i c (for a scratch re sponse in

th is case) analyzers . Kupa lov (5) also cons ide red the i nvo lvemen t of sensory

cor tex represen t ing tacti le r ecep t ive field for a scratch reflex. J a n k o w s k a and

Sol tys ik (4) on the b a s i s of the obse rva t ion that the t ra in ing resul ted in an

inc reased f requency of no t on ly the rewarded scratch reflex, bu t also o ther

reflexes from the s a m e recep t ive field (such as shak ing the head and pr ick ing

the ears) hypo thes i zed that the faci l i ta t ion mus t occur at the very low relay

s ta t ions in the afferent pa thway . It is wor thwh i l e to po in t out in th i s c o n n e c -

t ion that such "ope ran t s ens i t i z a t i on" occurr ing due to , as I n o w l ike to

be l i eve , " r ecep to r t u n i n g " m a y offer s o m e clues to b o t h pos i t ive and nega t ive

f indings in the e lus ive field of v iscera l operan t cond i t i on ing . T h e f indings of

Russ i an authors and ourse lves* s h o w that it is poss ib l e to b r ing under operan t

control an involuntary reflexlike r e sponse if the exper imenta l des ign favors the

deve lopmen t of "ope ran t s ens i t i z a t i on , " name ly , i f a reward fol lows a s t imu-

lus that el ici ts th is r e sponse . Dr . Old ' s concep t of " r ecep to r t u n i n g " ex tended

also, of course , to low afferent re lays , a l though no t ye t exp la in ing h o w it

occurs , g ives the ev idence of w h y and w h e r e in the C N S the sens i t iza t ion is

b rough t about . O n the o ther h a n d , failures in visceral cond i t i on ing of this

k ind m i g h t b e related to the exper imenta l des ign w h i c h ignores the s t imul i

control l ing a g iven " i n v o l u n t a r y " r e sponse .

To conc lude m y af ter thoughts , I wou ld l ike to express m y s incere admira-

t ion for the i n g e n i o u s w a y Dr . Olds executes s ingle neu ron recording dur ing a

2-day long learn ing procedure . It is not the sha rpness o f h i s e lec t rodes , bu t the

acui ty at the o ther end o f the record ing e q u i p m e n t that enab le s h i m to scan

the b ra in in search for plast ic changes on the level o f s ingle or a lmost s ingle

uni t s . Bu t admi t t ing that col lec t ing data wi th h is m e t h o d offers t r e m e n d o u s

assets , I wou ld l ike to express m y be l i e f that the t radi t ional w a y of ex-

tracellular s ingle un i t r ecord ing m a y also have its v i r tues . O n e sees less w i th a

* The credit for the first attempts of operant conditioning of a scratch reflex should be given to

Thorndike (7), who described behavior of cats which were allowed to escape from the cage by

scratching themselves.

12 A. "Operant Sensitization'' 375

sharp mic roe lec t rode , bu t m a y spot m o r e detai ls in b e h a v i o r of the same cell

t h roughout the record ing epoch . T o i l lustrate th is and to con t r ibu te to th is

m e e t i n g b y p resen t ing n e w data , let m e s h o w y o u jus t two un i t s , one from the

caudate nuc leus and o n e from a g lobus pal l idus of a m o n k e y w h i c h performs a

task invo lv ing a de lay of an ins t rumenta l r e s p o n s e (Figure 1) . T h e trial lasts

long e n o u g h to obse rve changes in the firing rate of a un i t dur ing several con-

secut ive epochs of the ent i re behav io ra l s e q u e n c e : (a) dur ing the control

per iod " C " pr ior to the onse t of a l ight s t imulus , (b) i m m e d i a t e l y after the

s t imulus onse t " S , " (c) dur ing the delay " D , " (d) dur ing the last 2 seconds

before a r e sponse w h e n the an t ic ipa tory or p re re sponse b e h a v i o r occurs

" P r e - R , " (e) dur ing the first s econd after the r e sponse " P o s t - R , " and finally, (f)

dur ing the feed ing b e h a v i o r " F . " B y averag ing the records of severa l t r ials , the

exact course and stat is t ical re l iabi l i ty of impu l se rate modu la t ions w a s as-

sessed . F igure 2 shows a caudate un i t [the dorsolateral part of the caudate at

the A 21 accord ing to the "S te reo tax ic A t l a s " of W i n t e r s , K a d o , and A d e y (8)]

averaged over 25 behav io ra l tr ials. Typica l ly , it e x h i b i t e d a ve ry l ow firing

rate, bu t s h o w e d an unusua l corre la t ion w i t h several phase s o f behav io r :

p robab ly an on - r e sponse to l ight , inc reased and level led firing rate dur ing the

delay, r i se of firing before the r e sponse and a h igh peak i m m e d i a t e l y after a

r e sponse . O n l y 2 0 % of our caudate un i t s have s h o w n such an in teres t in

more than two epochs . Hal f of the caudate neu rons in our m o n k e y s do

no t modu la t e firing rate dur ing the trial , and o f the r e m a i n i n g half , a great

major i ty s h o w modu la t ions assoc ia ted on ly w i t h one or two of the enumera t ed

epochs of the behav io ra l s e q u e n c e . T h e pall idal un i t s (Figure 3) are different

in several ways . T h e y have m u c h h ighe r firing rates and a lmos t all of t h e m

s h o w modu la t ion of firing f requency assoc ia ted w i th at least 2 or 3 phase s of

PRE- POST-

C S D R R F

ji_jT-joi_n_rL_n_ri

s 1 1

i i i i i i i i i i i i i 1 i i i i i i i i i i i i i i FIGURE 1. The schematic representations of a trial shows how the entire sequence of events was divided into epochs, or behavioral responses. The trial starts with presenting a light stimulus which continues until the animal pulls the bar; only the response performed after 8 seconds from the onset of light is rewarded. Typically, the monkey, after an orienting response to the light, waits 8 to 12 seconds and then responds. Somewhat arbitrarily, we enumerated 5 behavioral epochs: C, the control period prior to the light stimulus; S, 1 second after the onset of light; D, the delay; Pre-R, two seconds before the responses; Post-R, 1 second after the response; and F , drinking the juice. The deviation of firing rate (measured in 500 msec bins) exceeding three stan-dard deviations was considered as a significant response.

F -

R -

376 S. Stefan Soltysik

-3304U2RP05-

"1 \R~I

1 2 3 4 5 6 7 8

FIGURE 2. The firing rate changes during the trial in a caudate neuron. This is an atypical

caudate unit showing significant deviations from the slow intertrial firing level during several

epochs.

6U3SE13

o U \ , * - 8 - 7 - 6 - 5 - 4 - 3 - 2 -1 1 2 3 4 5 6 7 8 - 2 -1 1 2 3 4 5 6 7 8

SECS

FIGURE 3. Pallidal unit from the border of internal and external segment. Typically, this unit

shows changes of firing frequency during many epochs and particularly, a rhythmic oscillation

during drinking grape juice, delivered in 1/second jets into the mouth.

0N

OD

3S

213d saaid

s

SPIK

ES P

ER S

EC

ON

D

12A. "Operant Sensitization" 377

the same behav io ra l s e q u e n c e . Pe rhaps the m o s t s t r ik ing is the t e n d e n c y of

our pall idal un i t s to fol low faithfully the r h y t h m at w h i c h w e del iver the j e t s of

j u i ce in to the m o n k e y ' s m o u t h as a reward . No te that the un i t firing is

averaged over 40 trials and the osci l la t ions dur ing d r ink ing exceed 20 sp ikes

per s econd .

T h e po in t I wan t to m a k e is that caudate n e u r o n s reflect in the i r firing rates

only one or at m o s t two aspects of a complex behav io ra l s e q u e n c e , w h e r e a s

pall idal un i t s s h o w modu la t ions in the firing rate correlated w i t h several

phases of the s a m e behav io ra l s e q u e n c e . S i n c e b o t h caudate and pa l l idum

s h o w cons ide rab le he t e rogene i ty in neurona l firing, these detai ls of un i t

b e h a v i o r m i g h t eas i ly get lost w i th record ing t e c h n i q u e no t str ict ly l imi ted to

one un i t at a t ime .

A C K N O W L E D G M E N T

The data presented above are from work conducted jointly with N. A. Buchwald, C. D. Hull, T. Fekete, and A. Uyeda.

R E F E R E N C E S

1. Fedorov, V. K. Basic principles of mutual influences between various motor reactions. (In Rus-sian.) Fiziol. Zh. SSSR, 1952, 38: 559-565.

2. Galambos, R. Electric correlates of conditioned learning. In: The Central Nervous System and Behavior. First Conference. (M. A. B . Brazier, Ed.). Josiah Macy, Jr. Found., Madison Printing Co., Madison, New Jersey, 1959: 375-415.

3. Hernandez-Peon, R., Scherrer, H., and Jouvet, M. Modifications of electrical activity in coch-lear nucleus during "attention" in unanesthetized cats. Science, 1956, 123: 331.

4. Jankowska, E . , and Soltysik, S. Motor conditioned reflexes established from unconditioned motor reflexes reinforced by food. (In Russian.) In: Tsentral'nye i Perifericheskie Mekhanizmy DvigateVnoi Deyatel'nosti Zhivotnykh. Izd. Akad. Nauk SSSR, Moscow, 1960: 61-69.

5. Kupalov, P. S. Involuntary reactions to conditioned food stimuli. (In Russian.) In: Trudy Ob'edinennoi Sessii, Posv. 10-letiu so dnia Smerti LP. Pavlova. Izd. AMN SSSR, Moscow, 1948: 67-78.

6. Marsh, J . T. , Worden, F. G., and Hicks, L. Some effects of room acoustics on evoked auditory potentials. Science, 1962, 137: 280-282.

7. Thorndike, E. L. Animal Intelligence. Macmillan, New York, 1911. 8. Winters, W. D. , Kado, R. T., and Adey, W. R. A Stereotaxic Brain Atlas for Macaca nemestrina.

Univ. of California Press, Berkeley, 1969. 9. Yakovleva, V. V. Establishment of conditioned reflexes to a complex kinesthetic stimulus (to

the so-called involuntary movements). (In Russian.) Zh. Vssh. Nerv. Deiat., 1952, 2: 305-316.

13 Age, Sex, and Experience as Related to the

Neural Basis of Cognitive Development

PATRICIA S. GOLDMAN Laboratory of Psychology and Psychopathology,

National Institute of Mental Health,

Bethesda, Maryland

T h e process b y w h i c h an infant acqu i res the full c o m p l e m e n t of intel lectual

and emot iona l capac i t i es that typify the adult of the spec ies is one of the mos t

cha l l eng ing ques t i ons o f m o d e r n neu rob io logy . In spi te of the rap id progress

n o w b e i n g m a d e in a n u m b e r of different fields, w e still k n o w compara t ive ly

little abou t the neuro logica l ba s i s o f no rmal behav io ra l deve lopmen t , and

c o n s e q u e n t l y of the errors of centra l ne rvous sys t em matura t ion that can cause

ch i ldren to b e c o m e aut is t ic , dys lexic , s ch i zophren ic , or menta l ly re tarded.

W i t h regard to behav io r , no o ther o rgan i sm ' s cogni t ive ab i l i t i es have b e e n

s tudied as ex tens ive ly as those of the h u m a n ch i ld . T h e v o l u m i n o u s l i terature

in ch i ld deve lopmen t has b e e n a r ich source of behav io ra l f indings , bu t has

b e e n of m o r e l imi ted value w i t h respect to e luc ida t ing the neura l b a s i s of cog-

n i t ive growth . Equa l ly l imi t ed , h o w e v e r , have b e e n s tudies conce rned wi th

the ana tomica l , e lec t rophys io logica l , and b i o c h e m i c a l l andmarks in on togeny ,

as such s tudies have genera l ly neg lec ted behav io r . If w e are to learn m o r e

abou t the neura l subs t ra tes o f behav io ra l unfo ld ing , it wil l b e necessa ry to

con t inue efforts, h o w e v e r p re l iminary , at in tegra t ing w h a t is k n o w n abou t

b e h a v i o r and abou t the ne rvous sys t em. F i n d i n g s from neu robehav io ra l

s tudies in rhesus m o n k e y s as they relate to three u b i q u i t o u s factors in cogn i -

t ive deve lopmen t—age , sex , and exper i ence—are p resen ted in th is l ight .

379

380 Patricia S. Goldman

AGE

Stages o f Cogn i t i ve D e v e l o p m e n t

T h e concep t of s tages in h u m a n cogni t ive deve lopmen t is mos t c losely iden-

tified w i t h the work of J e a n P iage t (28 ,39) . A l though the obse rva t ion that

h u m a n b e i n g s demons t ra te inc reas ing c o m p e t e n c e for intel lectual ab i l i t i es

wi th age is pe rhaps as m u c h a s ta tement of universa l exper ience as of sc ien-

tific fact, there exis ts an ex tens ive exper imenta l l i terature on the subject . A

part icular ly in te res t ing example of age -dependen t changes in intel lectual func-

t ion ing is that of conse rva t ion—in P iage tan t e rms , the abi l i ty to ma in t a in a

concep t of equa l i ty in the face of sensory appearances . For ins t ance , if a quan-

ti ty of wa te r is poured from a con ta ine r of one s ize and shape in to another of

different d i m e n s i o n s , adults and chi ldren above the age of 6 or 7 wil l under-

s tand that the quan t i ty of wa te r in the second con ta ine r is exact ly the same as

in the first con ta iner . Q u i t e re l iably , howeve r , ch i ldren unde r the age of 6 or 7

will be l i eve that there has b e e n a change in quan t i ty (4 ,5 ,52 ) .

O n e n e e d not adhere to the concep tua l f ramework of P iage t to find ev idence

of chronologica l s teps in cogn i t ive deve lopment . Satz and V a n Nors t rand (46)

have recent ly r ev i ewed some of the ev idence ind ica t ing sequen t i a l and hierar-

chical deve lopmen t of perceptual , spat ial , and ve rba l skills w i th age. L e n -

nebe rg (34) has also p resen ted ev idence for on togene t i c mi le s tones in the

e m e r g e n c e of l anguage . A l though deve lopmenta l psycholog is t s differ great ly

on a n u m b e r of i s sues related to the chi ld ' s acqu i s i t i on of concep tua l skills

( 6 , 8 , 2 8 , 4 0 ) , the ev idence from a var ie ty of sources forces r ecogn i t ion of an

orderly and success ive a t t a inment of intel lectual a ch i evemen t s w i th age.

In contras t to the plentiful b o u n t y of s tudies on cogni t ive deve lopmen t in

chi ldren , very few s tudies have b e e n conce rned wi th th is subjec t in labora tory

an imals . For facts abou t cogn i t ive deve lopmen t in in f rahuman pr imates , w e

mus t turn to the p ionee r ing inves t iga t ions of Har low (25 ,26) . F r o m such

s tudies w e k n o w that infant m o n k e y s can b e cond i t i oned to avoid shock as

early as the fourth day of life and can learn to perform s imple spat ial and

visual d i sc r imina t ions w i t h i n the first few w e e k s of life. H o w e v e r , m o n k e y s

do no t demons t ra te full matura t ion of ob jec t d i sc r imina t ion capaci ty unt i l they

are 5 m o n t h s of age. At abou t th is s ame t ime , m o n k e y s b e g i n to perform

de layed- response p rob l ems bu t adult levels of prof ic iency apparent ly are not

reached unt i l the end of the first year of life and b e y o n d . Matura t ion of the

skills invo lved in l ea rn ing-se t pe r fo rmance m a y no t b e comple te unt i l the end

of the second year of life and even b y 3 years of age , m o n k e y s have no t yet

s h o w n adult- level eff iciency in lea rn ing still more complex oddi ty p rob lems .

Accord ing to Har low (25) , 4 or 5 years m a y not b e an un reasonab le es t imate

for the length of t ime requ i red for m o n k e y s to ach ieve full intel lectual matu-

ri ty. T h e s e f indings m a y b e regarded as ev idence for s tages of cogn i t ive devel-

o p m e n t in m o n k e y s paral le l ing those in man .

13. Neural Basis of Cognitive Development 381

Structura l Cons ide r a t i ons

It s e e m s r easonab le to a s s u m e , and i ndeed , has b e e n so a s s u m e d wide ly ,

that the matura t ion of the ne rvous sys t em under l ies the e m e r g e n c e of ever

inc reas ing ly complex men ta l ac t iv i t ies . Ye t , it is difficult to say prec ise ly

w h i c h paramete rs of the central ne rvous sys t em mature w h e n , and in wha t

s t ructures , to pe rmi t a chi ld to conse rve quan t i ty or to a l low a m o n k e y to

locate a r eward that has b e e n concea led from v i e w . M a n y s tud ies of central

ne rvous sys t em matura t ion qu i te r easonab ly per ta in to events e i the r of the

e m b r y o n i c or early postnata l per iods [e .g . , s tud ies of the tha lamus ( 1 1 , 4 4 , 5 6 ) ;

of the cor tex (9 ,31 ,41) of connec t i ons (10)] . A l though m o d e r n t e c h n i q u e s are

replac ing classic m e t h o d s in unrave l ing the in t r icac ies of n e u r o g e n e s i s , cell

migra t ion , and cell d i f ferent ia t ion ( 1 - 3 , 1 2 , 4 3 , 4 7 ) , t hese ana tomica l even ts

still an tedate funct ional matur i ty , at least in the p r ima te , b y m o n t h s and years .

Even t s , l ike mye l ina t ion , that do ex tend into the pos tna ta l years are k n o w n

only in b road out l ine . T h u s , F lechs ig (15) desc r ibed the bas i c p lan b y w h i c h

mye l ina t ion of the sensory and m o t o r reg ions of the p r imate b ra in p recede

mye l ina t ion of the assoc ia t ion areas—a plan w h i c h G e s c h w i n d (16) has related

to the phy logene t i c o r ig ins of l anguage . Yakov lev and Lecours (55) e labora ted

on cycles of m y e l i n o g e n e s i s . F i b e r sys tems of b ra in r eg ions m a y mye l ina te

early or late in fetal life and w h e t h e r o r ig ina t ing early or late , mye l i na t i on m a y

p roceed rapidly or s lowly. T h e cycle of m y e l i n o g e n e s i s , they sugges t , is an im-

portant pa ramete r o f reg ional matura t ion and m a y ex tend , in the case of in-

tracort ical neurop i l of assoc ia t ion cor tex, in to and past the th i rd decade of life.

W i t h respect to long- te rm deve lopment , it is difficult to say that w e have

gone far b e y o n d the b road ou t l ines layed d o w n b y F lechs ig (15) and Yakov lev

and Lecours (55) . M a n y gaps r ema in in the k n o w l e d g e of the structural fea-

tures that are e labora ted in the interval b e t w e e n b i r th and funct ional matur i ty

to confer u p o n the assoc ia t ion cor t ices , for example , the i r u l t imate funct ional

capac i t ies and ev idence of a neuro logica l ba s i s for s tages of cogn i t ive deve lop-

m e n t i s , as yet , unspeci f ic and indirec t .

Neura l B a s i s o f Cogn i t i ve D e v e l o p m e n t

S i n c e ch i ldren and m o n k e y s demons t ra t e inc reas ing ly complex cogni t ive

capaci ty at different s tages of deve lopment , it shou ld at least b e pos s ib l e to

demons t ra te correlat ive matura t ion of part icular neura l s t ructures w i th

advanc ing age. Behav io ra l obse rva t ions of m o n k e y s ra ised from infancy

w i thou t cer ta in parts of the i r frontal l obes have p rov ided s o m e ev idence that

cort ical areas of the p r imate b ra in do develop at different t imes after b i r th and

in re la t ion to the deve lopmen t of cogn i t ive skills ( 7 , 1 9 , 2 0 , 3 6 ) . It w o u l d appear

from these s tudies that the orbi ta l prefrontal cor tex, for example , b e c o m e s

funct ional ly s ignif icant at or before 1 year of age , wh i l e the dorsolateral

prefrontal cor tex s h o w s a s lower and m o r e prot rac ted course of deve lopment .


Evidence for the late deve lopmen t of the dorsolateral cor tex is s h o w n in F ig -

ure 1. T h e curves p resen t the m e a n scores (wi th ranges) for i n d e p e n d e n t groups

of opera ted and unopera ted m o n k e y s tested on de layed r e sponse for the first

t ime w h e n they were 1 2 , 2 4 , or an average of 3 1 m o n t h s of age . In all exper i -

menta l sub jec t s , the dorsolateral prefrontal cor tex as desc r ibed in Gold-

m a n (19) had b e e n r e m o v e d bi lateral ly at least 10 m o n t h s pr ior to tes t ing.

T h e l es ions were comple te ly comparab le in the two groups of m o n k e y s tes ted

at the earl iest and latest ages (19) . T h e m o n k e y s tes ted at 24 m o n t h s are still

alive and the i r l es ions have therefore not b e e n analyzed. C o m p a r i n g the per-

formance of the opera ted and unopera ted groups at 31 m o n t h s of age confirms

the we l l -known f inding that dorsolateral prefrontal l e s ions result in severe im-

pa i rmen t s on delayed r e sponse . H o w e v e r , the degree of i m p a i r m e n t p roduced

b y such l es ions dec l ines wi th dec reas ing age—at 24 m o n t h s of age , some

m o n k e y s w i t h dorsolateral prefrontal areas r e m o v e d in infancy perform as well

as unopera ted controls w h i l e o thers are severe ly impa i red . Still ear l ier , at 12

m o n t h s of age , the effect o f the l es ion vir tual ly d i sappears as opera ted

m o n k e y s perform as wel l as unopera ted controls . F igure 2 presen t s f indings

for ano ther related measu re , de layed a l ternat ion, g iven to the s a m e an imals

approx imate ly three m o n t h s later at 1 5 , 2 7 , and 34 m o n t h s o f age . Aga in , the

s ignif icance of the dorsolateral cor tex for b e h a v i o r changes w i th age. Whi l e

the scores of opera ted and unope ra t ed m o n k e y s over lap cons ide rab ly at 15

m o n t h s of age , b y 27 m o n t h s , there is comple te separa t ion b e t w e e n the scores

of the two groups . At 34 m o n t h s of age , the d ive rgence is m o r e p ronounced .

2 0 0 0 - 1

'—i—i—i—i—i—i—i—i—i—i—i—i—i— 3 6 9 12 15 18 21 24 27 30 33 36 39

A V E R A G E A G E IN M O N T H S

• D O R S O L A T E R A L

O UNOPERATED

FIGURE 1. Curves showing mean delayed response performance of operated and unoperated groups at 12, 24, and an average of 31 months of age. Each point is based on four monkeys except in the case of the 12-month unoperated control group where n = 9. Brackets indicate ranges.

TR

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— I — I — i — i — i — i — i — i — i — i — i — i — i — 3 6 9 12 15 18 21 24 27 30 33 36 39

A V E R A G E A G E IN M O N T H S

• D O R S O L A T E R A L

OUN0PERATED

FIGURE 2. Curves showing mean delayed alternation performance of the same groups as in Fig-

ure 1 approximately 3 months later at 15, 27, and an average of 34 months of age. Brackets again

indicate ranges.

At th i s last age , opera ted m o n k e y s , w i thou t excep t ion , fail to learn the task

w i t h i n the l imi ts of t ra in ing wh i l e the unopera ted m o n k e y s ach ieve cr i ter ion

per formance in an average of 170 trials. T h e f inding that the differences

b e t w e e n the two groups con t inues to inc rease b e y o n d 2 years of age ind ica tes

that the dorsolateral cor tex does not b e c o m e fully funct ional unt i l after 2

years of age. T h e addi t iona l fact that opera ted m o n k e y s s h o w inc reas ing ra ther

than static deficits w i th age further ind ica tes that normal ly s t ructures o ther than

the dorsolateral cor tex m a y med ia t e de layed- response b e h a v i o r be fore the dor-

solateral cortex matures .

S o m e sugges t ions as to w h i c h structures m a y b e impor tan t at ear l ier s tages

of deve lopmen t is g iven b y cons ide r ing the s tatus of b r a i n s t ructures o ther

than the dorsolateral cor tex at se lec ted po in t s a long the age c o n t i n u u m de-

sc r ibed in F igures 1 and 2 . M o n k e y s g iven orbi ta l prefrontal l e s ions in in fancy

have b e e n tes ted on de layed r e sponse and de layed a l ternat ion at 12 and 15

m o n t h s o f age , respec t ive ly (19 ,36) . M o n k e y s ra i sed from infancy wi th l e s ions in

the anterodorsa l sec tor o f the h e a d of the caudate nuc leus (22) and in the dorso-

media l nuc leus of the tha lamus (21) have s imi lar ly b e e n s tud ied unde r ident ica l

cond i t ions at the s a m e ages . All such groups we re impa i r ed on de layed al terna-

t ion and all bu t the group wi th tha lamic l es ions we re impa i r ed on de layed

r e sponse at 12 and 15 m o n t h s of age , w h e n m o n k e y s w i t h dorsolateral l es ions

were not . T h e s e f indings p rov ide ev idence that the orbi ta l prefrontal cor tex, the

head of the caudate nuc leus and the dorsal t ha lamus are funct ional ly ma tu re at

the 1-year s tage of deve lopmen t and could m e d i a t e de layed r e sponse funct ions

TR

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(De

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at that age. Later in on togeny there appears to b e a shift in the relat ive impor -

tance of these ana tomica l ly related frontal l obe s t ructures s ince in the juven i l e

and adult m o n k e y , de layed r e sponse per formance cannot b e m a i n t a i n e d b y

res idual s t ructures in the a b s e n c e of the dorsolateral cortex. W e are far from

under s t and ing the m e c h a n i s m s of these changes in central ne rvous sys t em

organ iza t ion wi th age. S o m e i ssues of in terpre ta t ion assoc ia ted wi th these

f indings have recent ly b e e n d i scussed e l sewhere in greater detai l (21) . H o w -

ever , the p resen t f indings do s e e m to project an i m a g e of a neura l ba s i s for

h ierarchica l s tages in cogni t ive deve lopmen t that future research will hopefully

b r ing in to sharper focus.

S E X

Sexua l D i m o r p h i s m in N o n c o g n i t i v e B e h a v i o r s

T h e l i terature of compara t ive and phys io log ica l p sycho logy con ta ins abun-

dant ev idence of behav io ra l differences b e t w e e n the sexes , no t on ly wi th

regard to reproduct ive behav io r , bu t in m a n y nonreproduc t ive behav io r s as

wel l , e .g. , in food preferences (51) , emot iona l i ty (17 ,24) , aggress ion (14) , and

play (23) . S i n c e differences b e t w e e n the sexes in such behav io r s as aggress ion

and play m a y b e cons t rued as in t imate ly b o u n d up wi th sex- typical ac t iv i t ies ,

it is no t surpr is ing that these behav io r s , as wel l as the more p la in ly ev iden t

reproduct ive act ivi t ies of the spec ies can b e inf luenced b y gonadal h o r m o n e s

at cri t ical per iods in deve lopmen t (14 ,23 ,24 ) . M o r e o v e r , s ince gonadal hor-

m o n e s are thought to affect b e h a v i o r b y inf luenc ing the neura l different ia t ion

of hypo tha lamic m e c h a n i s m s , it w o u l d no t b e further surpr i s ing if the neural

s tructures subse rv ing these behav io r s were to differ in males and females .

Indeed , morpholog ica l differences b e t w e e n the sexes have recent ly b e e n re-

por ted for the neurop i l of the rats ' p reopt ic area (42) . Dif ferences b e t w e e n the

sexes have also b e e n repor ted in r e sponse b o t h to hypo tha lamic injury and to

septal damage (38 ,50) .

T h e Q u e s t i o n o f S e x u a l D i m o r p h i s m in Cogn i t i ve B e h a v i o r

A l though subs tant ia l ev idence m a y b e found for sex differences in intel lec-

tual measu re s at a lmost all ages of the deve lopmenta l spec t rum [for r ev iew see

Jarv ik (29)] inc lud ing , inc identa l ly , sex differences in the age at w h i c h ch i ldren

mas te r conserva t ion tasks (27) , it is no t k n o w n w h e t h e r neura l s t ructures s u b -

serving intel lectual or cogn i t ive b e h a v i o r are also sexual ly d imorph ic . As

Jarv ik po in t s out, " i t is difficult, i f no t i m p o s s i b l e to sort the cultural inpu t in

such sex differences from the phys io log ica l i npu t . " H o w e v e r , some sugges t ive

f ragmentary ev idence exis ts . For example , K i m u r a (32,33) repor ted differences


b e t w e e n the sexes in the abi l i ty to p rocess d ichot ica l ly p resen ted digi ts b y the

s p e e c h - d o m i n a n t h e m i s p h e r e at early ages . M o r e recent ly , W i t e l s o n and Pall ie

(54) have repor ted that the morpho log ica l a s y m m e t r y b e t w e e n the r ight and

left t empora l lobes of adults w a s m o r e m a r k e d in females ear l ier in o n t o g e n y

than in males . T h e result , w h i c h m u s t b e in terpre ted caut ious ly o w i n g to the

small sample s izes , w a s related to differences b e t w e e n the sexes in language

deve lopmen t and h e m i s p h e r i c la teral izat ion for l anguage . F ina l ly , e p i d e m i o -

logical ev idence for expec t ing neurologica l differences b e t w e e n the sexes m a y

b e m e n t i o n e d . Four to n i n e t imes as m a n y b o y s as girls are d i agnosed as

hav ing m i n i m a l b ra in dysfunct ion (53) ; the i nc idence of cer ta in forms of cere-

bral pa lsy is 2 - 8 t imes greater in males than in females (49) and specific

read ing d isab i l i t i es are 6 t imes m o r e f requent in ma les than females (46) .

In th is area, as in the area of s tages in cogn i t ive deve lopmen t , it is ins t ruc-

t ive to look at s tud ies of in f rahuman pr imates . He re , h o w e v e r , there i s ve ry

little ev idence pe r ta in ing to sex differences in cogn i t ion and lea rn ing in b ra in -

damaged sub jec t s . Genera l ly speak ing , th is m a y b e b e c a u s e the effects of cen -

tral ne rvous sys t em les ions are over r id ing , p r imate s tudies f requent ly involve

small n u m b e r s of cases , inves t iga tors have no t as a rule looked for sex dif-

fe rences , or sex differences are s imp ly not present . It is o f in teres t in th is con-

text, t hen , that sex differences in l ea rn ing skil ls have e m e r g e d in the course of

s tudies c o n c e r n e d w i t h the deve lopmen t o f the orbi ta l prefrontal cor tex.

E v i d e n c e for S e x u a l D i m o r p h i s m in Cogn i t i ve B e h a v i o r o f M o n k e y s

M o n k e y s g iven orbi ta l prefrontal l es ions at 50 days of age were tes ted on an

objec t d i sc r imina t ion reversal task at 75 days of age . T h e resul ts revealed a

subs tant ia l over lap b e t w e e n the error scores o f the opera ted m o n k e y s and

those of the unope ra t ed cont ro ls , l ead ing to the conc lus ion that the orbi ta l

cor tex was prefunct ional at that early s tage of deve lopmen t (21) . H o w e v e r ,

upon c loser examina t ion , there appeared to b e a close l ink b e t w e e n the sex of

the m o n k e y and the degree of effect p roduced b y the l e s ion , accoun t ing for

mos t of the var iab i l i ty b e t w e e n subjec t s w i t h i n groups . A s ind ica ted in

Tab le I , the orbi ta l l e s ions p roduced a greater effect in m a l e s t han in females .

W h e r e a s the opera ted males m a d e more errors (and took longer to learn) than

the unopera ted ma les , there were no differences b e t w e e n opera ted and un-

Table I Performance on Object Reversal at 2i Months of Age'

Unoperated Operated

Males

Females 168 ± 22.6 219 ± 18.9

277 ± 34.7 202 ± 29.1

" Mean errors over 6 reversals ± standard error.


opera ted females . A m o n g m o n k e y s opera ted as j uven i l e s or adults in the

same s tudy no such t rend was mani fes t , a f inding cons i s t en t w i th the lack of

repor ted sex differences in the l i terature on adult b r a i n - d a m a g e d m o n k e y s .

T h e s e f indings in the 2 i - m o n t h - o l d m o n k e y sugges ted that it w o u l d b e of

in teres t to r eexamine data col lected in o ther expe r imen t s invo lv ing orbi tal

prefrontal l e s ions . A large n u m b e r of cases were avai lable from s tudies in

w h i c h the orbi ta l prefrontal cor tex had b e e n r e m o v e d bi lateral ly w i t h i n the

first 50 days of age and the m o n k e y s had first b e e n assessed on delayed

response at 12 m o n t h s of age (19 ,36 ,22a ) . F igure 3 presen ts the results

of compar i sons b e t w e e n males and females , wi th and wi thou t orbi tal l e s ions ,

on th is task at that age . C o m p a r i n g the opera ted and unopera ted ani-

mals w i t h i n each sex ind ica ted that again , the orbi ta l l es ions resul ted in

impa i rmen t s in males and not in females . A l so , s imi lar differences b e t w e e n

opera ted males and females we re ev iden t on a de layed al ternat ion test g iven

to the same m o n k e y s at a round 15 m o n t h s of age (22a) . H o w e v e r , b y 18

m o n t h s of age and b e y o n d , no sex differences on learn ing measu re s were o b -

ta ined e i ther in early or late opera ted m o n k e y s (22a) . In v i e w of the lack of

ev idence for differences b e t w e e n males and females at the later s tages of

deve lopment , the results were regarded as ev idence for a deve lopmenta l

difference in the rate at w h i c h the orbi ta l cortex develops in the two sexes . As

such, p rev ious conc lus ions (19 -21 ) w i t h regard to the funct ional status of the

orbi ta l cor tex at different s tages of deve lopmen t should b e qual if ied in relat ion

to gender . It n o w appears that the orbi ta l cortex develops on togene t ica l ly ear-

l ier in males than in females .

A sex-related difference in the deve lopmen t of cort ical r eg ions could have

major impl ica t ions for the o rgan iza t ion of b e h a v i o r dur ing the format ive years

2000n

1500-

500H

J 0 ORBITAL

• U N O P E R A T E D

MALES FEMALES

FIGURE 3. Delayed response performance at 1 year of age in males and females with and without

orbital prefrontal lesions induced in infancy. Reproduced, with permission, from Goldman

et al, 1974.

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and qu i t e pos s ib ly for behav io ra l differences that outlast these years . D e v e l o p -

men ta l di f ferences in neura l ma tura t ion m a y also have a bea r ing on the h i g h e r

i nc idence of m a n y ne rvous sys t em disorders in male ch i ldren . F ina l ly , the

present f indings ra ise the poss ib i l i ty that deve lopmen ta l d i f ferences wil l b e

found for o ther cort ical r eg ions as wel l , a l though the d i rec t ional i ty o f the dif-

ferences m a y vary w i t h the cogn i t ive skills invo lved . O n the o ther h a n d , it is

poss ib l e that on ly the orb i ta l r eg ion b e c o m e s funct ional ly mature accord ing to

different t ime tables in males and females due to i ts especia l ly in t ima te con-

nec t ions w i t h the l i m b i c s y s t e m and hypo tha l amus (30 ,37 ) . All o f these poss i -

b i l i t i es are sub jec t s for future research .

E X P E R I E N C E

T h e Effec ts o f Ea r ly Expe r i ence on B r a i n and B e h a v i o r

T h e effects o f ear ly expe r i ence on b e h a v i o r and o n structural pa ramete rs o f

central ne rvous sys t em matura t ion are w i d e l y r ecogn ized ( 1 3 , 1 8 , 3 5 , 4 5 ) . T h e r e

is also cons ide rab le ev idence to suppor t the no t ion of cri t ical pe r iods for the

effects of expe r i ence u p o n the ne rvous sys t em as in the i m p r i n t i n g p h e n o m e -

n o n s h o w n b y the offspring of precocia l b i rds (48) . S tud ie s of early expe r i ence

and cri t ical pe r iods in on togene t i c deve lopmen t have for the m o s t part b e e n

conduc ted o n intact or no rma l o rgan i sms and little in format ion is avai lable on

the s igni f icance of exper i ence dur ing format ive per iods on the s u b s e q u e n t

behav io r o f b ra in - in ju red an imals . S u c h s tudies wou ld have the mer i t o f ex-

pos ing the neura l b a s i s for the in tegra t ion of early expe r i ence (for example ,

wha t neural s t ructures med ia t e impr in t ing? ) in addi t ion to a s ses s ing its role as

a modif ier o f a b n o r m a l deve lopmen t . In th i s last sec t ion , I repor t b e l o w pre-

l iminary ev idence from a s tudy conce rned wi th the effect of expe r i ence on

recovery of funct ion fo l lowing early b ra in l e s ions .

Ev idence for Exper i en t i a l Factors in R e c o v e r y o f Func t ion

As p rev ious ly repor ted , m o n k e y s g iven orbi ta l l es ions in in fancy we re i m -

pai red on a n u m b e r of cogn i t ive tasks w h e n they were tes ted b e t w e e n the

ages of 12 and 18 m o n t h s of age . It shou ld b e added that toward the e n d of

th is pe r iod , deficits on m e a s u r e s o f orbi ta l funct ion are found in females as

wel l as males . H o w e v e r , w h e n these s ame m o n k e y s we re re tes ted on one of

the measu re s—de layed a l ternat ion—at 24 m o n t h s of age , they exh ib i t ed

remarkab le recovery o f funct ion (36) . T h a t i s , the opera ted m o n k e y s that were

e i ther severe ly impa i r ed or failed to learn the task at the earl ier age per formed

as wel l or near ly as wel l as unopera ted an imals at the later age . T h e recovery

exh ib i t ed at the 2 -year s tage of deve lopmen t has b e e n a t t r ibu ted to the matu-


ra t ion of intact s t ructures dur ing the second year of l ife, and is d i scussed more

fully in G o l d m a n (20) . E v e n though the ini t ia l t ra in ing w a s not sufficient to in-

duce s imi lar recovery in m o n k e y s opera ted as j uven i l e s and had different and

poss ib ly nega t ive effects in m o n k e y s g iven dorsolateral l e s ions in in fancy (19) ,

it was of in teres t to de t e rmine w h e t h e r or no t recovery could b e o b t a i n e d at

the 2-year s tage of deve lopmen t fo l lowing orbi ta l l es ions in infancy in the ab -

sence of formal exper i ence in t e rven ing at the 1 - l i - y e a r per iod . In o ther

words , was the long- range recovery exh ib i t ed b y the early opera ted m o n k e y s

solely dependen t on the matura t ion of the ne rvous sys t em?

In order to answer th is ques t i on , four m o n k e y s g iven orbi ta l prefrontal

l es ions in infancy we re m a i n t a i n e d in the labora tory unt i l t hey were 2 years of

age w i thou t b e i n g tes ted at any pr ior t ime . T h e y we re compared wi th

opera ted and unopera ted controls ra i sed under ident ica l cond i t ions and wi th

comparab le groups g iven in t e rven ing t ra in ing . All groups of m o n k e y s were

tes ted b e g i n n i n g at 24 m o n t h s of age on the s ame bat te ry and order of tests as in

p rev ious research . F igure 4 s h o w s the scores o b t a i n e d on delayed al ternat ion

WITH P R E V I O U S E X P E R I E N C E

jDin UNOPERATED

I / /

WITHOUT PREVIOUS

UNOPERATED

E X P E R I E N C E

V7X

11 SUBJECTS

FIGURE 4. Delayed alternation performance measured after operated and unoperated monkeys were 2 years old. Upper: monkeys given previous training at the 1-year stage of development. Lower: monkeys given no previous training.

TRIA

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'V ASY A w i n ORBITAL

ORBITAL


b y the m o n k e y s w i t h orbi ta l l es ions and b y the unopera ted cont ro ls , w i th and

w i thou t p rev ious expe r i ence . M o n k e y s opera ted at 50 days of age , tes ted in i -

tially at 1 yea r of age , and then re tes ted at 2 years were able to solve the

p r o b l e m and m o s t pe r fo rmed as wel l as the re tes ted unope ra t ed controls .

Ope ra t ed m o n k e y s first t es ted after 2 years h a d e lapsed , h o w e v e r , un i formly

failed to learn the task at all. T h e s tudy is i ncomple t e in that the m o n k e y s

tes ted ini t ia l ly at 2 years wil l b e re tes ted once aga in w h e n they are 3 years old.

Fai lure to ob t a in recovery at 3 years will ind ica te the degree to w h i c h formal

t ra in ing m u s t b e g iven at cri t ical t imes in on togeny . H o w e v e r , the presen t

resul ts do p rov ide ev idence that recovery from bra in in jury is great ly facil i-

ta ted b y expe r i ence , and , i ndeed , expe r i ence w a s m o r e s ignif icant for the

opera ted an imals than it w a s for the unopera ted cases . It is of in teres t that the

in te rven ing t ra in ing n e e d no t b e successful , s ince m o n k e y s that demons t ra t e

recovery after p rev ious t ra in ing did so in spi te of the i r past fai lures. F r o m the

po in t of v i e w of r emed ia l educa t ion , unsuccessfu l t r a in ing w o u l d appear to b e

be t te r than no t ra in ing at all.

S U M M A R Y

S tud i e s o f rhesus m o n k e y s ra ised from infancy w i thou t por t ions of the i r

central ne rvous sys tems p rov ide ev idence regard ing the t i m i n g of funct ional

matur i ty of specific r eg ions . S o m e f indings have b e e n p re sen ted w h i c h s h o w

that deficits fo l lowing l e s ions of specific cort ical areas e m e r g e at that age w h e n

abi l i t ies d e p e n d e n t upon the cort ical area in ques t i on take on adult form in

unopera ted m o n k e y s . H o w e v e r , age is b u t a c o n v e n i e n t abs t rac t ion for cond i -

t ions that vary over t ime . O n e such cond i t ion is the p resence and t i ter of

gonada l h o r m o n e s . T h e f inding that orbi ta l funct ions m a y deve lop at different

t imes in ma les and females is o f in teres t from a n u m b e r of pe r spec t ives , bu t it

is espec ia l ly s t imula t ing to cons ide r the poss ib i l i ty that the deve lopmen t of

cort ical t i s sue m a y b e regula ted b y n e u r o e n d o c r i n e factors in a fash ion analo-

gous to that e n v i s i o n e d for different ia t ion of hypo tha l amic m e c h a n i s m s .

Final ly , exper ien t ia l factors m a y d e p e n d great ly on the matura t iona l s tatus o f

those b ra in r eg ions de s igned to b e rec ip ien t s o f that s t imula t ion . T h e fact that

young ch i ldren acqu i re s econd languages w i th far greater ease than adults

or converse ly that l anguage fails to deve lop before 1 8 - 2 8 m o n t h s of age

regardless of t ra in ing are features of h u m a n expe r i ence cons i s t en t w i t h the in-

t e rdependence of expe r i ence and neuro logica l matura t ion . T h e p resen t f inding

that t ra in ing at re la t ively early per iods of deve lopmen t faci l i ta ted recovery

from bra in in jury ind ica tes further that b r a i n - d a m a g e d ind iv idua ls can b e

even m o r e sens i t ive to the effects o f p rev ious expe r i ence than in tact cases .

A g e , sex , and expe r i ence are factors that m a y b e i so la ted for expe r imen ta l pur-

poses and for the pu rpose o f d i scuss ion . H o w e v e r , it i s p rec i se ly the complex


in te rac t ions of these var iab les that cons t i tu te the subjec t mat te r of future

research in neu rob io logy , to w h i c h s tudies of infant m o n k e y s m a y con t r ibu te

an ima l mode l s of no rma l and d isordered h u m a n deve lopment .

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18. Goldman, P. S. The relationship between amount of stimulation in infancy and subsequent emotionality. Ann. N.Y. Acad. Sci., 1969, 159: 640-650.

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24. Gray, J . A. , Levine, S., and Broadhurst, P. L. Gonadal hormone injection in infancy and adult emotional behavior. Anim. Behav., 1965, 13: 33-45.

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13A Discussion: Effects of Caudate Nuclei Removal

versus Frontal Cortex Lesions in Kittens

JAIME R. VILLABLANCA




I wil l br ief ly repor t on our resul ts in k i t tens wi th large b ra in l e s ions , e i ther

cort ical or subcor t ica l . It d id no t take us ve ry long to b e c o m e aware of the

m a n y technica l difficulties and potent ia l sho r t comings of the l e s ion m e t h o d

as appl ied to the s tudy of the deve lop ing b ra in ; the m a n y difficulties and

unavo idab le ha rdsh ips pe r ta in ing to the research in th is area r ende r Dr .

G o l d m a n ' s s tud ies even m o r e mer i to r ious .

O u r in teres t has focused on s tudy ing the effects of cort ical ve r sus subcor t ica l

b ra in l e s ions in n e w b o r n an ima l s , as well as in compar ing the m a g n i t u d e and

character is t ics of recovery of the resul t ing defects w i t h those of s imi la r l e s ions

inf l ic ted in the adult an imal . In the long range , our a i m is the be t t e r under -

s tand ing of the m e c h a n i s m s invo lved in recovery p rocesses fo l lowing early

in jury to the b ra in .

A r e v i e w of the l i terature repor t ing the effects o f cort ical l e s ions in n e w b o r n

an imals ind ica tes , in genera l , that such in jur ies cause m u c h less severe func-

t ional deficit and al low m u c h m o r e recovery than s imi lar l e s ions infl icted in

adul thood ( 1 , 3 , 4 , 7 , 1 1 , 1 2 , 1 8 , 2 0 , 2 2 , 2 4 - 2 6 ) .

T h e knowledge regard ing the effects of subcor t ica l exper imen ta l b ra in le -

s ions in the n e w b o r n is more l imi ted s ince far fewer reports dea l ing w i t h

th is p r o b l e m have b e e n p u b l i s h e d ( 1 0 , 1 3 , 1 5 , 1 7 , 2 3 ) . A n inf luent ia l p i ece of

work in th is area has b e e n the repor t b y Kl ing and Tucke r (17) that l es ions

des t roy ing 1 0 - 5 0 % of the caudate nuc le i in n e w b o r n m o n k e y s p roduced

devas ta t ing neu robehav io ra l effects. Fu r the rmore , the de ter iora t ion of the

393

394 Jaime R. Villablanca

m o n k e y s was p rogress ive , s ince four o f the an imals d ied w i t h i n 6 w e e k s and

the r e m a i n i n g three , w i th the smal les t l e s ions , were m a i n t a i n e d wi th great

difficulties for up to 3 m o n t h s . S i n c e l e s ions o f comparab le ex t ens ion have

b e e n per formed in adult m o n k e y s (2 ,6 ,21) w i thou t the de le ter ious con-

s e q u e n c e s obse rved in the n e w b o r n , the conc lus ion resul t ing from th is exper i -

m e n t was that subcor t ica l b r a i n l e s ions inf l ic ted early in life have a m u c h

worse ou t come than s imi la r l e s ions infl icted in adul thood and are also rela-

t ively m o r e de t r imenta l t han early cort ical l e s ions .

Dr . G o l d m a n (9) , as well as o ther authors ( 1 4 , 1 6 , 1 8 ) , has p re sen ted ev idence

ind ica t ing that the t ime course of the effect of early b r a in in jury m a y no t b e as

s imple as that sugges ted above , i . e . , s teady progress ive recovery or s teady

progress ive de ter iora t ion of funct ion. Ins tead , accord ing to the i r data, a re-

versal of the t rend prevalent up to a g iven po in t in t ime m a y occur a long wi th

further deve lopment . T h i s impor tan t obse rva t ion s t rongly e m p h a s i z e s the need

for a long fol low-up of the exper imenta l an imals , in to adu l thood i f pos s ib l e , in

order to b e able to m a k e mean ingfu l s t a tements . Unfor tunate ly , no t comply ing

wi th th is r e q u i r e m e n t m a y b e an impor tan t d r a w b a c k in m a n y o f the deve lop-

menta l b ra in l es ion s tud ies up to n o w .

W e have a t t empted to test in n e w b o r n k i t t ens the poss ib i l i ty ra i sed b y the

Kl ing and Tucke r expe r imen t s quo t ed above that the early l es ion of a subcor -

tical s t ructure, l ike the caudate nuc leus , m a y have a m o r e marked impac t upon

neurobehav io ra l deve lopmen t than pure cort ical damage . T h e cort ical area

selected for compar i son w a s the frontal reg ion . T h e resul ts of s imi lar l e s ions to

these s tructures in adult ca ts , repor ted e l sewhere in th is vo lume (27) , have

p rov ided us w i t h a base l ine for eva lua t ing the k i t tens l es ion effects as wel l as

of the age var iable in the final o u t c o m e of the in jur ies . O u r s t ra tegy has b e e n

to ablate as m u c h of the cauda te or frontal cort ical areas as pos s ib l e in order

to avoid m a s k i n g c o m p e n s a t o r y effects b y r e m a i n i n g por t ions o f t hese struc-

tures and wi th the in t en t ion o f ini t ia l ly d i sc los ing gross funct ional defects .

Hopeful ly , the eventual ly larger , u n e q u i v o c a l defects resul t ing from ex tens ive

ab la t ions wil l gu ide us in the future in a finer grain analys is of the con-

s e q u e n c e s of local ized les ions .

T h e surgical t e c h n i q u e s used to abla te the caudate nuc le i , as well as the

frontal cort ical areas in k i t t ens , we re s imi lar to those used in adult cats (27) .

A ba t te ry of neuro logica l tests w a s appl ied per iodica l ly to all opera ted

k i t tens and to the i r l i t termate controls to assess the i r neurobehav io ra l status. In

addi t ion to the i t ems i n the descr ip t ion o f ou r s imi la r s tud ies in adult cats

(27) , the fo l lowing aspects we re also assessed : tail pos i t ion , o p e n i n g of

eye l ids , b l i nk ref lexes, audi tory s tar t l ing and o r ien t ing , roo t ing reflex, p lay

act ivi ty , thermoregula tory sh ive r ing , and pendula r head m o v e m e n t s . T h e

k i t t ens ' gross b e h a v i o r has b e e n as sessed dur ing f requent obse rva t ions in

the i r h o m e cages , w i th the i r l i t termates and mo the r , and in an open field in

the laboratory. In addi t ion , the deve lopmen t of the k i t t ens ' m o t o r act ivi ty has

b e e n quant i f ied (19) b y record ing the i r m o v e m e n t s in an open field (by m e a n s

13A. Caudate Nuclei Removal versus Frontal Cortex Lesions 395

of a sens i t ive force t ransducer , from b i r th to 21 days , or b y u s ing mul t ip le

photocel l b e a m s , from days 21 to 62) and the i r bas i c l ea rn ing ab i l i t i es have

b e e n evaluated in a T - m a z e w h e r e the an imals we re t ra ined to m a k e a black-

w h i t e d i sc r imina t ion and to perform a pos i t ion reversal task. S o m e of the

k i t tens are n o w b e i n g tes ted in a pas s ive -avo idance pa rad igm and the older

ones are b e g i n n i n g the ba r p r e s s ing t ra in ing , p rev ious ly u sed to s tudy adult

B A c or B F r cats (28) .

V e r y br ief ly , the results are as fol lows:

1. Bi la tera l r emova l of the caudate nuc le i has b e e n successful ly accompl i shed

in 14 k i t t ens ( B A c ) , and bi la teral removal of the frontal cort ical areas has b e e n

per formed i n five k i t t ens (BFr) . T h e average age of all l e s ioned an ima l s at the

t ime of surgery was 18 .3 days , w i th a m i n i m u m of 7 and a m a x i m u m of 35

days. Four of the B A c k i t tens d ied at 6 8 , 8 8 , 105 , and 115 days after surgery ,

respec t ive ly , b e c a u s e of in tercurrent d i seases s imi lar ly affecting the i r l i t ter-

ma tes . T h e r e m a i n i n g ten B A c k i t t ens , as wel l as all the B F r k i t t ens , are still

alive and in good heal th . To date , the average survival t ime of all an imals is 98

days , w i t h three of the B A c k i t tens h a v i n g r eached pube r ty (over 7 - 1 0 m o n t h s

of age) .

2 . T h e only h is to logica l mater ia l avai lable to date be longs to the four de-

ceased k i t tens m e n t i o n e d above . T w o of these b ra ins s h o w e d a lmost comple te

r emova l of the caudate nuc le i (95 and 1 0 0 % of caudate t i s sue r e m o v e d , respec-

t ive ly) , w i th little d a m a g e to o ther b r a in areas (Figure 1 ) , w h e r e a s the o the r

two b ra ins had a part ial , a symmet r i c ab la t ion .

3. All k i t tens we re up , wa lk ing , and nur s ing , e i ther the s ame day of surgery

or the fo l lowing day; on ly five of t h e m requ i red supp lementa l t ube feed ing for

1-3 days .

4 . T e n of the B A c k i t tens exh ib i t ed a b e h a v i o r s imi la r to the " c o m p u l s o r y

approach ing s y n d r o m e " (CAs) s een i n adult B A c cats (27) . T h i s w a s an i m -

press ive c h a n g e , par t icular ly in the smal les t k i t t ens , s ince pr ior to surgery they

s tood and wa lked hes i tan t ly and weak ly , and ye t the day fo l lowing surgery

they w o u l d eager ly a n d s t rongly approach a n d follow the inves t iga tor or an

ob jec t in a s te reo typed m a n n e r for m a n y m i n u t e s ; they wou ld also c o m e back

to the inves t iga to r w h e n repea ted ly p u s h e d away , as d id the adult B A c cats .

T h e m a i n dif ference b e t w e e n the C A s i n the k i t t en w h e n compared w i t h s imi -

lar p h e n o m e n o n in the adult cat w a s i ts shor ter durat ion; i n d e e d , in the k i t ten

the C A s w a s usual ly clearly p resen t from 1 to 4 days and thereafter progres-

s ively dec reased such that b y the ten th pos topera t ive day it was no longer

present . A fainter , shor ter las t ing , audi tory approach cou ld b e e l ic i ted ,

w h e r e a s no man i fes t a t ions of tacti le approach ing could b e i nduced in the B A c

k i t t ens .

In the four k i t t ens for w h o m the b ra ins w e r e avai lable for h i s to logy , there

was a clear , pos i t ive corre la t ion b e t w e e n the a m o u n t o f caudate r emova l and

the m a g n i t u d e of the C A s ; o n e of t h e m , w i t h on ly abou t 3 0 % of caudate nuclei

r e m o v e d , d id no t exh ib i t any C A s at all, w h e r e a s the syndrome w a s very pro-


FIGURE 1. Histological sections of the brain of a kitten at 133 days of age with bilateral removal of the caudate nuclei performed at 18 days of age. Weil-stained frontal sections at frontal (A-B), septal-fornix (C-D), and anterior thalamic (E-F)levels.

A

B

'C D

F E


n o u n c e d in the two k i t tens w i th a lmost total removal . T h e B F r k i t tens did not

exh ib i t any s te reo typed approach ing ; h o w e v e r , m o s t k i t t ens in b o t h groups

d isp layed s te reo typed v i sua l t racking of pendu la r m o t i o n , s imi la r to B A c and

BFr adult cats (27) , w h i c h also pers i s ted for a short per iod days) .

5. Ne i the r the B A c nor the B F r k i t t ens have s h o w n any gross n e u r o b e h a v -

ioral abnormal i t i e s after the early pos topera t ive per iod had e lapsed . H o w -

ever , there is s o m e ind ica t ion that the matura t ion of several neuro logica l

parameters unde r obse rva t ion m a y b e de layed; t hese f indings are b e i n g ver i -

fied in o n g o i n g s tudies .

T h e c o m p a r i s o n wi th thei r l i t termates at each tes t ing sess ion has b e e n the

m o s t re l iable cr i ter ion to evaluate the per formance of the b ra in - l e s ioned

k i t t ens , s ince part o f our no rma t ive data on in tact k i t t ens w a s in d i sc repancy

wi th s imilar data from other inves t iga tors (1 ,8) . T h e deve lopmen t of the l i m b

placing reac t ions , part icular ly the contact p lac ing reac t ion , dese rves specia l

m e n t i o n in this respect . O u r exper i ence has b e e n that th is is a funct ion w h i c h

develops progress ive ly in normal k i t tens and does not b e c o m e fully mature

unt i l the th i rd or fourth m o n t h of age. A s imi lar , a l though de layed t ime

course , occurred in the opera ted k i t tens . T h i s f inding, w h i c h is cons i s t en t

wi th the m a r k e d inab i l i ty of y o u n g k i t tens to wa lk o n p lanks or approach

edges w i thou t fal l ing, is in contras t w i th reports from o ther authors (1 ,5 ,8)

that the contact p lac ing reac t ion is deve loped dur ing the first days of life.

6. T h e B A c k i t t ens readi ly l ea rned the bas i c T - m a z e d i sc r imina t ion

task. H o w e v e r , they b e h a v e differently from the i r l i t termates in that t hey had

difficulties in lea rn ing the reversal aspects o f the task, as demons t ra t ed b y

the i r s t rong t e n d e n c y to pers i s t in the r e sponse p roduc ing the last re inforce-

men t , a persevera t ive defect also e x h i b i t e d b y the adult B A c cats (28) . T h e

p re l iminary data on m o t o r act ivi ty m e a s u r e m e n t s have no t revealed gross dif-

ferences in the deve lopmen t of the mo to r act ivi ty pat terns o f B A c and B F r

k i t tens in re la t ion to the i r l i t te rmates (19) .

In s u m m a r y , the above data sugges t that in the cat: (a) subcor t ica l b ra in

l es ions in in fancy do not p roduce any m o r e severe defects than early cort ical

in jur ies do; (b) a l though b o t h cort ical and subcor t ica l b r a in l e s ions are fol-

l owed b y a s t r ik ing recovery , our o n g o i n g obse rva t ions ind ica te that there

m a y b e a re tardat ion in neuro logica l matura t ion ; and (c) finally, t hese exper i -

m e n t s demons t r a t e that , un l ike the f indings in m o n k e y s (17) , b i la tera l , a lmost

total ab la t ion of the caudate nuc le i can b e done in k i t t ens and the se an imals

can b e m a i n t a i n e d in g o o d heal th for indef ini te pe r iods of t ime .


Supported by USPHS Grant Nos. MH-07097, HD-05958, and HD-04612. This research was carried on in collaboration with Drs. R. J. Marcus, C. E. Olmstead, and M. S. Levine, Department of Psychiatry, UCLA.


R E F E R E N C E S

1. Amassian, V. E . , Ross, R., and Donat, J. Development of contact placing and thalamocortical organization in kittens. Fed. Proc., 1971, 30: 434.

2. Battig, K., Rosvold, H. E . , and Mishkin, M. Comparison of the effects of frontal and caudate lesions on delayed response and alternation in monkeys. / . Comp. Physiol. Psychol., 1960, 53: 400-404.

3. Beach, F. A. The neural basis of innate behavior: Relative effects of partial decortication in adulthood and infancy upon the maternal behavior of the primiparous rat. / . Genet. Psychol, 1938, 53: 109-148.

4. Benjamin, R. M. , and Thompson, R. F. Differential effects of cortical lesions in infant and adult cats on roughness discrimination. Exp. Neurol, 1959, 1: 305-321.

5. Bignall, K. E . , and Schramm, L. Behavior of chronically decerebrated kittens, Exp. Neurol, 1974, 42 : 519-531.

6. Davis, G. D. Caudate lesions and spontaneous locomotion in the monkey. Neurology, 1958, 8: 135-139.

7. Doty, R. W. Functional significance of the topographical aspects of the retino-cortical projec-tion. In: The Visual System: Neurophysiology and Psychophysics. (R. Jung and H. Kornhuber, Eds.). Springer-Verlag, Berlin and New York, 1961: 228-245.

8. Fox, M. W. Reflex development and behavioral organization. In: Developmental Neurobiology. (W. A. Himwich, Ed.). Thomas, Springfield, Illinois, 1970: 553-580.

9. Goldman, P. S. Functional development of the prefrontal cortex in early life and the problem of neuronal plasticity. Exp. Neurol, 1971, 32: 366-387 .

10. Goldman, P. S., and Rosvold, H. E. The effects of selective caudate lesions in infant and juve-nile rhesus monkeys. Brain Res., 1972, 43: 53 -66 .

11. Goldman, P. S., Rosvold, H. E . , and Mishkin, M. Selective sparing of function following prefrontal lobectomy in infant monkeys. Exp. Neurol, 1970, 29: 221-226.

12. Harlow, H. F . , Akert, K., and Schlitz, K. A. The effects of bilateral prefrontal lesions on learned behavior of neonatal, infant and preadolescent monkeys. In: The Frontal Granular Cortex and Behavior. (J. M. Warren and K. Akert, Eds.). McGraw-Hill, New York, 1964: 126-148.

13. Isaacson, R. L. , Nonneman, A. J . , and Schmaltz, L. W. Behavioral and anatomical sequelae of damage to the infant limbic system. In: The Neuropsychology of Development. (R. L. Isaacson, Ed.). Wiley, New York, 1968: 41-78.

14. Kennard, M. A. Reorganization of motor function in the cerebral cortex of monkeys deprived of motor and premotor areas in infancy. / . Neurophysiol, 1938, 1: 477-496.

15. Kling, A. Behavioral and somatic development following lesions of the amygdala in the cat. / . Psychiatr. Res., 1965, 3: 263-273.

16. Kling, A. The effect of cerebral ablation in infant monkeys on motor and cognitive function. In: Physical Trauma in Mental Retardation. (C. R. Angls and E. A. Baring, Eds.). U.S. Dept. of Health, Education, and Welfare, Washington, D. C , 1970: 197-205.

17. Kling, A., and Tucker, T. J . Effects of combined lesions of frontal granular cortex and caudate nucleus in the neonatal monkey. Brain Res., 1967, 6: 428-439.

18. Kling, A., and Tucker, T. J . Sparing of function following localized brain lesions in neonatal monkeys. In: The Neuropsychology of Development. (R. L. Isaacson, Ed.). Wiley, New York, 1968: 121-145.

19. Levine, M. S., Hull, C. D. , and Buchwald, N. A. Development of motoric activity in kittens. Proc. Soc. Neurosci., 1974, 4: 305.

20. Raisler, R. L. , and Harlow, H. F. Learned behavior following lesions of posterior association cortex in infant, immature and preadolescent monkeys. / . Comp. Physiol Psychol, 1965, 60: 167-174.

21. Rosvold, H. E . , and Delgado, J . M. R. The effect of delayed-alternation test performance of


stimulating or destroying electrically structures within the frontal lobes of the monkey's brain. /. Comp. Physiol. Psychol., 1956, 49: 365-372.

22. Scharlock, D. P., Tucker, T. J . , and Strominger, N. L. Auditory discrimination by the cat after neonatal ablation of temporal cortex. Science, 1963, 141: 1197-1198.

23. Schneider, G. E. Mechanisms of functional recovery following lesions of visual cortex or supe-rior colliculus in neonate and adult hamsters. Brain, Behav. Evol., 1970, 3: 295 - 3 2 3 .

24. Tsang, Y. -C. Maze learning in rats hemidecorticated in infancy. / . Comp. Psychol, 1 9 3 7 2 4 : 221-253.

25. Tucker, T., and Kling, A. Differential effects of early vs. late brain damage on visual duration

discrimination in cat. Fed. Proc, 1966, 25: 207.

26. Tucker, T. J . , and Kling, A. Differential effects of early and late lesions of frontal granular

cortex in the monkey. Brain Res., 1967, 5: 377-389.

27. Villablanca, J . , and Marcus, R. J . Effects of caudate nuclei removal in cats. Comparison with

effects of frontal cortex ablation. Chapter 10, this volume.

28. Villablanca, J . R. , Olmstead, C. E . , and Marcus, R. J . Perseverative instrumental behavior in

caudatectomized cats. Proc. Soc. Neurosci., 1974, 4: 461.

14 Prenatal Nutrition and Neurological

Development

JOHN DOBBING Department of Child Health,

University of Manchester,

Manchester, England

I N T R O D U C T I O N

T h e dec i s ion to inves t iga te the effects of infant malnu t r i t ion on the deve lop-

ing b ra in i m p o s e s on the researcher an unusua l ly h i g h s tandard of social and

scientif ic r e spons ib i l i t y w h e n it c o m e s to in te rpre t ing h i s resul ts ; for the s u b -

jec t invo lves h i m in the in ter re la t ion b e t w e e n m a n ' s m o s t impor tan t h u m a n

faculty, that of h i g h e r men ta l funct ion, and our greates t m a n m a d e scourge : the

malnu t r i t ion of m o s t of the wor ld ' s ch i ldren . T h e fol lowing remarks are put

forward in an a t t empt to counterac t s o m e of the m o r e ext ravagant c la ims of

an imal expe r imen te r s in th i s u n i q u e l y h u m a n field.

Expe r imen ta l pro jec ts wil l i nev i t ab ly b e conduc ted on an ima l spec i e s ,

usual ly the rat, and exper i ence shows that it is no t difficult to p roduce results

w h i c h can eas i ly b e gross ly mi s l ead ing w h e n uncr i t ical ly in te rpre ted for pol i -

t i c ians , p l anner s , and gran t -g iv ing b o d i e s . T h u s an imals underfed dur ing fetal

and neona ta l life w h o s e b ra ins are pe rmanen t ly small and w i t h too few cells

have found the i r h u m a n counterpar ts in the fertile i m a g i n a t i o n of m a n y w h o

have not ever cons ide red the real s ignif icance (or o the rwise ) of the f inding.

T h e ext rapola t ion has often b e e n m a d e wi thou t even w o n d e r i n g w h e t h e r a

m o d e s t r educ t ion of total b ra in cells or of b r a in w e i g h t m a y b e of any real s ig-

ni f icance , espec ia l ly if that b ra in res ides in a smal ler b o d y . A d d to th i s the

very great uncer ta in t i es o f s tud ies of an imal b e h a v i o r from w h i c h rats are hap -

pi ly p r o n o u n c e d to b e " e m o t i o n a l , " of " sho r t m e m o r y " o r w i t h " l ea rn ing

401

402 John Bobbing

d i sab i l i t i e s , " and a w e b of t enden t ious ext rapola t ion can b e spun w h i c h ap-

pears inf ini tely p laus ib le to the u n w a r y h u m a n .

DEFICITS AND DISTORTIONS

V e r y few, if any of these no t ions w i th s t and the scrut iny of e i ther neurob io l -

ogis ts or behav io ra l sc ien t i s t s , b y w h o m they are ve ry rarely put forward or ,

in prac t ice , j udged . It cannot , for example , b e a s s u m e d that a s o m e w h a t

smal ler b r a in in a s o m e w h a t smal ler an imal is necessa r i ly a b a d th ing , unless ,

for example , one ho lds to the ex t raord inary doct r ine that the s o m e w h a t

smaller b r a in of w o m e n is the cause of the i r a l leged men ta l infer ior i ty to m e n .

Defici ts in b ra in cell n u m b e r s w h i c h have at tracted great a t tent ion have often

b e e n glial cell defici ts; and even w h e n they have b e e n neurona l , as in the

n e w b o r n rat, t hey m a y wel l have b e e n funct ional ly un impor tan t , as well as

p roduced b y maneuve r s gross ly ou t s ide any h u m a n exper ience . Unfor tunate ly

w e do no t k n o w the phys ica l b a s i s of h i g h e r men ta l act ivi ty , bu t it i s m u c h

more l ikely to res ide in the c i rcui t ry—the sys tem of dendr i t ic b r a n c h i n g and

synapt ic connec t iv i ty—than in cell n u m b e r s ; and th is phys ica l sys t em has so

far res is ted ser ious quant i f ica t ion. Indeed the funct ional reserve in n u m b e r s of

cells is l ikely to b e vast , a n d quan t i t a t ive deficits o f 1 0 - 2 0 % overall are

ex t remely unl ike ly to b e direct ly s ignif icant . Stil l less is it l ikely that small

reduc t ions in the n u m b e r of l aminae in the mye l i n shea ths wil l affect m a n ' s

des t iny .

T h e above scep t i c i sm abou t the type of exper imenta l ev idence so c o m m o n l y

adduced is not m e a n t in any w a y to imp ly any var ia t ion in the author ' s

profound and long-s t and ing be l i e f that env i ronmen ta l factors dur ing deve lop-

men t do so often play powerful roles in res t r ic t ing u l t imate h u m a n ach ieve -

ment . Indeed it is the very se r iousness of th is be l i e f w h i c h p rompts the

p resen t homi ly .

Gran ted that the phys ica l make -up of the pa ranormal b ra in is i m p o s s i b l e to

interpret funct ional ly , it can still b e conjec tured that i t is d is tor t ions of its

archi tec ture rather than un i fo rm deficits in it w h i c h are m o r e l ikely to b e s ig-

nif icant for its d i sordered intel lectual funct ion (13) . T h e b ra in w h i c h is smal ler

than is appropr ia te to the b o d y s ize (11) ; the ce rebe l lum smal ler than is appro-

priate to the b ra in s ize (6) ; the differential loss of specific neurona l types in

cer ta in areas in ce rebe l lum and cor tex (8) ; pe rmanen t ly al tered enzymat ic or

o ther cons t i tuen t re la t ionsh ips ra ther than mere ly al tered quan t i t i e s (1) : these

should p r o b a b l y g ive m o r e cause for concern . A n d the h u n t shou ld b e on for

be t te r and more m a n a g e a b l e quant i ta t ive ind ices o f dendr i t ic and synapt ic

s tructure, so that d i s tor t ions of these should b e sought .

In the behav io ra l field, ma te rna l - in fan t in te rac t ion m a y wel l b e the m o s t

impor tan t factor as wel l as the m o s t neg lec ted . H o w does the growth- re ta rded

n e w b o r n affect even the we l l -nour i shed foster m o t h e r in ways that " feed

b a c k " to the chi ld and pe rmanen t ly affect h i s future character?

14. Prenatal Nutrition and Neurological Development 403

CROSS-SPECIES EXTRAPOLATIONS

T h e mos t se r ious t r ansgress ions , h o w e v e r , of those w o r k i n g in th is gen-

eral field h a v e re la ted to the genera l ext rapola t ion of data o b t a i n e d in rats

and o ther spec ie s to m a n . It is th is w h i c h has m a d e our sub jec t a lmost d is rep-

utable to the po in t w h e n faith in the usefulness of an ima l work has b e g u n

to b e a b a n d o n e d b y ped ia t r i c ians and m a n y o thers . E v e n nut r i t ion is t s in

m a k i n g in te r spec ies ext rapola t ions have often s t rangely i gno red thei r o w n

three " u n i t i e s , , of deve lopmenta l nut r i t ion: i ts sever i ty , i ts dura t ion , and its

t iming .

Never the le s s , p rov ided these three var iab les are taken into account , the

deve lopmen t of the h u m a n b ra in is essent ia l ly so s imi la r to that of an ima l s

that it i s r ea sonab le to expect he lp from the s tudy even of rats in e luc ida t ing

the h u m a n cond i t ion . T h e bas i c s tages of b ra in deve lopmen t are vir tual ly

ident ica l in all m a m m a l s (3) . A s imi la r embryo log ica l pe r iod is fo l lowed b y a

per iod of p r edominan t l y neurona l cell d iv i s ion so that the adult n u m b e r of

neu rons is a lmost comple te ly ach i eved qu i t e early (9) . T h i s is fo l lowed b y an

a lmost explos ive pe r iod of o l igodendrogl ia l mul t ip l ica t ion w h o s e products

p roceed to syn thes i ze and lay d o w n mye l in . Unde r ly ing and con tempora ry

w i th th i s " g l i a l - m y e l i n a t i o n " phase , w h i c h also co r responds to the b r a in

" g r o w t h spur t , " are the in t r ica te process o f dendr i t ic g rowth and b r a n c h i n g

and the e s t ab l i shmen t of synapt ic c o n n e c t i o n s , as wel l as a hos t o f dramat ic

n e u r o c h e m i c a l t ransformat ions p r e sumab ly assoc ia ted w i th the deve lopmen t

in s tructure.

All the spec ies go th rough this s ame deve lopmen ta l s e q u e n c e , and the ana-

tomica l uni t s o f w h i c h the b ra in is c o m p o s e d are also r emarkab ly s imi lar in

chemica l c o m p o s i t i o n and in me tabo l i c and e lec t rophys io logica l p roper t ies

from one spec ies to ano ther .

T h e major spec ie s di f ference, apart from the degree of complex i ty of the

final product , is in the t im ing of these p rocesses in re la t ion to b i r th (7) . T h u s

in rats b i r th occurs toward the end of neu rona l mul t ip l ica t ion , w h i l e the b ra in

is still ve ry i m m a t u r e . In the gu inea p ig the b ra in g rowth spurt is a lmos t over

at b i r th and the n e w b o r n an ima l is r emarkab ly mature . B y contras t w i th bo th ,

h u m a n neurona l mul t ip l i ca t ion is the m a i n c o m p o n e n t o f b ra in g rowth in the

second in t rau te r ine t r imes te r (10) , the " g r o w t h spur t " p roper ex t end ing from

the b e g i n n i n g of the th i rd t r imes ter unt i l toward the end of the s econd pos t -

natal year (12) .

STAGES OF BRAIN GROWTH, NOT AGES

It is therefore a r id icu lous t ravesty to equa te "fetal b ra in g r o w t h " in any one

of these spec ies w i t h that in any other . Norma l b i r th has no m e a n i n g for the

g rowth and d e v e l o p m e n t of the b ra in , w h i c h p rogresses smoo th ly th rough

404 John Dobbing

bi r th w h e t h e r the latter occurs at the immatu re s tage (rat), the mature (gu inea

p ig ) , or at an in te rmed ia te (human) s tage of b r a in g rowth (Figure 1) . T h u s

express ions such as " t h e fetal b r a i n " or " t h e neona ta l b r a i n " are va lue less

un less the spec ies b e specif ied and the b ra in g rowth character is t ics of that

spec ies k n o w n .

A part icular ly h e i n o u s t ravesty is f requent ly perpe t ra ted w h e n the effects of

malnu t r i t ion on neurona l cell d iv i s ion are b e i n g cons ide red . Exper imenta l ly it

is ex t remely conven ien t that b i r th in the rat marks the close of the m a i n p h a s e

of cerebra l neu rona l mul t ip l i ca t ion , before glial mi tos i s has ser ious ly begun .

Th i s m e a n s , in genera l t e rms , that any effect of materna l malnu t r i t ion in th is

spec ies on the mi to t ic rate o f b r a in cells wil l se lect ively affect neu rons ra ther

than gl ia ; and the level o f D N A in a n e w b o r n rat b ra in wil l b e qu i te direct ly

related to n u m b e r of neu rons .

T h e t empta t ion has therefore b e e n great , and for s o m e i r res is t ib le , to use

rats for expe r imen t s to accentua te the impor tance of mate rna l malnu t r i t ion on

b ra in deve lopment . Va r ious nu t r i t iona l r e g i m e n s have led to smal le r n e w b o r n

rat b r a in s w i t h smal ler n u m b e r s of neu rons , and the genera l ext rapola t ion has

b e e n m a d e that h u m a n materna l malnu t r i t ion wil l b e s imilar ly impor tan t for

the h u m a n neurona l mul t ip l ica t ion .

T h i s conc lus ion is l ikely to b e comple te ly false and p r o b a b l y cons t i tu tes a

ser ious and very mi s l ead ing misapp l i ca t ion , to h u m a n s , of the exper imenta l

f indings in an ima l s , e v e n suppos ing that small r educ t ions in neurona l n u m b e r

mat ter .

\ A

V \ \ \ \ \ '\ \

/ \ / /

i \ \ i \ \ i \ \

/ An

-30 -20 A G E

-10 A BIRTH

20 30 A G E

FIGURE 1. Velocity of human brain growth (wet weight) compared with that in other species.

Prenatal and postnatal age is expressed as follows: human ( ) in months (12); guinea pig

(-- ) in days (9); pig ( ) in weeks (5); rat (—•— — •— — — — — )

in days (11).

PE

RC

EN

T O

F

AD

UL

T W

EIG

HT

rr—

•

——

—


R A T C O M P A R E D W I T H H U M A N F E T A L L I F E

T h e error a r i ses qu i t e s imply from the fo l lowing cons ide ra t ions . It i s wel l

k n o w n that fetal g rowth suscep t ib i l i ty to mate rna l malnu t r i t ion i n b o t h rat

and m a n is vir tual ly conf ined to the last th i rd of p regnancy . W h e t h e r b e c a u s e

the fetus is too small re lat ive to the m o t h e r in the midd le third o f p r egnancy or

for o ther less o b v i o u s reasons the " m a l n o u r i s h e d " h u m a n fetus , w h e t h e r in

mul t ip le p r egnancy or o the rwi se , s e e m s to g row qu i t e wel l un t i l abou t 28 ges -

ta t ional w e e k s . At th is t ime it b e g i n s to lose g round rapidly as it a t tempts to

inc rease i ts s ize relat ive to b o t h the m o t h e r and to the p lacenta . It i s dur ing

the th i rd t r imes te r on ly that g rowth res t r ic t ion is de tec tab le in the o the rwi se

normal m a l n o u r i s h e d fetus. F r o m th is it is c lear that a l though neu rona l mu l -

t ip l ica t ion in the rat occurs in the h igh ly vu lne rab le last one- th i rd of fetal l ife,

in the h u m a n it occurs in the h igh ly pro tec ted s econd t r imes te r w h e n g rowth

processes are ex t remely unl ike ly to b e i n c o n v e n i e n c e d b y placenta l insuf-

f iciency. If th is b e t rue, w e are therefore abso lu te ly p reven ted from c o m p a r i n g

h u m a n fetal ma lnu t r i t ion w i th rat fetal malnu t r i t ion in t e rms of neu rona l mul -

t ip l ica t ion.

T h e fa lseness of the c o m p a r i s o n can b e p u s h e d still further. It m u s t b e

r e m e m b e r e d that the equ iva len t h u m a n b r a in age of the rat b r a in at b i r th

canno t b e m u c h later than 18 w e e k s of h u m a n ges ta t ion (see la ter) , and that

the equ iva len t rat b r a in age to that at b i r th , at h u m a n full t e rm, i s abou t 5 - 7

rat pos tnata l days . It m u s t fol low that n e w b o r n rats s h o w i n g the 20 or 3 0 %

b o d y w e i g h t deficits so c o m m o n in the expe r imen ta l l i terature wou ld b e

equ iva len t to h u m a n fe tuses at 18 w e e k s ges ta t ion s h o w i n g s imi la r b o d y

w e i g h t deficits . S u c h severe deficits of h u m a n fetal g rowth in the s econd

t r imes ter have neve r b e e n repor ted , even in ve ry unusua l h i g h l y pa tho log ica l

c i r cums tances ; and th is m a k e s these rat " m o d e l s " va lue less except to the spe-

cific inves t iga t ion of rats . T h e very lowes t full-term b i r th w e i g h t o f a h u m a n

nutr i t ional ly " sma l l - fo r -da te s" b a b y is abou t hal f the no rma l average we igh t ,

and any g rowth re tardat ion due to a failure o f suppl ies in these cases has oc-

curred dur ing the th i rd t r imes ter , after the e n d of the m a i n stage of neurona l

mul t ip l ica t ion . A m o r e correct mode l to s tudy the h u m a n m a l n o u r i s h e d fetus

wou ld therefore b e to take n e w b o r n rats of no rmal w e i g h t and reduce the i r

g rowth rate dur ing their first 5 - 7 postnata l days , to a degree no greater than is

necessa ry to ach ieve ha l f the normal w e i g h t at that t ime . T h i s w o u l d clearly

not d is turb the major pe r iod of mul t ip l ica t ion of neu rons in the i r b r a i n s , any

more than w o u l d b e l ikely in h u m a n s , bu t it m a y wel l affect the m o r e impor -

tant s u b s e q u e n t s teps of dendr i t ic and synapt ic g rowth ou t l ined above .

A s t rong plea m u s t therefore b e m a d e that un less one is e rec t ing mode l s for

those severe nonnu t r i t i ona l cond i t i ons w h i c h s low fetal g rowth in the s econd

h u m a n t r imes te r , one shou ld p r o b a b l y no t ma lnou r i sh the rat fetus i n order to

s tudy the effects of mate rna l ma lnu t r i t ion on h u m a n fetal b ra in growth . It

406 John Dobbing

mus t aga in b e s t ressed that early undernu t r i t ion at cer ta in s tages of h u m a n

growth a lmost cer ta in ly does affect the phys ica l b r a in g rowth p rocesses per-

manen t ly . It i s also n o w b e y o n d ques t ion that ch i ldren g rowing in a total

e n v i r o n m e n t w h i c h inc ludes undernu t r i t ion at these cri t ical t imes are less well

e n d o w e d as h u m a n b e i n g s in later life. Fur the rmore it is m y be l i e f that an imal

expe r imen t s proper ly conduc ted and r e spons ib ly extrapolated do i l luminate

inves t iga t ion of these mat ters in w a y s w h i c h w o u l d b e i m p o s s i b l e in our o w n

spec ies . Bu t b e c a u s e of the profound se r iousness of these mat te rs , w e mus t all

b e m u c h m o r e careful abou t our in te r spec ies ext rapola t ions .

THE HUMAN BRAIN GROWTH SPURT

T h e b ra in growth spurt pe r iod has b e e n s h o w n in exper imenta l an imals to

b e the t ime w h e n the g rowing b ra in is mos t vu lnerab le to undernut r i t ion ,

bo th con t emporaneous ly wi th the under feed ing and ul t imately . A pre requ is i t e

for t ransla t ing th i s in format ion from the exper imen ta l an ima l to the h u m a n is

a descr ip t ion of the later s tages of h u m a n growth in the same quant i ta t ive

te rms as have b e e n used to inves t iga te the effects of res t r ic t ion on deve lop ing

an imal b ra ins . In th i s , the v o l u m i n o u s pa thologica l l i terature is very little

he lp . It is even difficult to const ruct a s m o o t h growth curve for b ra in we igh t in

w h i c h fetal m e r g e s conv inc ing ly w i t h pos tna ta l b r a in we igh t . T h e fol lowing is

a n e w a t tempt to desc r ibe normal h u m a n b ra in g rowth quant i ta t ive ly , and es -

pecial ly to de l inea te the age l imi t s of the h u m a n b ra in g rowth spurt . T h i s is a

part icularly necessa ry s tep in the a rgument b e c a u s e of the spec ies differences

in the t iming of b ra in g rowth m e n t i o n e d above .

T h e first p r o b l e m , w h i c h is less difficult to o v e r c o m e than m i g h t at first ap-

pear , is h o w to d raw a normal g rowth curve from data o b t a i n e d from dead

h u m a n b ra ins . T h e mater ia l m u s t necessa r i ly b e c ross -sec t iona l ra ther than

longi tudina l , and th is can on ly part ly b e c o m p e n s a t e d for b y col lect ing a suf-

ficient n u m b e r of s p e c i m e n s to por t ray the m a g n i t u d e of the var iance at each

age. In our work , b r a in s were carefully selected from fetuses and ch i ldren w h o

appeared to s h o w n o ser ious dev ia t ions from norma l somat ic g rowth , and no

b ra in w a s accepted w h i c h s h o w e d any consp icuous neuropa tho logy .

Fetal b ra ins o f the late first and early second t r imes ter were from induced

abor t ions w h i c h can b e p r e s u m e d normal . Th i rd t r imes ter b r a in s were ac-

cep ted i f the b o d y w e i g h t fell w i t h i n one s tandard devia t ion of the expec ted

w e i g h t for ges ta t ional age . Pos tnata l b ra ins were from chi ldren dy ing acutely

fol lowing acc idents or ve ry b r i e f i l lness . Fur the rmore on ly those parameters

were measu red w h i c h s h o w no pos tmor t em al terat ion in the inev i tab le in-

terval b e t w e e n death and the avai labi l i ty of the spec imen . O n e h u n d r e d forty-

e igh t comple te h u m a n b ra in s , r ang ing in age from 10 ges ta t ional w e e k s to

adul thood, were d iv ided in to three gross r eg ions , " f o r e b r a i n / ' " c e r e b e l l u m , "


and " s t e m / 7 we re w e i g h e d , and inves t iga ted for D N A , wa te r , and choles terol

content . T h e deta i led f indings are repor ted e l s ewhere (12) .

BRAIN WEIGHT

G r o w t h in w e i g h t is s h o w n in F igure 2 . T h e famil iar s i g m o i d g rowth curve

c o m m e n c e s i ts upward inf lect ion in m i d p r e g n a n c y and s e e m s to b e g i n to level

out b e t w e e n 1 and 2 years of pos tnata l age . Def in i t ion o f the end po in t of the

g rowth spurt of h u m a n b ra in is difficult in th i s and in s u b s e q u e n t figures

part ly b e c a u s e of an ex t reme scarci ty of s p e c i m e n s from chi ldren over 1 year of

age, and part ly b e c a u s e the t rans i t ion b e t w e e n the s teep part of the g rowth

spurt and its l eve l ing out is ra ther gradual . For these reasons the t empta t ion to

draw sub jec t ive l ines th rough the data has b e e n res i s ted . H o w e v e r , it s e e m s

clear from F igure 2 that , i n te rms of w e i g h t a lone , the g rowth spurt of h u m a n

bra in runs from m i d p r e g n a n c y to abou t 18 m o n t h s of pos tnata l age . O n th is

calculat ion no t m u c h m o r e than one - s ix th of it is fetal, and in cases w h e r e fetal

g rowth res t r ic t ion is conf ined to the last t r imes ter , at least s ix - seven ths of

the vu lnerab le pe r iod are postnata l .

BRAIN WEIGHT/BODY WEIGHT RATIO

The ratio of b r a in w e i g h t to b o d y w e i g h t w a s once cons ide red to b e a useful

guide to w h e t h e r or not a b i r th w e i g h t w a s small for the ges ta t iona l age . T h i s

1.5

HUMAN WHOLE BRAIN

B I R T H 2 4 6 8 A D U L T

AGE (years)

FIGURE 2. Whole human brain weight from 10 gestational weeks to adulthood (12).

WEI

GH

T (K

g)

In

o

408 John Bobbing

HUMAN BRAIN

10 20 30 B IRTH 6 12 A D U L T

WEEKS MONTHS

FIGURE 3. Ratio of brain weight to body weight in developing human brain (12).

was w h e n all small-for-dates b a b i e s we re though t to b e " m a l n o u r i s h e d "

dur ing the third t r imester , and w a s b a s e d on the fact (and the f inding in s o m e

such b a b i e s ) that undernu t r i t ion affects the b o d y w e i g h t m o r e than the bra in

we igh t . T h i s is the effect of " b r a i n spa r i ng , " wel l k n o w n to deve lopmenta l nu -

tr i t ionis ts . It shou ld b e clearly b o r n e in m i n d , howeve r , that a l though the

bra in , l ike b o n e and geni ta l deve lopmen t , cer ta inly is less affected than o ther

t i s sues , it is still affected, and its degree of affection is m o r e impor tan t than i ts

" s p a r i n g . "

Unfor tunate ly the ratio o f b ra in w e i g h t to b o d y w e i g h t is of little use in

m a k i n g cl inical j u d g m e n t s , even in those b a b i e s w h o s e in t rauter ine growth

has b e e n s lowed b y a failure o f supply . T h i s is b e c a u s e , as s een in F igure 3 , it

is subjec t to too great a var iance in a normal popula t ion . T h e ratio is one of the

b ra in parameters w h i c h falls w i t h inc reas ing age in all spec ie s . It does so more

gradual ly in the s lowly g rowing h u m a n than in s o m e o thers .

C E L L U L A R I T Y

Cellulari ty is a concen t ra t ion express ion and m a y m e a n n u m b e r o f cells pe r

mic roscope field or a m o u n t o f D N A per un i t w e i g h t of t i ssue . It is to b e dis-

t i ngu i shed carefully from total n u m b e r o f cells pe r ana tomica l reg ion .

T h e cel lulari ty o f the h u m a n forebra in (and h e n c e of the w h o l e b ra in , s ince

forebra in is m u c h the largest propor t ion) falls w i th inc reas ing fetal age , even

though the total n u m b e r of cells is r i s ing rapidly at the s ame t ime (Figure 4 ) .

T h i s over r id ing di lu t ion effect m u s t b e due to the relat ively faster arrival of

cell subs tance (and noncel lu lar mye l in ) ra ther than the already fast rate of cell

mul t ip l ica t ion; and the s teep fall in cel lulari ty m a k e s this pa ramete r a difficult

BR

AIN

W

EIG

HT

I B

OD

Y

WE

IGH

T

x

100


HUMAN FOREBRAIN

8 •

B I R T H 2 4 6 8 A D U L T

AGE ( y e a r s )

FIGURE 4. Concentration of DNA-P per unit fresh weight, equivalent to cellularity (see text) in

human forebrain (12).

one to cons ider . T h e difficulty is c o m p o u n d e d b y the concur ren t paradoxica l

r ise in cel lulari ty o f the ce rebe l lum (Figure 5 ) . Th i s is ent i re ly cons i s t en t w i t h

the ext raordinar i ly rapid rate o f g rowth o f the ce rebe l lum relat ive to that of the

b r a in as a w h o l e , and the compara t ive ly small average s ize of adult cerebel lar

cells; so that the d i lu t ion effect seen in the forebra in i s doub ly defeated. T h e

mix ture of apparent ly oppos i t e g rowth character is t ics w i t h i n the b ra in m a k e s

cellulari ty a difficult pa ramete r to cons ide r .

HUMAN CEREBELLUM

B I R T H 2 4 6 8 A D U L T

AGE ( y e a r s )

FIGURE 5. Concentration of DNA-P per unit fresh weight, equivalent to cellularity (see text) in

human cerebellum (12).

(UJ6

/aio

ujH

) d

'VN

Q

DN

A-P

(f

tmo

le/g

m)

4 1 0 John Bobbing

T O T A L C E L L N U M B E R

T h i s is a m u c h eas ie r pa ramete r to under s t and , a l though as before , the

who le b r a in or a w h o l e ana tomica l ly def ined reg ion m u s t b e avai lable for i ts

quant i f ica t ion . His to logica l he t e rogene i ty forbids the use of f ragments or

b i o p s y s p e c i m e n s . F igure 6 s h o w s the total D N A in the h u m a n forebra in ,

from w h i c h it can b e e s t ima ted that cell mul t ip l ica t ion does not b e g i n to draw

to a c lose unt i l wel l in to the s econd postnata l year . A t th is t ime total cell

n u m b e r is still subs tant ia l ly less than its adult va lue . P rev ious conc lus ions

b a s e d on reports that adult b ra in cell n u m b e r is a t ta ined b y 5 postnatal

m o n t h s (17) therefore r equ i re drast ic rev i s ion .

Cell mul t ip l ica t ion in the three major b ra in reg ions is compared in Figure 7.

In th is ins tance s m o o t h e d l ines have b e e n drawn subjec t ive ly th rough the

po in t s . T h e excep t iona l g rowth charac ter i s t ics of the h u m a n ce rebe l lum, c o m -

parable wi th the s a m e p h e n o m e n a in o ther an imal spec i e s , are readi ly seen. It

is f requent ly and e r roneous ly s tated that the ce rebe l lum grows " l a t e r " than the

rest of the bra in . F igure 7 s h o w s on the cont rary that a l though i ts cells do

b e g i n to mul t ip ly later , it ach ieves i ts o w n growth spurt m u c h earl ier . T h e cer-

ebe l lum therefore g rows very m u c h faster th rough a shor ter pe r iod bu t

b road ly concur ren t ly wi th the o ther parts of the bra in . Indeed it has ach ieved

its adult n u m b e r of cells b y abou t 15 postnata l m o n t h s , an age w h e n the rest of

the b ra in is on ly abou t 6 5 % of the w a y toward adult quan t i t i e s . T h e same

pecul iar i t ies of cerebel lar g rowth can b e seen in i ts rate of mye l ina t ion and i ts

g rowth in we igh t . It is these s imi lar i t ies w i th cerebel lar g rowth in o ther

S

HUMAN FOREBRAIN

FIGURE 6.

B I R T H 2 4 6 8 A D U L T

AGE (years)

Total DNA-P, equivalent to total cell number in human forebrain (12).

DN

A-P

(m

mo

le)

3

14. Prenatal Nutrition and Neurological Development 4 1 1

100 .. /

/ HUMAN BRAIN

/ /

/ -

/ i Y

i f 1 FOREBRAIN / ' / STEM

i/ CEREBELLUM / /

/ / (1

F

C BIRTH

AGE (years)

FIGURE 7. Comparative values for total DNA-P, equivalent to total numbers of cells, in three

human brain regions. Values have been calculated as a percentage of the adult value (12).

an imals w h i c h lead to the ques t i on w h e t h e r the s ame differential vu lnerab i l i ty

exis ts i n h u m a n s , w i t h the s ame funct ional c o n s e q u e n c e s .

M Y E L I N A T I O N

There are several chemica l ind ices w h i c h are useful for m e a s u r i n g the

process of mye l ina t i on quant i ta t ive ly . T h e mos t specific for mye l in are the

30

HUMAN FOREBRAIN

A

FIGURE 8.

BIRTH 2 4 6 8 ADULT

AGE (years)

Cholesterol content of human forebrain, an index of myelination (see text) (12).

DN

A-P

( p

erc

en

t a

du

lt valu

e)

CH

OL

ES

TE

RO

L

(g)

4 1 2 John Bobbing

hexose -con ta in ing l ip ids , ce reb ros ide and sulfat ide (4) . Choles te ro l , howeve r ,

i s a m u c h eas ie r l ip id to e s t ima te re l iably in ve ry large n u m b e r s of samples ,

and a l though it i s m u c h less specif ic , its accumula t ion does bea r a cons tan t

re la t ion to that of ce reb ros ide , ar r iv ing before the ce reb ros ide does . A s m e n -

t ioned earl ier , the spurt in choles terol syn thes i s follows that of glial cell mul -

t ip l ica t ion , s ince it is syn thes i zed b y t h e m , and it therefore occup ies the later

phase s of the b ra in g rowth spurt .

Figure 8 shows its inc rease in h u m a n forebra in , and it wil l b e seen that

a l though its end po in t is no t very wel l def ined, there is still qu i t e a rapid r ise

a lmost to 4 years o f age , fo l lowed b y a very gradual r ise to adult levels . T h e

growth spurt proper , w h i c h inc ludes mye l ina t ion , m a y therefore in th is sense

b e cons ide red to con t inue wel l in to the fourth year .

N E U R O N S A N D G L I A

It has already b e e n m e n t i o n e d in desc r ib ing the genera l sequen t ia l pat tern

of m a m m a l i a n b ra in g rowth that the ma jo r phase of neurona l mul t ip l ica t ion

occurs before the numer ica l ly larger phase of glial mul t ip l ica t ion , so that an

adult n u m b e r o f neu rons is a ch i eved very early, except for specia l cases in cer-

ta in reg ions . Theore t ica l ly , therefore , any growth curve s h o w i n g the increase

in n u m b e r s of cells of all types should exh ib i t two consecu t ive phases of cell

d iv is ion . S u c h a p h e n o m e n o n has no t b e e n demons t ra t ed in any of the small

laboratory spec ies s tud ied , a l though it was in these spec ies that the p h e n o m e -

n o n was first desc r ibed b y h is to logis t s . It n o w appears that the failure to s h o w

the two phases in " D N A c o u n t s " m a y b e due to the shor t l i fe-span and the

c o n s e q u e n t rapid i ty o f deve lopmenta l progress in the smal ler spec ies , in

w h o m such even ts take only a few hours or days to occur . It wou ld thus

requi re a lmost hour ly mon i to r ing of concep t ion t i m e , and m u c h more

f requent obse rva t ions on aspects of later deve lopmen t to reveal the two phases

of cell mul t ip l ica t ion . H o w e v e r , in h u m a n s and o ther s lowly g rowing spec ies ,

such as the cow, the t ime scale is m u c h m o r e ex tended , and it so h a p p e n s that

h u m a n s are the first s lowly g rowing spec ies w h o s e b ra in growth has b e e n

quant i ta t ive ly desc r ibed us ing " D N A c o u n t s . " T h u s the two phases of b ra in

cell d iv i s ion have b e e n quant i ta t ive ly revealed for the first t ime in ourse lves .

F igure 9 shows a " h i g h p o w e r v i e w " of the earl ier s tages of the data already

s h o w n in F igure 6. Cell mul t ip l ica t ion clearly occurs in two qu i t e d is t inc t

phases w i th a r emarkab ly sharp cut-off po in t at 18 w e e k s of ges ta t ion. It s eems

a lmost cer ta in that the pe r iod from 10 to 18 w e e k s is the major pe r iod o f

h u m a n neurob las t mul t ip l ica t ion , di f ferent ia t ion to n o n d i v i d i n g neu rons oc -

curr ing toward the end of th is t ime . Gl ia l d iv i s ion t hen takes over and oc-

cup ies the r ema inde r of the mul t ip l ica t ive phase unt i l wel l in to the second

postnata l year .


10 20 30 BIRTH 2 4 10 20 30 BIRTH 2 4 A WEEKS MONTHS B WEEKS MONTHS

FIGURE 9. A, Total DNA-P, equivalent to total cell number in the human forebrain from 10 ges-

tational weeks to 4 postnatal months, showing the two-phase characteristics of 4 prenatal cell mul-

tiplication. Figure 9B is a semilogarithmic plot of the same data as appears in Figure 9A to show

the sharp separation of the two phases at 18 gestational weeks. Regression lines with 9 5 % con-

fidence limits are added (12).

T h i s f inding ra ises n e w i s sues conce rn ing h y p o t h e s e s o f deve lop ing b ra in

vulnerabi l i ty . T h e r e can b e no ques t i on that mate rna l env i ronmen ta l factors

such as h e r undernu t r i t ion dur ing p r egnancy w o u l d no t b e g i n se r ious ly to re-

tard fetal g rowth unt i l the early th i rd t r imester . T h e s ame m a y b e so of m a n y

of the obs te t r ic pa tho log ies w h i c h affect p lacental suff iciency, so that in gen-

eral i t m a y wel l b e that fetal g rowth re tardat ion o f the k i n d w h i c h is due to a

res t r ic t ion of supp l ies to the fetus wi l l a lways spare the m u c h ear l ier neu rona l

mul t ip l ica t ion phase in h u m a n s . S u c h later res t r ic t ions will equa l ly cer ta inly

not spare the s u b s e q u e n t dendr i t ic g rowth o f the n e w l y arr ived n e u r o n s no r

the e s t ab l i shmen t o f the ne twork synapt ic connec t iv i ty ; and , as has a l ready

b e e n sa id , these m a y b e funct ional ly m o r e impor tan t p rocesses than the mere

a c c o m p l i s h m e n t of neurona l n u m b e r .

W A T E R

Jus t as in o ther deve lop ing t i s sues , the wa te r con ten t of the b ra in falls

dur ing deve lopmen t . Its fall i s s imi la r to the rec iprocal of the concomi t an t

increase in l ip ids and i s i l lustrated in F igure 10 .

T h e c l in ical impor t ance of F igure 10 is re lated to the con t roversy over

w h e t h e r o r not t rue cerebra l e d e m a can occur i n the h u m a n n e w b o r n b a b y .

Cer ta in ly it c anno t b e sat isfactori ly d i agnosed cl inical ly a l though it is often

p r e s u m e d in b a b i e s s h o w i n g var ious neuro log ica l s igns . Unfor tuna te ly the

DN

A-P

(m

mo

le)

I HUMAN FOREBRAIN

HUMAN FOREBRAIN

4 1 4 John Bobbing

HUMAN WHOLE BRAIN

90

80

B I R T H 2 4 6 8 A D U L T

AGE (years)

FIGURE 10. Water content of human brain during development (12).

pos tmor t em d iagnos i s is equal ly ques t i onab l e , largely due to the s teepness of

the fall s h o w n in the figure. N o pa thologis t can carry in h i s m i n d a m e m o r y

for such rapidly chang ing b ra in cons i s t ency at th is age ; and even i f he m e a -

sured b ra in wa te r he would find it difficult w i th th is degree of s t eepness to

p ronounce on a g iven b ra in ' s normal i ty : the var iance can b e so eas i ly in-

creased b y small errors in es t ima t ing per inata l age. Fur ther , the o ther aids to

pos tmor t em d iagnos i s in adult b ra in e d e m a are of little use in the n e w b o r n .

F la t ten ing of gyri w h e n the cons i s t ency is so w e t a n y w a y and swel l ing of the

forebra in wh i t e mat te r w h e n there is so little of it at th is age are bo th un-

helpful. Q u i t e a sens i t ive i ndex in exper imenta l e d e m a o f deve lop ing b ra in is

the altered sod ium:po t a s s ium rat io , and the ques t ion migh t b e explored

w h e t h e r an e s t ima t ion of these ca t ions in ce rebrosp ina l fluid migh t b e a useful

d iagnos t ic test in the l iv ing . T h e al tered rat io is p r e s u m a b l y a reflect ion of im-

pa i rmen t of homeos ta t i c p u m p m e c h a n i s m s for s o d i u m and po t a s s ium and

the sens i t iv i ty of the rat io is due to the b ra in levels m o v i n g in oppos i t e direc-

t ions dur ing asphyxia l b ra in edema .

HAZARDS OR NEURONAL MULTIPLICATION IN HUMANS

H o w can neurona l mul t ip l ica t ion b e th rea tened in the h u m a n fetus? W e can

p robab ly a s sume that such threats wil l have to b e subs tant ia l if they are to b e

impor tan t . Fur ther , s ince the neurona l n u m b e r is u l t imate ly heav i ly out-

w e i g h e d b y glial n u m b e r , even a subs tant ia l neurona l n u m b e r deficit wil l b e

WA

TE

R

(perc

en

t)

CO


difficult or i m p o s s i b l e to d i s t ingu i sh b y total cell " D N A c o u n t s " in the mature

b ra in and w o u l d on ly b e seen after ve ry l abor ious quan t i t a t ive h i s to logy . It

wou ld no t b e o b v i o u s on s tandard neuropa tho log ica l examina t i on .

T h e fo l lowing is an i m c o m p l e t e l ist of the k inds of adverse factors , m o s t of

t hem compara t ive ly u n c o m m o n , w h i c h m a y b e expec ted to defeat the h igh

degree of p ro tec t ion normal ly su r round ing the fetus dur ing the per iod of

h u m a n neu rona l mul t ip l ica t ion .

1. X- I r rad ia t ion and o ther rad ioac t ive exposure . It is r emarkab le that the

large n u m b e r s of cases of severe mic rocepha ly w i t h men ta l re tardat ion i n p o p -

ula t ions surv iv ing the a tomic b o m b s d ropped on H i r o s h i m a and Nagasak i

were near ly all exposed to the radioac t iv i ty at a c i r cumsc r ibed phase of fetal

life: b e t w e e n 10 and 18 ges ta t ional w e e k s (15) ; and a l though it m u s t b e ad-

mi t ted that th i s is p robab ly a per iod of impor tan t deve lopmen t a long m a n y

parameters o ther than neurona l cell mul t ip l ica t ion , the r e s e m b l a n c e b e t w e e n

the sens i t ive per iod of fetal exposure to severe a tomic i r radia t ion and the data

in F igure 9B is sufficiently s t r ik ing to ra i se the ques t i on of a causal rela-

t ionsh ip . T h e same que ry m u s t also, therefore , ar ise regard ing d iagnos t ic and

especia l ly therapeut ic X- i r rad ia t ion .

2. Vira l in fec t ions of the fetus, pr inc ipa l ly rubel la , are often p resen t and ef-

fect ive th roughou t th is t ime and m a y lead to mic rocepha ly .

3. C h r o m o s o m a l anomal i e s are obv ious ly present , w h e n they occur ,

t h roughou t th is pe r iod . The re has b e e n a sugges t ion (2) that two factors as-

socia ted w i t h c h r o m o s o m a l de r angemen t m a y lead to se r ious al terat ion of

neurona l cell n u m b e r : first, the e longa t ion of mi to t ic in terval and h e n c e the

s lowing of mi tos i s in cer ta in c h r o m o s o m e anomal i e s , toge ther wi th , s econd ,

the strictly chronolog ica l de te rmina t ion of neurob las t d i f ferent ia t ion at a cer-

ta in age , an even t w h i c h normal ly b r ings the phase of neurob las t mul t ip l ica-

t ion to an end . At least in theory these two factors could c o m b i n e to reduce

neurona l n u m b e r s , bu t it i s , of course , qu i t e u n k n o w n w h e t h e r such reduc-

t ion is ever s ignif icant .

4 . O t h e r congen i ta l anomal i e s are assoc ia ted w i th a var ie ty o f fetal g rowth

re tardat ion that , un l ike the "ma lnu t r i t i ona l " type , p r o b a b l y opera tes

th roughou t ges ta t ion and wil l therefore b e p resen t even dur ing the first and

second t r imes ters . I ndeed any such modif ica t ion of the who le " g r o w t h pro-

g r a m " m a y p o s s i b l y lead to s o m e reduc t ion in neurona l n u m b e r .

5. T h e occas iona l m o t h e r w i th h igh p l a sma levels o f pheny la l an ine or o ther

me tabo l i c error wi l l expose he r fetus to he r o w n d e r a n g e m e n t at this t ime . T h e

poor out look for such p regnanc i e s m a y b e related to the c o m m o n failure to in-

st i tute d ie tary control unt i l later in the 1 0 - 1 7 - w e e k per iod . In the p resen t state

of knowledge it w o u l d clearly b e w i s e to control ma te rna l pheny la l an ine levels

from an earl ier s tage of a p l anned p regnancy .

6. Mate rna l m e d i c a t i o n at th is t ime m u s t also b e cons ide red a pos s ib l e haz-

ard to d iv id ing neurob las t s , especia l ly p ro longed s teroid therapy w h i c h is

4 1 6 John Bobbing

k n o w n in exper imenta l an imals to reduce b ra in cell d iv i s ion at comparab le

s tages of deve lopment .

7. T h e ques t i on of m u c h "unc l a s s i f i ed" men ta l re tardat ion is ent i re ly open ,

and it m a y finally b e w o n d e r e d w h e t h e r its o r ig in m a y not b e found in th is

" m i d d l e " pe r iod of b ra in growth .

T h u s there are at least two pos s ib l e pe r iods of b ra in vu lnerab i l i ty to g rowth

res t r ic t ion: the first, and p robab ly less c o m m o n , w h i c h has ju s t b e e n dis-

cussed , related to the 1 0 - 1 7 - w e e k pe r iod of neurona l mul t ip l ica t ion ; and sec-

ond , the m u c h more r ecogn ized later pe r iod of the b ra in g rowth spurt dur ing

w h i c h the p rocesses at r isk p r o b a b l y inc lude dendr i t ic a rbor iza t ion , es tab l i sh-

m e n t of synapt ic connec t iv i ty , and mye l ina t ion .

C E L L S I Z E A N D C E L L N U M B E R

A n ext remely useful concep t re la t ing to ca tch-up poten t ia l fo l lowing g rowth

re tardat ion has b e e n enunc i a t ed b y W i n i c k and N o b l e (18) . It is b a s e d on the

genera l t ruth that all t i s sues pass th rough two over lapping phases of growth:

an early hyperplas t ic phase of cell mul t ip l ica t ion and a later hyper t roph ic one

of g rowth in cell s ize . W i n i c k and N o b l e (18) sugges ted that g rowth res t r ic t ion

dur ing the first phase led to an i r recoverable reduct ion in cell n u m b e r , bu t that

reduct ion in cell s ize caused b y res t r ic t ion dur ing the second phase was

recoverable . The re s e e m s lit t le doub t abou t the genera l appl icabi l i ty of th is

idea , a l though it m a y poss ib ly have b e e n too uncr i t ical ly appl ied to such

t i ssues as ad ipose t i ssue dur ing b a b y h o o d . Cer ta in ly it i s difficult to apply to

the b ra in , largely due to th i s o rgan ' s ex t reme his to logica l he te rogene i ty . It is

no t clear , for example , w h a t cell s ize m e a n s to a neu ron . D o e s it apply to the

cell b o d y ? A n d if so does it mat te r? O r does it apply to the w h o l e cell inc lud ing

b o t h i ts complex dendr i t ic t ree and its s o m e t i m e s e n o r m o u s l y long axon? It

p r e sumab ly does no t apply to axonal length , so w o u l d axonal th ickness b e in-

volved? T h e usual ind ices for m e a s u r e m e n t o f cell s ize , the amoun t of pro te in

per uni t quant i ty of D N A , are also mani fes t ly unrea l in the b ra in w h e r e the

(axonal) pro te in in any g iven area such as the s t em is in no w a y anatomica l ly

related to m u c h of the D N A of the s a m e area, bu t to the re levant cell b o d i e s in

another part of the b ra in a l together . N o r can the exc lus ion of all bu t cell

n u m b e r from p e r m a n e n t reduc t ion , as sugges ted b y a strict in terpre ta t ion of

the W i n i c k and N o b l e (18) hypo thes i s , b e he ld to account for such demons t ra -

b le u l t imate deficits in the b ra in as those in noncel lu lar mye l in or synapt ic

connec t iv i ty . It i s , of course , ve ry l ikely that the i r concep t m a y apply to glial

cell n u m b e r and glial cell s ize in ma lnou r i shed h u m a n infants . I ndeed the def-

ici t in b ra in cell n u m b e r in such few s p e c i m e n s as have b e e n e x a m i n e d from

underpr iv i l eged c o m m u n i t i e s is p robab ly exclus ive ly a glial deficit due part ly

to the later appearance of glial cells and part ly to the compara t ive ly late t iming

of the nut r i t ional g rowth res t r ic t ion in these chi ldren .


T h e h is to logica l he t e rogene i ty of the b ra in and sp ina l cord p r o b a b l y de-

pr ives the s imple cell n u m b e r - c e l l s ize concep t of m u c h of the usefulness for

w h i c h it ha s b e c o m e r e n o w n e d in such h o m o g e n e o u s t i s sues as l iver and

musc le .

GENERAL COMMENTS ON BRAIN GROWTH SPURT VULNERABILITY

So far vu lne rab i l i ty has b e e n d i scussed a lmost en t i re ly in re la t ion to nutr i -

t ional g rowth res t r ic t ion. O n e ques t i on w h i c h ar ises is w h e t h e r lack of any

part icular nu t r i en t is m o r e l ikely than any o ther to p roduce the u l t imate defi-

cits and d is tor t ions ou t l ined earl ier ; or w h e t h e r it is l ikely to b e the mere fact

o f g rowth res t r ic t ion at such a c o m p l e x and rap id pe r iod of g rowth . T h e

chances s e e m very h i g h that it is the latter.

If th i s b e t rue, there can b e a good deal of reconc i l i a t ion b e t w e e n the

var ious r e sea rch ing fac t ions , each wi th its o w n favorite nut r i t iona l culpri t af-

fect ing b ra in g rowth . W h e t h e r the die tary deficit b e one of me re quan t i ty of

food, or one specifically of pro te in , a par t icular a m i n o ac id , a range of e ssen-

tial fatty ac ids , i ron , z inc , or folic ac id , it is an at tract ive uni fy ing h y p o t h e s i s

that the final c o m m o n pa th feature bea r ing on the deve lop ing b ra in i s the

g rowth res t r ic t ion that all these d ie tary deficits m a y produce .

S u c h a un i fy ing h y p o t h e s i s fur thermore admi t s in to the e t io logy of poor

b r a in g rowth all the nonnu t r i t iona l or secondar i ly nut r i t ional g rowth re tarding

cond i t ions , p rov ided they opera te on the g rowth p rogram for a subs tan t ia l

part of the b ra in g rowth spurt per iod .

It is poss ib l e , in favor of th is un i fy ing h y p o t h e s i s , that nu t r i t ion i s t s have

great ly exaggera ted the d e p e n d e n c e of the g rowing b ra in on s ingle art icles of

diet . As has b e e n e m p h a s i z e d before , the b r a in has a r emarkab le facil i ty for

abs t rac t ing i ts n e e d s from the c i rcula t ing pool of precursor subs t ances , often

qui te normal ly aga ins t a large unfavorable gradient . M o r e o v e r , the b r a in ' s

n e e d s are quan t i t a t ive ly ve ry smal l c o m p a r e d w i t h the s ize of the total pool ,

and it s e e m s m o s t un l ike ly that i ts o w n growth wi l l often b e l imi ted b y the

avai labi l i ty to it of any s ingle nu t r imen t . H o w e v e r , the b ra in is an in tegral

part o f the w h o l e somat ic growth plan, and it s e e m s a m u c h m o r e acceptab le

concep t that it is secondar i ly at r isk w h e n e v e r the w h o l e p lan is res t r ic ted

dur ing the ex t remely del icate per iod of i ts g rowth spurt . In th is w a y the c o m -

parat ively early t im ing of this pe r iod , c o m p a r e d w i t h that in o ther t i s sues ,

m a y b e the m o s t impor t an t factor to cons ide r .

SMALL-FOR-DATES BABIES, MALNOURISHED OR OTHERWISE

W h e r e these b a b i e s are g rowth re tarded on ly dur ing the last t r imes ter ,

inc lud ing those re tarded b y materna l s m o k i n g , and w h e r e t hey e x h i b i t good

4 1 8 John Bobbing

ca tch-up growth on b e i n g l ibera ted b y b i r th from the i r restraint , the general

vu lnerab le per iod hypo thes i s wou ld predic t n o very dele ter ious effect on final

ou t come . W h e r e , h o w e v e r , such growth- re ta rded b a b i e s are bo rn into an ex-

ternal e n v i r o n m e n t (or in to any con t inu ing restraint pa thology) w h i c h leads to

further g rowth res t r ic t ion dur ing the first two pos tnata l years , then poor b ra in

growth m a y b e expec ted .

T h u s , for example , it m a y b e unprof i table for ep idemio log i s t s to look into

the intel lectual ou t come s imply of " smal l - fo r -da tes" b a b i e s . Ra the r a group

should b e separate ly and part icular ly cons ide red in w h o m a major part of the

bra in g rowth spurt pe r iod w a s b l anke t ed b y growth res t r ic t ion. S u c h restr ic-

t ions wou ld inc lude (see be low) res t r ic t ions on s t imulus of an emot iona l or

" in te l l ec tua l " nature .

B y a s imi lar r ea son ing , it could wel l b e mi s l ead ing to look for d i m i n i s h e d

intel lectual per formance in all ch i ldren underfed at some t ime dur ing thei r in-

fancy. T h e key to the p r o b l e m wil l a lmost cer ta in ly and once aga in involve the

three " u n i t i e s " of deve lopmenta l undernu t r i t ion m e n t i o n e d earl ier: they

should have b e e n g rowth re tarded for a subs tant ia l part of the w h o l e vulnera-

ble per iod , to a g iven degree o f sever i ty , bu t above all at the correct ch rono-

logical age before they are expec ted to s h o w ul t imate deficit. A d d to these

var iab les the e n o r m o u s and we l l -documen ted nonnut r i t iona l factors in the ex-

ternal e n v i r o n m e n t w h i c h m a y greatly add to or c o m p e n s a t e for the nut r i t ional

difficulty, and it wil l no t b e surpr i s ing that the l i terature a b o u n d s w i th con-

fusing and confl ic t ing ev idence from differently se lected samples in different

parts of the wor ld .

M A T E R N A L S L I M M I N G

At tempts to reduce mate rna l w e i g h t or materna l we igh t ga in dur ing preg-

n a n c y b y dietary m e a n s are w i d e s p r e a d in m a n y affluent soc ie t ies . T h e obste t -

ric mot ives are var ious and confused . For s o m e obs te t r i c ians the extra we igh t

gain in toxemic cond i t i ons , w h i c h is ma in ly due to abnorma l wa te r re ten t ion ,

is mi s t aken ly equa ted wi th that usual ly due to overea t ing ; and it i s wrong ly

supposed that it can b e " c o u n t e r e d " b y reduc ing food in take in a m o t h e r

w h o s e fetus m a y b e already suffering from an insufficient supply. For o thers ,

the oppor tun i ty afforded b y the mo the r ' s " c a p t i v i t y " dur ing p regnancy for

cur ing her of all he r v ices is i r res is t ib le . T h e s m o k i n g m o t h e r is adv i sed to

s top, and th is s eems sens ib l e in the in teres t of the fetus; bu t the ove rwe igh t

m o t h e r i s , for s imi lar sorts of r easons , put on a reduc ing diet at the very t ime

she needs to b e well fed, and this is p robab ly to b e deplored. In s o m e affluent

cultures it is even still cus tomary to reduce mate rna l in take w i th the express

in ten t ion of r educ ing fetal g rowth so that del ivery m a y b e more comfor table ,

and th is mus t b e r ep rehens ib l e . In underpr iv i l eged c o m m u n i t i e s w h e r e the c o m m o n e s t cause of s lowed in-


t rauter ine g rowth is w i d e s p r e a d materna l malnu t r i t ion , it is easy to s h o w that

adequa te mate rna l nu t r i t ion wil l res tore " n o r m a l " b i r th w e i g h t in the same

small m o t h e r w h o has a l ready b o r n several low b i r th w e i g h t b a b i e s . E thn ic

inf luences o n b i r th w e i g h t are therefore far from exclus ive and should not b e

accepted as major ones unt i l s h o w n to pers is t in the p re sence of an adequa te

nut r i t ional e n v i r o n m e n t dur ing p regnancy .

C O N C L U S I O N S

T h e case has b e e n argued for a par t icular k ind of vu lne rab i l i ty of the devel-

op ing b ra in to g rowth res t r ic t ion dur ing its g rowth spurt pe r iod ; and for a

specia l var iant of th i s , w h i c h is l ikely to b e m u c h less c o m m o n , bu t pe rhaps

more severe , dur ing a separa te , still ear l ier s tage of b r a in g rowth w h e n

neurona l mul t ip l i ca t ion is occurr ing.

N o n e of the demons t r ab l e phys ica l c o n s e q u e n c e s of g rowth res t r ic t ion at

this t ime w o u l d b e of any s igni f icance i f they had n o behav io ra l or funct ional

c o n s e q u e n c e s ; bu t ev idence for the latter has accumula ted and is n o w compe l -

l ing (14) . In spi te of the academic hand i cap of our i gnorance of the phys ica l

b a s i s o f h i g h e r men ta l funct ion , there s eems little doub t that it does have a

cons ide rab le phys ica l b a s i s in the b ra in and that i ts d e v e l o p m e n t can b e s ig-

nif icantly spo i led b y poor env i ronmen ta l cond i t i ons dur ing cer ta in g rowth

per iods . N o n e of th is den ie s the supreme con t r ibu t ion of the nonnu t r i t iona l ,

less t ang ib le e n v i r o n m e n t of the g r o w i n g fetus and b a b y w i t h i n the family,

the relat ive impor t ance of w h i c h is n o w b e g i n n i n g even to b e m e a s u r e d (16) .

A c h i e v e m e n t m a y b e cons ide red to b e the a lgebra ic s u m of pos i t ives and neg -

at ives in the total deve lopmenta l env i ronmen t . T h e demons t r a t ion that the

h u m a n b ra in g rowth spurt is m u c h more pos tnata l than w a s formerly though t

creates a n e w oppor tun i ty to ensure one impor tan t pos i t ive in the calcula t ion,

b y act ively p romot ing good bod i ly g rowth at the t ime w h e n th is m o s t impor -

tant organ is pa s s ing th rough its o w n vu lnerab le per iod of g rowth .


I am grateful to the Medical Research Council and the National Fund for Research into Crip-pling Diseases (both of Great Britian) for supporting this work; and to my colleagues Dr. B. P. F. Adlard, Miss Jean Sands, and Dr. J . L. Smart for their continuing collaboration. Figures 2-10 are reproduced by permission of the editors, Archives of Disease in Childhood.

R E F E R E N C E S

1. Adlard, B . P. F . , and Dobbing, J . Permanent changes in the activity and subcellular distribu-tion of acetylcholinesterase and lactate dehydrogenase in adult rat cerebellum after X-irradia-tion in infancy. Exp. Neurol, 1972, 35: 547-550.

420 John Bobbing

2. Barlow, P. The influence of inactive chromosomes on human development. Humangenetik, 1973, 17: 105-136.

3. Davison, A. N. , and Dobbing, J . The developing brain. In: Applied N euro chemistry. (A. N. Davison and J . Dobbing, Eds.). Blackwell, Oxford, 1968: 253-286.

4. Davison, A. N., and Peters, A. Myelination. Thomas, Springfield, Illinois, 1970. 5. Dickerson, J . W. T., and Dobbing, J . Prenatal and postnatal growth and development of the

central nervous system of the pig. Proc. R. Soc. Lond. [Biol.], 1967, 166: 384-395. 6. Dobbing, J . Undernutrition and the developing brain. In: Developmental Neurobiology. (W. A.

Himwich, Ed.) , Thomas, Springfield, Illinois, 1970: 260-261. 7. Dobbing, J . Undernutrition and the developing brain: The relevance of animal models to the

human problem. Am. J. Dis. Child., 1970, 120: 411-115.

8. Dobbing, J . , Hopewell, J . W., and Lynch, A. Vulnerability of developing brain. VII. Perma-nent deficit of neurons in cerebral and cerebellar cortex following early mild undernutrition. Exp. Neurol, 1971, 32: 4 3 9 ^ 4 7 .

9. Dobbing, J . , and Sands, J . Growth and development of the brain and spinal cord of the guinea pig. Brain Res., 1970, 17: 115-123.

10. Dobbing, J . , and Sands, J . Timing of neuroblast multiplication in developing human brain. Nature (Lond.), 1970, 226: 639-640.

11. Dobbing, J . , and Sands, J . Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth-spurt. Biol Neonate, 1971, 19: 363-378.

12. Dobbing, J . , and Sands, J. Quantitative growth and development of human brain. Arch. Dis. Childhood, 1973, 48: 757-767.

13. Dobbing, J . , and Smart, J . L. Early undernutrition, brain development and behaviour. In: Clinics in Developmental Medicine. No. 47. (S. A. Barnett, Ed.). Heinemann, London, 1973: 16-36.

14. Hertzig, M. E . , Birch, H. G., Richardson, S. A. , and Tizard, } . Intellectual levels of school children severely malnourished during the first two years of life. Pediatrics, 1972, 49: 814-824.

15. Miller, R. W. , and Blot, W. J . Small head size after in utero exposure to atomic radiation. Lancet, 1972, 2: 784-787.

16. Richardson, S. A. Ecology of malnutrition: Non-nutritional factors influencing intellectual and behavioural development. Pan American Health Organization, Scientific Publication, 1972, No. 251: 101-110 .

17. Winick, M. Changes in nucleic acid and protein content of the human brain during growth. Pediat. Res., 1968, 2: 352-355.

18. Winick, M., and Noble, A. Cellular response in rats during malnutrition at various ages. / . Nutr., 1966, 89: 300-306.

15 The Futility of Comparative IQ Research

JOHN GARCIA Department of Psychology,



T h i s confe rence i s en t i t led " B r a i n M e c h a n i s m s in M e n t a l R e t a r d a t i o n , " bu t

paradoxical ly there has b e e n little d i s cus s ion of o n e centra l i s sue . P r e s u m a b l y

the t e rm " m e n t a l r e ta rda t ion" refers to a r educed " m e n t a l a g e " or M A , the

t roub le some numera to r in that w e l l - k n o w n equa t i on M A / C A = IQ w h e r e C A

is the chronolog ica l age and IQ is the in te l l igence quo t i en t o f the sub jec t

unde r cons ide ra t ion . T h i s equa t i on has the e legant s impl ic i ty o f the E ins t e in -

i an £ = M C 2 and i ts soc iopol i t ica l impl i ca t ions are a b o u t as exp los ive as a nu-

clear reac t ion . Neve r the l e s s , w e m u s t deal w i t h the concep t s o f men ta l age

and I Q and the ope ra t ions b y w h i c h t h e y are m e a s u r e d , i f w e are to under -

s tand the m e a n i n g of the te rm " m e n t a l r e t a rda t ion" and its s igni f icance or lack

of s igni f icance in our research p rograms .

To avoid ano the r end less con t roversy w e m u s t e s c h e w the b road i s sues such

as the o n e ra i sed b y J e n s e n at the very onse t o f h i s 1969 pape r in the Harvard

Review (15 ) , i . e . , " C o m p e n s a t o r y educa t ion has b e e n t r ied and i t has apparent ly

f a i l ed . " T h e p rovoca t ive form of the s t a t emen t leads to d i spu tes w h i c h canno t

b e dec ided b y scient i f ic i nqu i ry . S u b s t i t u t i o n of any ca tch ph ra se , such as N a -

t ional De fense , Pol ice Pro tec t ion , A u t o m o t i v e Transpor ta t ion , Env i ronmen ta l

Conse rva t ion , for C o m p e n s a t o r y Educa t ion w o u l d start an endless deba te on

any topic . T o reach a dec i s ion , w e n e e d to ra ise m o r e m u n d a n e q u e s t i o n s ,

such as: Specif ical ly h o w is in te l l igence m e a s u r e d ? H o w is an I Q test m a d e up?

W h a t sort o f r e s p o n s e s inc rease (or decrease) the I Q tes t scores?

421

422 John Garcia

OPERATIONAL DEFINITION OF INTELLIGENCE

P. W . B r i d g m a n (4) decreed that w h e n cont roversy ar ises c o n c e r n i n g a con-

cept , scientif ic clarif ication can b e ach ieved only b y examina t ion of the fun-

damenta l opera t ions u p o n w h i c h that concep t i s b a s e d . J e n s e n ' s r e v i e w of the

b io log ica l and socia l de t e rminan t s of in te l l igence (15) r e n e w e d a confus ing

debate w h i c h can b e clarified on ly i f the a priori a s sumpt ions w h i c h under l ie

the m e a s u r e m e n t o f " i n t e l l i g e n c e " are so e x a m i n e d . M o s t o f the po in t s w e

w i s h to m a k e he re have b e e n m a d e in o ther contex ts and , i ndeed , a close

examina t ion of J e n s e n ' s t reat ise and the c r i t iques and rebut ta ls that fol lowed

wel l reveal the i s sues at least a l luded to in the d iscurs ive sec t ion of the papers .

Howeve r , the m a i n effect of th i s con t roversy is to g ive " i n t e l l i g e n c e " a con-

ceptual ex i s tence i n d e p e n d e n t of the specific con ten t o f the tests and the real

l imi ta t ions of men ta l m e a s u r e m e n t t e chn iques . A s a result , readers w h o are

not thoroughly famil iar w i t h the a s sumpt ions and opera t ions of men ta l m e a -

su remen t are at a loss as to h o w to evaluate the data p resen ted b y e i ther the

hered i ta r ians or the env i ronmenta l i s t s .

T h e fundamenta l ques t ions concern the very na ture of " i n t e l l i g e n c e " and

h o w it is measu red . It is often a rgued that " t rue i n t e l l i gence" cannot b e

measu red b y the bes t of tests bu t , i f this is t rue, then the i s sue is w i thou t

empir ica l con ten t and n e e d no t conce rn us from a scientif ic po in t of v i ew .

M o r e often, as J e n s e n po in t s out , in te l l igence is def ined as " w h a t the in-

te l l igence test m e a s u r e s . " Le t us accept th is as the first s tep and e x a m i n e wha t

in te l l igence tests measu re and h o w they m e a s u r e it.

Robe r t C. T ryon ' s " T h e o r y o f Psycholog ica l C o m p o n e n t s " (27) is an ex-

cellent po in t of depar ture . H e po in t s out that the no t ion of genera l in te l l igence

(g) rests u p o n comple te ly arbi t rary a s sumpt ions of men ta l testers w h o select

the tests and analyze t h e m for " f ac to r s . " J e n s e n also testifies to the e lus ive na-

ture o f g s ta t ing, " A s tests c h a n g e , the nature of g wil l also change , and a test

w h i c h is loaded , say .50 ong w h e n factor ana lyzed a m o n g one se t of tes ts , m a y

have a load ing of .20 or . 80 , or s o m e o ther va lue , w h e n factor analyzed a m o n g

other sets of t e s t s . " H e goes on to ci te h i s o w n ev idence to s h o w that no

mat te r h o w uni ta ry a test m a y appear to b e , it i s a lways poss ib l e to fract ionate

the var iance in the scores in to smal ler subfactors .* D e p e n d i n g u p o n the predi-

lec t ions of the factor analyst , in te l l igence can b e concep tua l i zed as a s ingle

factor exer t ing i ts inf luence in all p r o b l e m s , as seven p r imary intel lectual ab i l i -

t ies each wi th a res t r ic ted sphe re of in f luence , or as a vir tual ly infini te n u m b e r

of assoc ia t ive b o n d s wi th on ly na r row specific inf luence upon per formance .

* In all fairness we must admit that Jensen seems to be expressing another point of view on page 9, when he writes "Despite numerous theoretical attacks on Spearman's basic notion of a general factor, g has stood like a rock of Gibraltar in psychometrics. . . . " This apparent contradiction is quickly resolved when one considers how stone masons cut big rocks into little ones or how sculptors can shape any rock to their own ends.

15. The Futility of Comparative IQ Research 423

The re is no i n d e p e n d e n t w a y to dec ide w h i c h of these no t ions is the m o s t

accurate .

H o w e v e r , T r y o n po in t s out that the actual opera t ions of men ta l tes ters is in

accordance w i t h the latter a s s u m p t i o n , for in genera l , they use a large n u m b e r

of d ispara te test i t ems often a r ranged in sub tes t s . T o avoid excess ive dupl ica-

t ion they seek i t e m s and sub tes t s w h i c h y ie ld l ow in tercorre la t ions w i t h i n the

test ba t te ry bu t a r ea sonab le corre la t ion w i t h the total score on the bat tery .

T h e y act as i f the re w e r e a large n u m b e r of i n d e p e n d e n t en t i t i es to b e sam-

pled.

In any case , there exis ts vir tual ly an infini te n u m b e r of i t e m s , i . e . , tasks or

p rob l ems w h i c h can b e wr i t ten and used for m e a s u r i n g in te l l igence . T h e

menta l tes ter m u s t select a r easonab le sample from th is m y r i a d of i t ems . H e

mus t m a k e dec i s ions conce rn ing (a) the l anguage and the i d i o m in w h i c h they

are phrased , (b) the cultural and social d o m a i n from w h i c h they are d rawn,

and (c) the sample of peop le on w h i c h they are to b e tes ted . In m a k i n g these

pragmat ic , a priori dec i s ions he defines the concep t " i n t e l l i g e n c e " as m e a s -

ured b y h i s test . It m a y b e verba l , quan t i t a t ive , or m e c h a n i c a l in te l l igence ;

it m a y b e W h i t e , B lack , or C h i c a n o in te l l igence ; or it m a y b e female or male

in te l l igence d e p e n d i n g u p o n h o w he c h o o s e s to wr i te h i s i t ems , on w h o m

h e c h o o s e s to val idate h i s i t e m s , and w h a t va lue j u d g m e n t h e p laces u p o n

the w i d e var ie ty of h u m a n ta lents . B u t i t is no t a genera l in te l l igence scale

w h i c h can b e u sed to compare b iosoc ia l g roups .

THE FAILURE OF THE HEREDITARIAN APPROACH TO MEASURE INTELLIGENCE

Histor ica l ly , the first scientif ic a t tempt to dev i se in te l l igence sprang from

D a r w i n ' s no t i ons of adapta t ion and S p e n c e r ' s no t ion of " surv iva l of the fit-

t e s t . " D a r w i n ' s cous in , Franc i s Gal ton , or iginal ly des igned test i t ems to assess

adapt ive a t t r ibutes and potent ia l i t ies of ind iv idua l h u m a n s dea l ing w i t h

s imple labora tory tes ts . T h u s from its very incep t ion menta l t es t ing had a

hered i ta ry b i a s , for Ga l ton w a s c o n v i n c e d of the gene t ic de t e rmina t ion of in -

te l l igence , as e v i d e n c e d b y "Hered i t a ry G e n i u s " (8) p u b l i s h e d in 1869 . Never -

the less , he w a s also s t rongly in f luenced b y the Br i t i sh empi r ica l t radi t ion that

the m i n d w a s totally d e p e n d e n t u p o n env i ronmen ta l inpu t s . T h u s h e , and

later J a m e s M c K e e n Cattel l (6) , concent ra ted u p o n the capaci ty o f ind iv idua l s

to deal w i th s imple s ignals qu ick ly and correct ly and cons t ruc ted tes ts of sen-

sory acui ty , perceptua l j u d g m e n t s , and reac t ion t ime . T h i s approach s e e m e d

to have the v i r tue of ref lect ing the psychob io log ica l po ten t ia l of an ind iv idua l ,

i n d e p e n d e n t of a cultural ly def ined socia l n i c h e . H o w e v e r , the very no t ion of

deve lop ing in te l l igence test i t ems w h i c h do not reflect the effects o f differen-

tial exposure to expe r i ence and lea rn ing p roved to b e i l lusory, s ince acqu i s i -

424 John Garcia

t ion of in format ion and c o m p l e x skills is w h a t mos t people cons ide red in-

te l l igence to b e . In the final ana lys i s , h o w e v e r , in te l l igence as defined b y

i t ems selected from the an th ropomet r i c labora tory b y the hered i ta r ians were

d e e m e d a fai lure, because they were no t pred ic t ive of success in the a c a d e m y

or the marke t p lace , h o w e v e r wel l t hey m a y have fared in the p lay ing field or

the ba l l room.

CORE CURRICULUM AND STANDARD PUPILS AS s ine qua n o n FOR IQ

Envi ronmenta l i s t s , i . e . , the e s t ab l i shed school au thor i t ies , we re in effect

a l lowed to define " i n t e l l i g e n c e " in the " succes s fu l " approach to men ta l tes t ing

as in i t ia ted b y Alfred B i n e t in France and carr ied on in th is count ry b y L e w i s

T e r m a n and M a u d Merr i l l at S tanford Un ive r s i t y (25) . T h e S tanford-Bine t test ,

b y v i r tue of the m o n u m e n t a l a m o u n t o f appl ied research , b e c a m e the defini-

t ive measu re of IQ agains t w h i c h mos t o ther IQ tests are evaluated. It is

impera t ive that w e e x a m i n e w h a t k ind o f in te l l igence th is test measu res .

T h e first pract ical s t ra tagem dev i sed b y B i n e t and e m p l o y e d b y T e r m a n was

to select i t ems predic t ive of progress in school . Fur the rmore , the i t ems re-

flected on ly that s e g m e n t of the cur r icu lum w h i c h the school au thor i t ies

d e e m e d to b e impor tan t , n a m e l y the read ing , wr i t ing , and ar i thmet ic of the

" c o r e " curr iculum. T h e effect o f th i s arbi t rary dec i s ion w a s to exclude i t ems

drawn from the " s p e c i a l " c lasses such as the art s tud io , mus ica l conserva tory ,

and the m a c h i n e s h o p from the IQ test thus re legat ing these ab i l i t i es forever

in to the sphe re of " s p e c i a l " ab i l i t i es , and in effect r educ ing the m e a s u r e d in -

te l l igence of people w h o s e skills and voca t ions lay in these spheres . Ar t i s ts ,

m u s i c i a n s , and m a c h i n i s t s , l ike Black A m e r i c a n s , n e e d no t b e in te l l igent ; they

only n e e d " s p e c i a l i z e d " ta lents to b e successful . Y e t in j u d g i n g the intel lectual

level of prehis tor ic m a n , cave pa in t ings , mus ica l i n s t rumen t s , and handicraf ts

w e r e accepted w i thou t ques t i on as marks o f advanced in te l l igence . A s i l lus-

trated in F igure 1, th i s p rocess has g iven us the popular concep t ion of the

h u m a n m i n d .

T h e second pract ical s t ra tagem dev i sed b y the men ta l testers w a s the " s t an-

dardiza t ion g r o u p . " T h e y r ecogn ized that A m e r i c a n school ch i ldren at the

turn of the cen tury formed a mot ley group w i t h paren ts o f different social ,

e thn ic , and language g roup ings . S i n c e one could on ly b e sure that ch i ldren of

wh i t e Eng l i sh - speak ing paren ts had the full oppor tun i ty to b e c o m e conversan t

in the language and p rob l ems used in the core cur r icu lum, these ch i ldren were

selected as the sample on w h i c h the test i t ems were val ida ted or s tandardized .

Ch i ld ren from o ther b io soc i a l g roups w e r e exc luded , m a k i n g the or ig ina l

S tanford-Bine t a midd le class " A n g l o " I Q , to use that loose b iosoc ia l ca tegory

wi thou t pre judice . It shou ld no t b e surpr i s ing that vocabu la ry i t ems b a s e d on

15. The Futility of Comparative 1Q Research 425

Special Talent

FIGURE 1. The structure of the turn-of-the-century school curriculum, the construction and

labeling of tests, and the popular conception of the child's mind reflect one and the same cultural

phenomenon. This myth assumes a central role for general intelligence and peripheral roles for

special (motor) abilities and special (sensory) talents without independent neurophysiological

verification. The same prejudice tracks the minority child into special courses deemed appropri-

ate for his inherited characteristics without regard for the evidence necessary for genetic infer-

ence.

c o m m o n Eng l i sh usage p roved to b e the m o s t efficient sub tes t in th i s IQ test.

To use th is test and o the r group tests b a s e d pr imar i ly on Ang lo vocabu la ry on

o ther m ino r i t y g roups for compara t ive pu rposes , o n e m u s t m a k e the a s s u m p -

t ion that all g roups have h a d equa l oppor tun i ty and mot iva t ion to learn the

same words . Need le s s to say , these a s sumpt ions invar iab ly lead to con-

troversy.

T h e thi rd pract ical s t ra tagem e m p l o y e d b y the men ta l tes ters w a s to wr i te

i t ems de s igned to y ie ld a direct m o n o t o n i c re la t ionsh ip to i nc reas ing age ,

w h i c h is to say, that an i t em m u s t b e passed b y progress ive ly larger n u m b e r s

of o lder ch i ldren . N o w m a n y i t ems have an inverse or U - s h a p e d funct ion. For

example , " B l a c k is a color ; t rue or f a l se?" is apt to b e a difficult and confus ing

i t em for very y o u n g ch i ldren w h o have no t mas t e r ed read ing or for col lege

s tudents w h o have mas te red the d i s t inc t ion b e t w e e n h u e and b r igh tnes s .

Grade school ch i ld ren , famil iar w i th the crayon b o x , wou ld have no difficulty

in m a r k i n g the i t em " t r u e . " S u c h an i t em wou ld have to b e re jec ted or scored

in several ways in order to force a direct m o n o t o n i c funct ion wi th age n e c e s -

sary for the m e a s u r e m e n t of in te l l igence .

It is m o s t ins t ruc t ive to e x a m i n e i t ems a n d the scor ing gu ide l ines in the

manua l for the S tanford-Bine t . The re o n e can see h o w IQ scores are genera ted .

ITEM SELECTION AND THE CRITERION GROUP

426 John Garcia

T o paraphrase a few examples , a ch i ld m i g h t b e asked , " W h a t w o u l d y o u do if

y o u lost s o m e t h i n g that b e l o n g e d to your f r i end?" T h e key ins t ructs us that the

correct answer mus t invo lve an apology or res t i tu t ion or bo th . T h i s i s wha t

midd le class ch i ld ren are taught and this is the answer w h i c h increases in

f r equency w i t h age. All o ther r e sponses , inc lud ing an a t tempt to deal w i th the

f r iend 's fee l ings , n o mat te r h o w insightful , on ly reduce the sub jec t ' s I Q score .

Not all the correct answers are so in tu i t ive ly s imple . For example , the subjec t

migh t b e asked " H o w are loud and soft a l i k e ? " T h e r e sponse , " B o t h affect the

ea r , " is scored correct . O n the o ther h a n d , " Y o u can hear bo th of t h e m " is

incorrect . A t ano ther poin t , a kni fe can b e e i ther " s h a r p " or " v e r y sha rp , "

howeve r , a mi le can only b e " l o n g . " T h e answer " too l o n g " reduces the sub -

jec t ' s m e a s u r e d I Q .

T h e correct an swer m a y appear to b e arbi t rary to anyone w h o be l i eves that

an I Q test should direct ly measu re r ea son ing capaci ty o r inte l lectual power ,

bu t the IQ test mere ly de t e rmines w h e t h e r the ch i ld ' s a n s w e r r e s e m b l e s the

answer of the average u rban A n g l o chi ld of h i s age . If it r e s emb le s that of an

older ch i ld , he is super ior in I Q . T h i s general t e c h n i q u e of compar ing the s u b -

jec t ' s answer to the mos t f requent answer g iven b y a cr i ter ion group is

employed in m a n y tes ts . Mul t iphas i c psychia t r ic tests do no t m e a s u r e thought

d i s tu rbance to ach ieve thei r d iagnos t ic e n d s , they mere ly compare the sub -

jec t ' s answers wi th that of groups d i agnosed as s ch izophren ic , m a n i c , para-

no id , etc. Voca t iona l tes ts do no t measu re the sub jec t ' s capaci ty or even inter-

est in a voca t ion or p rofess ion , they mere ly ma tch h i s an swer to those g iven

b y the e s t ab l i shed m e m b e r s o f the voca t ion or profess ion . A n y i t e m , w h e t h e r

it deals wi th va lues , h o b b i e s , appet i tes , or wha teve r , is accep tab le j u s t as long

as it d i s t i ngu i shes one in san i ty f rom ano ther , or one profess ion from the

o thers . O f course , if the m e m b e r s of a g iven profess ion h a p p e n to b e of

another sex or e thn ic g roup , the sub jec t ' s chances to b e counse l led in to that

profess ion are great ly r educed , no t necessa r i ly b e c a u s e h e has n o in teres t o r

potent ia l in the profess ion , bu t b e c a u s e he does not r e spond to i r relevant

i t ems in the same w a y that the profess ional does (1) .

W H Y N O T M E A S U R E L E A R N I N G D I R E C T L Y ?

S i n c e p rev ious exposure to the IQ con ten t and the tes t ing s i tua t ion is such a

crucial var iab le in IQ per fo rmance , o n e is inc l ined to ask " W h a t h a p p e n s

w h e n these pract ice effects are con t ro l l ed?" J e n s e n prov ides s o m e ev idence in

h i s ra ther r emarkab le r ev iew. H e po in t s out that in conven t iona l I Q tes ts , the

ch i ld ' s per formance depends u p o n w h a t he has learned at h o m e or e l sewhere

before h e c o m e s in to take the test , and that i t is pos s ib l e to dev ise i t ems that

s h o w " h o w fast a chi ld can learn s o m e t h i n g n e w and unfami l ia r r ight in the

test s i tua t ion . " C o m m e n t i n g on such per formance u p o n var ious tasks (e .g . ,

m e m o r y for digi ts p resen ted a loud, l ea rn ing l ists of famil iar ob jec t s , associa t -


ing pairs o f ob j ec t s , recal l ing n a m e s or ob jec t s p re sen ted r andomly) , J e n s e n

wr i t e s , c i t ing a hal f dozen of h i s o w n pub l i ca t ions , " . . . l ower class ch i ld ren ,

w h e t h e r w h i t e , neg ro or M e x i c a n - A m e r i c a n , per form as wel l on these direct

l ea rn ing tests as do midd le -c lass ch i ldren . L o w e r class ch i ld ren in the I Q range

of abou t 60 to 80 do marked ly be t t e r than midd le class ch i ld ren w h o are in th is

range of I Q . "

H o w e v e r , J e n s e n does no t go on to exploi t a n e w l ine of r ea son ing . Ins tead

he deprec ia tes h i s o w n lea rn ing s tudies for the inc red ib le r eason that the data

have a l ow corre la t ion w i t h IQ w h i c h , of course , is p rec ise ly w h a t y o u wou ld

expec t if IQ per fo rmance favors pr ior l ea rn ing to w h i c h the ch i ld ren were dif-

ferential ly exposed . H e then c i tes data from h i s o w n labora tory to s h o w that

if tes t ing cond i t i ons are c h a n g e d , the lower class ch i ldren are aga in d isad-

van taged . In e s s e n c e , ch i ldren are n o w presen ted w i t h ob jec t s w h i c h were

se lected so that they could b e classif ied in to o n e of four ca tegor ies : an ima l s ,

furni ture, c lo th ing , or foods . N o w the midd le class ch i ld ren u t i l i z ing th i s cat-

egorical c lus ter ing se lec ted b y midd le class tes ters w e r e super io r in recall per-

formance . J e n s e n goes on to no te that the recall of ca tegor ized l is ts correlates

wi th I Q , i . e . , w i t h w h a t they k n e w before t hey c a m e in to the test s i tua t ion.

L o w e r class ch i ld ren s h o w " s o m e id iosyncra t ic c lus t e r ing" bu t do no t " s p o n -

t a n e o u s l y " use the appropr ia te ca tegor ies in recall , desp i te the fact that pos t -

test i n t e rv iews ind ica ted that they k n e w the n a m e s of the ca tegor ies .

O b v i o u s l y , ca tegor ica l c lus ter ing i s a m e m o r i z i n g s t ra tegy that can b e

learned b y assoc ia t ion , bu t in th is r ev i ew there w a s n o repor t o f a t t empts to

teach n e w and unfami l ia r ca tegor ical c lusters to b o t h c lasses o f ch i ldren . In-

stead J e n s e n re legates the l ea rn ing of n e w and unfami l ia r tasks to Leve l I (as-

socia t ive l ea rn ing abi l i ty) and the spon taneous use of " A n g l o " ca tegor ies in

recall to Leve l II (p rob lem solv ing abi l i ty) solely on the b a s i s of corre la t ion

wi th I Q data. T h u s the syl logis t ic opera t iona l trap is sprung. In te l l igence is

wha t IQ tests measu re . I Q tests m e a s u r e w h a t is l earned before o n e c o m e s to

the test s i tua t ion . L e a r n i n g in the test s i tua t ion is no t a measu re of t rue in-

te l l igence .

In s u m m a r y , IQ i t ems are no t va l ida ted on an abst ract no t i on of " t r u t h " or

because they measu re " in t e l l ec tua l " capac i ty . T h e y are va l ida ted b y a correla-

t ional t e c h n i q u e d e s i g n e d to reflect p rogress of Ang lo ch i ld ren th rough a core

cur r icu lum des igned b y Ang lo educa tors . T h i s corre la t ion d e p e n d s no t on ly

on the test i t ems bu t also u p o n the subjec t s w h o w e r e tes ted . T h e r e is n o

reason to a s s u m e that such a test wou ld b e su i t ab le to m e a s u r e the s a m e func-

t ion in ano the r b iosoc ia l g roup . For example , i f the p rogress of Black ch i ld ren

is to b e m e a s u r e d , then the i t ems m u s t b e se lec ted for t h e m in the s a m e

empi r ica l w a y that they we re se lec ted for the W h i t e group. T h e failure to select

and search d i l igent ly for i t ems w h i c h empi r ica l ly reflect p rogress ive c h a n g e

w i t h age in Black A m e r i c a n s m a k e s any test , even those w h i c h n o w inc lude

Black A m e r i c a n s in s tandard iza t ion g roups , of d u b i o u s val id i ty .

Natural ly the Ang lo educa tors and men ta l tes ters se lec ted the i r o w n subcul -

428 John Garcia

ture and the progress o f the chi ldren of the i r o w n b iosoc ia l group as the sine

qua non for the def ini t ion of in te l l igence . All b iosoc ia l g roups , c iv i l ized as wel l

as p r imi t ive , t end to v i e w themse lves as h u m a n and others as s o m e w h a t less

than h u m a n . At the turn of the cen tury , m o s t s egmen t s of Eu ropean and

A m e r i c a n soc ie ty t ended to v i e w the n o n - E u r o p e a n s as the " W h i t e m a n ' s

b u r d e n " and even the m o s t p rogress ive A m e r i c a n educators w e r e confident

that the school , an essent ia l part of the A m e r i c a n " m e l t i n g p o t , " w o u l d in

t ime conver t the ou ts iders in to core A m e r i c a n s . Apparen t ly th i s o p t i m i s m is

w a n i n g as it b e c o m e s apparen t that b iosoc ia l groups tend to ma in t a in cultural

con t inu i ty and in tegr i ty over cen tur ies . It i s surpr i s ing that w i th today ' s me th -

odologica l and technica l soph i s t i ca t ion , data ga thered wi th tes ts des igned to

place ind iv iduals w i t h i n the i r o w n social group are u sed to draw compar i sons

b e t w e e n groups .

WHY MEN AND WOMEN ARE EQUAL IN INTELLIGENCE

T h e d i l e m m a w h i c h faces a n y o n e w h o a t tempts to compare in te l l igence of

two b iosoc i a l groups can bes t b e i l lustrated b y compar ing the test per formance

of ma les and females . O n the average , males excel on i t ems invo lv ing speed

and coord ina t ion of gross m o t o r ac t iv i t ies , spat ial tasks , m e c h a n i c a l p rob -

l ems , and ar i thmet ic r eason ing ; females fare be t t e r on tasks invo lv ing fine

motor ski l ls , perceptual skil l , m e m o r y , numer ica l computa t ion , and verba l

f luency (2) .

Bo th the b io log ica l and the social a rgument are called forth to account for

these differences. T h e b io logica l ly inc l ined po in t to differences in b o d y struc-

ture, muscu la tu re , and h o r m o n a l factors. T h e natural ch i ld -bea r ing and rear-

ing funct ions dic ta ted that w o m e n were b io logica l ly b o u n d to the pro-

tected cen te r of the p r imi t ive vi l lage whi l e the s t ronger and faster males were

more effective in dea l ing wi th dangers of h u n t i n g and f ight ing at the pe r ime-

ter of the tr ibal terr i tory. T h u s evolu t ion can b e v i e w e d as favoring sexual dif-

ferences . T h e social ly inc l ined po in t to the o b v i o u s differences in social

t ra in ing . W o m e n are r ewarded for accep t ing the f emin ine role and r id iculed

for a t t empt ing to acqu i re mechan i ca l and quant i t a t ive skil ls . T h e y are den i ed

educa t iona l and profess ional oppor tun i t i es in the m a n ' s wor ld b y law as wel l

as cus tom. S i n c e n o o n e can define in te l l igence as an ent i ty i ndependen t ly of

test resul ts , th is p i q u a n t con t roversy goes on and on .

T h e men ta l testers dealt w i th th is p rob l em in the i r character is t ic practical

fashion. F igure 2 m a k e s it clear that i f too m a n y " m a l e i t e m s " we re inc luded

in a test ba t te ry , males w o u l d have a h i g h e r in te l l igence score on the average

and i f too m a n y " fema le i t e m s " were inc luded , females w o u l d have the h ighe r

in te l l igence . In k e e p i n g w i t h the social t rends toward equa l i ty of the sexes ,

L e w i s T e r m a n and M a u d Merr i l l (25) a s s u m e d that the in te l l igence of males and

females was on the average equa l , and ba l anced the n u m b e r and we igh t of

15. The Futility of Comparative IQ Research 4 2 9

1 d>9 1

TESTS

FIGURE 2. A virtually infinite number of items can be written which presumably measure in-telligence. Some favor men, some favor women, and some favor neither sex as the overlap in the two spheres indicates. Lewis Terman and Maud Merrill balanced their tests to equate the sexes by arbitrary selection of items. The same rationale holds for Black versus White differences in re-sponding to test items. Since the gene pools underlying races overlap and contain enormous numbers of genes affecting intellectual performance, a similar value judgment would be necessary if a small finite sample of genetic factors were to be selected as a eugenic criterion for intellectual improvement.

the male and female i t ems . For in s t ance , in the 1937 S tanford-Bine t , i t ems that

s h o w e d a large sex difference we re exc luded w h i l e i t e m s w h i c h on ly s l ight ly

favored one sex were ba l anced b y i t ems favor ing the oppos i t e sex to an equa l

degree . T h e dev ia t ion in te l l igence procedure m a i n t a i n s th i s equa l i ty th rough

the use of separa te n o r m s for males and females .

T h e s e sex dif ferences cons i s t en t ly appea r in the Gradua te R e c o r d E x a m i n a -

t ion (GRE) w h e r e m e n do be t te r in "quan t i t a t i ve s u b t e s t s " and w o m e n do

be t te r in "ve rba l s u b t e s t s . " Here aga in compara t ive group j u d g m e n t s are

avo ided b y a s s u m i n g equa l we igh t s for quan t i t a t ive and ve rba l sub tes t s and

b y m e a s u r i n g each ind iv idua l ' s pe r fo rmance in re la t ion to the pe r fo rmance of

a group of the same sex (17) . T h u s the IQ of w o m e n is equa l to that o f m e n on

the average b e c a u s e in te l l igence tes ters des ign the i r tests that way .

O n e could treat o ther b io soc i a l differences in the same way . W h a t if for ex-

ample , C h i c a n o ch i ldren score lower than Ang lo ch i ld ren on a test c o m p o s e d

of " A n g l o i t e m s . " W e n e e d no t agon ize abou t he red i ty and e n v i r o n m e n t . W e

mere ly n e e d to wr i te s o m e " C h i c a n o i t e m s " w h i c h reflect the progress of

C h i c a n o ch i ldren . W e can t h e n ba lance the C h i c a n o i t ems w i t h the Ang lo

i t ems in a s ingle test , or w e can m a k e up a C h i c a n o tes t and s tandard ize it for

C h i c a n o progress , then pres to , C h i c a n o s w o u l d b e equa l to A n g l o s in in-

te l l igence .

N o w s o m e p r o p o n e n t s of men ta l t e s t ing p ious ly express a conce rn that such

inc lus ion of C h i c a n o i t ems and Black i t ems wi l l a t tenuate the pred ic t ive va lue

430 John Garcia

of the IQ test. It is jus t as conce ivab le that the pred ic t ive value m a y b e ulti-

mate ly e n h a n c e d w h e n equa l oppor tun i ty i s afforded to mino r i t y people . In any

case , it i s aga in ins t ruc t ive to look at th is in te rms of ma les versus females . In

the grade schools girls on the average surpass b o y s of the same age b y a w i d e

marg in in academic pe r fo rmance , ye t m e n are accepted in to graduate un iver -

si ty t r a in ing a lmost to the exc lus ion of w o m e n . T h e b io logica l ly inc l ined say

that girls mature faster than b o y s , bu t that y o u n g m e n are less emot iona l and

more adapt ive thus a be t te r r isk in graduate w o r k than w o m e n . T h e socially

inc l ined say that the grade school cul ture is m o r e compa t ib l e w i th the little

gir ls ' social role wh i l e the graduate school admi s s ions are b i a s e d in favor o f

the adult male social role. H o w e v e r , the men ta l tes ters a s s igned the same

average I Q and the s ame d i s t r ibu t ion of I Q s to b o y s , gir ls , m e n , and w o m e n

wi thou t apparent conce rn for its p red ic t ive value in grade school or univer-

sity. Unfor tunate ly , they we re no t in the pos i t ion to ass ign equa l pay for equa l

work , or equa l oppor tun i ty to place equal ly in te l l igent w o m e n into profes-

s ional schools . A s a resul t I Q scores are m o r e predic t ive o f ma le success than

female success (26) .

THE ASSUMED "GROWTH" AND "DECLINE" OF INTELLIGENCE WITH AGE

W h e n an ind iv idua l in te l l igence test is p re sen ted to g roups o f sub jec t s of

differing ages it is found that the n u m b e r of i t ems passed inc reases w i t h age ,

level ing off b e t w e e n the ages of 16 to 26 yea r s , and then there is a s teady

decl ine w i th advanc ing years . T h i s i s not a direct obse rva t ion of the g rowth

and dec l ine of intel lectual funct ion. In te l l igence tests were d e s i g n e d to y ie ld

th is p ic ture of intel lectual g rowth b e c a u s e it fitted the a priori no t i ons of intel-

lectual g rowth he ld b y the men ta l tes ters and school author i t ies .

O b v i o u s l y , an in te l l igence tes t c o m p o s e d of i t ems se lec ted to reflect school

progress should y ie ld a p e a k in intel lectual deve lopmen t at the prec ise age

w h e n people are l eav ing school . T h e longer the sub jec t s are out of school the

worse they should do on school tests b e c a u s e peop le forget some skills and

acqu i re o thers as t hey en te r n e w fields. T h i s decl ine in the s lope of in-

te l l igence can b e as logical ly a t t r ibuted to the d i scon t inu i ty of the i r env i ron-

men t , as to s o m e loss in inte l lectual funct ion. B y o ther s tandards , ab i l i ty to

deal w i t h the ext raschool e n v i r o n m e n t s e e m s to con t inue to grow. For ex-

ample , mos t auto in su rance c o m p a n i e s h a v e dec ided on an empi r ica l actuarial

ba s i s that abi l i ty to deal w i th the p rob l ems of survival on the h i g h w a y s is not

fully deve loped unt i l age 2 5 . If o n e searched for i t ems w h i c h reflected inc reas -

ing abi l i ty to deal w i t h the ext raschool e n v i r o n m e n t and " b l e n d e d " these

i t ems in w i th the school i t e m s , in te l l igence wou ld s e e m to con t inue to g row

monoton ica l ly b e y o n d age 16 (see F igure 3) (29) .


20 40 60

Age

FIGURE 3 . The solid line is the "curve of average decline of mental ability with age" presented by

Wechsler ( 2 9 ) . Significantly it closely approximates the curves obtained with tests of school activi-

ties: block design, price arrangement, digit span, and digit symbol. The dashed line indicates the

same function obtained with tests more meaningful to adults: information, vocabulary compre-

hension, and arithmetic. The dotted line is our speculation on the probable results of a test as-

sessing the adaptive responses to the highway patrol and internal revenue service, to credit and

property demands, and to irksome social responsibilities at work and home.

A n e x a m i n a t i o n of the or ig ina l S tanford-Bine t tes t ing ki t reveals that th i s

b l e n d i n g tact ic w a s e m p l o y e d to deal w i th the d i scon t inu i ty o f e n v i r o n m e n t

w h e n the ch i ld l eaves h o m e and enters school . I t ems invo lv ing s enso ry -mo to r

tasks ref lect ing the h o m e e n v i r o n m e n t , such as n a m i n g toys and fol lowing in-

s t ruc t ions , are qual i ta t ive ly different from the scholas t ic i t ems . T h e s e i t ems

we re se lec ted and s tandard ized on preschool ch i ldren so as to y ie ld a s m o o t h

m o n o t o n i c funct ion w i t h age dur ing the t rans i t ion from preschoo l years to

school years w h e n m e a n men ta l age is p lot ted. H o w e v e r , the d i scon t inu i ty of

e n v i r o n m e n t m a k e s the p red ic t ion of future in te l l igence from these p reschool

tests to the scholas t ic tes ts less re l iable than pred ic t ion w i t h i n the scholas t ic

test se r ies .

T h e s imple fact is that adults do less wel l w i th inc reas ing age on tests

de s igned for ch i ld ren and that adults w i t h relat ively low IQ scores are often

successful even in academic p rofess ions , because long- t e rm adapt ive pers i s -

tence to ob ta in ha rd -won skills and goals in the adult wor ld canno t b e pre-

dic ted from a test m a d e up from " p a p e r and p e n c i l " i t ems to b e solved w i t h i n

m i n u t e s . For "p r ac t i c a l " r easons , test i t ems have to conform w i t h the seg-

m e n t e d educa t iona l rec i ta t ion act ivi t ies in a c lass room set t ing . I t ems have to

b e b r i e f so that m a n y i t ems can b e g iven and the IQ o b t a i n e d in one s i t t ing.

Q u i t e natural ly it has b e e n found that I Q s o b t a i n e d in th is w a y are m o r e

closely re la ted to ac t iv i t ies of an accountan t than the ac t iv i t ies of a l u m b e r -

jack ,* m o r e c losely re la ted to the skill of a b r idge player than that of a quar ter -

* Herrnstein ( 1 2 ) presents a list ranking civilian occupations according to I Q . At the top of the list are accountants and auditors ( I Q = 1 2 7 ) who work in a situation on tasks which resemble the I Q situation. At the bottom of the list are lumberjacks and teamsters ( I Q = 9 1 ) whose skills, abilities, and technical language are considered too "specialized" for I Q test items. Ob-viously, both groups are highly specialized, i.e., adapted to their particular niche. The ques-tion of which group is more likely to survive successfully depends whether the survival test is conducted in the urban counting house or the north woods. Such considerations forced the mental testers to abandon the conceptionalization "intelligence is adaptive" in favor of "in-telligence is whatever our test measures."

Mea

n S

core

432 John Garcia

back . S i n c e these i t ems reflect the subcul ture of the s tudent , t eachers and professors are apt to v i e w the i r mas te ry as mer i to r ious , b u t except for T V q u i z p rograms , there are few occupa t ions w h i c h r e s e m b l e the pecul ia r n i c h e of the s tudent tak ing an in te l l igence test.

THE INVENTION OF THE STABLE IQ

It was i m m e d i a t e l y o b v i o u s to the men ta l tes ters that infants , ch i ldren ,

you ths and adults b e l o n g e d to separa te b iosoc ia l g roups w h i c h differed in

b io log ica l matura t ion and level o f socia l iza t ion . Appropr ia te tests were de-

s igned for each group and a scale w a s sough t w h i c h wou ld m a k e all groups

equ iva len t in in te l l igence desp i te age differences . T h e men ta l testers w a n t e d a

numer ica l score w h i c h w o u l d r e m a i n cons tan t for each ind iv idua l as h e grew

older and accumula ted m o r e years and educa t ion .

First the in te l l igence quo t i en t ( IQ) was dev i sed b y the s imple a s sumpt ion

that the m e a n men ta l age (MA) of each group was equa l to i ts actual ch rono-

logical age (CA) . W h e n the M A is d iv ided b y the C A and mul t ip l ied b y 100

the resul t i s the I Q . Fo r any age group the m e a n I Q is equa l to 100 . T h e chi ld

w h o performs at a h i g h e r M A than the average for h i s group rece ives an IQ

propor t ional ly h i g h e r than 100 , and the chi ld w h o performs lower than h i s

age group rece ives an IQ lower than 100 . O b v i o u s l y th is lower score could

e i ther b e due to " b i o l o g i c a l i m m a t u r i t y " or " in fe r io r t r a in ing . "

T h e IQ dev ice appears ra t ional unt i l t he m e a s u r e d M A reaches a level ing off

after age 16. After th is po in t if C A con t inues to increase then IQ b e c o m e s

progress ive ly lower . T h i s w a s solved in a bo ld fash ion b y the men ta l testers

w h o set a ce i l ing on actual age (CA) to conform to the ce i l ing on m e a s u r e d

M A . Never the less peop le con t inue to g row older and do worse o n I Q tests .

T h i s depress ing state of affairs w a s hand led in a m o r e soph i s t i ca ted w a y w h e n

the M A / C A ratio w a s d ropped in favor o f the dev ia t ion IQ w h e r e the IQ of any

age group is a s s u m e d to b e 100 and the var ia t ion of ind iv idua ls o f any group

is a s s u m e d to b e symmet r ica l ly d i s t r ibu ted wi th a s tandard dev ia t ion of 16.

H o w e v e r , w h e n people are tes ted and re tes ted IQ scores t end to fluctuate

w ide ly desp i te these efforts to s tab i l ize t h e m over age . O n e s tudy found that

b e t w e e n the ages of 6 to 18 years approx imate ly 6 0 % of the ind iv idua ls re-

tes ted had changed 15 I Q po in t s or more . Approx ima te ly 1 0 % had changed 30

IQ po in t s or m o r e (13) . A m o r e recent s tudy ind ica tes that the average IQ

c h a n g e from infancy to ado lescence is near ly 30 po in t s or e n o u g h to change

the classif icat ion o f a ch i ld f rom " n o r m a l " e i the r up to " g e n i u s " o r d o w n to

" m o r o n " (19) .

THE DILEMMA OF THE CULTURE-FREE TEST

Let us n o w cons ide r the d i l e m m a w h i c h faces any researcher w h o a t tempts

to compare the in te l l igence o f different cultural and socia l groups . T h e no t i on


of a "cu l tu re - f ree" test is i l lusory, b e c a u s e each i t e m in such a test w o u l d have

to b e tes ted on m e m b e r s o f at least two different cultural g roups . If, on the

average , the pe r fo rmance of o n e group o n a g iven i t em i s supe r io r to that of

the o ther g roup , o n e m a y conc lude that the i t em is culturally b i a s e d in favor of

that group and , therefore , should b e e l im ina t ed from the "cu l tu re - f ree" test . If

th is cr i ter ion is accep ted then no i t e m w h i c h different iates b e t w e e n the two

groups is acceptab le and , in effect, the two groups are declared to b e equa l in

in te l l igence as m e a s u r e d b y the test . W i d e s p r e a d tes t ing of the i t em in a var i -

e ty of cul tures does no t ass is t us , s ince w h e n e v e r differences are found , "cu l -

tural b i a s " is a lways a pos s ib l e exp lana t ion . T h e a l ternat ive conc lus ion that a

g iven i t em reflects a true difference in in te l l igence b e t w e e n groups requ i res an

i n d e p e n d e n t cr i ter ia o f men ta l capaci ty . S u c h cri ter ia are prec luded b y the

circular opera t iona l def ini t ion " in t e l l i gence is w h a t the in te l l igence test m e a -

s u r e s . " Each i t e m in an in te l l igence test reflects the env i ronmen ta l deve lop-

m e n t of a skill comple te ly con founded w i t h hered i ta ry capaci ty to acqu i re that

skill . N o a m o u n t o f socia l research and n o level o f s tat is t ical ana lys i s can ever

unravel that b io soc i a l confound ing .

THE DILEMMA OF HEREDITY VERSUS ENVIRONMENT STUDIES WITH HUMAN SAMPLES

A s imi lar d i l e m m a faces the researcher w h o a t tempts to de t e rmine the rela-

t ive inf luence of he red i ty or e n v i r o n m e n t w i t h i n a s ingle b iosoc i a l group.

Ideal ly the researcher w o u l d l ike to a t t r ibute 1 0 0 % of the dif ference in IQ

scores of h i s sample of ch i ld ren propor t iona l ly e i ther to the inf luence of " f am-

i l y " and or to the inf luence of " h o m e . " * In m o s t cases , family and h o m e are

confounded , that is ch i ld ren are ra i sed in the family h o m e , so the re-

searcher usual ly deals w i th foster ch i ldren w h e r e ch i ldren o f o n e fami ly are

ra ised in the h o m e of another . T h e first p r o b l e m that ar ises is that the foster

chi ld has b e e n nur tured w i t h i n the e n v i r o n m e n t of the mo the r ' s b o d y and

env i ronmen ta l in f luences of d i sease , nu t r i t ion , and t rauma dur ing th is per-

iod have a l ready left the i r mark on h i m forever. Sub t l e depr iva t ions of pro-

te in , h o r m o n e s , oxygen , or minera l s dur ing th is pe r iod could reduce a po-

tent ial gen ius to an i m b e c i l e . T h e s e are env i ronmen ta l in f luences w h i c h the

foster ch i ld b r i n g s to h i s n e w h o m e . T h i s con found ing of he red i ty and env i ron-

m e n t can neve r b e comple te ly unrave led b y s tudies o f the foster ch i ld .

T h e s e c o n d p r o b l e m ar ises from the p l acemen t prac t ices o f adop t ion

agenc ies . T h e y select appropr ia te h o m e s for the i r foster ch i ldren . Fo r exper i -

* While this notion may be defended by appeals to the statistical procedures of analysis of variance, it does not make any sense genetically speaking, to say as Herrnstein does . . . "that the genetic factor is worth 80 percent and that only 20 percent is left to everything else." Estimates of the heritability of a trait do not yield estimates of how much that trait will be influenced by environmental changes.

434 John Garcia

menta l r easons the cond i t ions of h o m e rear ing should b e a l lowed to vary from

very poor to ve ry good cond i t ions . If w e ra ise b io logica l ly h e t e r o g e n e o u s

chi ldren in un i form e n v i r o n m e n t s , then hered i ty wil l s e e m more impor tan t . If

w e ra ise b io logica l ly h o m o g e n e o u s chi ldren in differential ly s t imula t ing env i -

r o n m e n t s , t hen e n v i r o n m e n t will s e e m m o r e impor tan t in de t e rmin ing the

ind iv idua l va r iance in IQ scores . In all such s tudies the researcher m u s t m a k e

practical dec i s ions and work wi th the par t icular sub jec t s and socia l se t t ings

that are avai lable , and these dec i s ions fix the ou t come of h i s resul ts .

For example , w h e n Cyri l Bur t (5) chose to s tudy t w i n s , s ib l ings , and unre -

lated Eng l i sh ch i ldren .in L o n d o n foster h o m e s , the ou tcome w a s de t e rmined

b y the legal and social cons t ra in ts i m p o s e d : (a) u p o n Eng l i sh ch i ldren d e e m e d

e l ig ib le for adop t ion , (b) u p o n L o n d o n h o m e s d e e m e d sui table for adopt ing

t h e m , and (c) upon a t tempts o f the socia l agency to ma tch foster chi ld and

parent . Therefore , he red i ty w a s found to b e m u c h more impor tan t than env i -

r o n m e n t unde r these specific cond i t ions . Inev i tab ly , co - twin s tudies have

b e c o m e controvers ia l on th is ve ry poin t . K a m i n (16) po in t ed out that in co -

twin s tudies the ident ica l tw in pa i rs w e r e often p laced in h o m e s m a t c h e d b y

n e i g h b o r h o o d or famil ial r e la t ionsh ip , thus env i ronmen ta l in f luences and

o ther se lect ive factors opera ted along wi th b io logica l factors to p roduce s imi lar

IQ scores i n ident ica l tw ins . C o n s i d e r h o w m u c h m o r e impor tan t e n v i r o n m e n t

wou ld have appeared if ha l f o f the chi ldren had b e e n ra ised in pover ty

s t r icken h o m e s and the r ema inde r in intel lectual ly en r i ched k indergar tens . A n

example of th is k ind of s tudy w a s carr ied out b y R ick H e b e r and h i s assoc ia tes

in M i l w a u k e e (11) . T h e y se lec ted 4 0 Black chi ldren w h o s e mo the r s tes ted

b e l o w 80 IQ and w h o s e fa thers ' I Q s we re es t imated to b e abou t the same .

B e g i n n i n g vir tual ly at b i r th , 20 chi ldren w e r e reared in an en r i ched academic

nursery w i t h vir tual ly a one - to -one teacher/pupi l ra t io . V e r b a l facil i ty and

p rob l em solving w e r e s t imula ted and social ly rewarded . T h e 20 control

chi ldren we re a l lowed to r e m a i n at h o m e . B o t h groups we re tes ted regularly.

B y age 3 , the expe r imen ta l group averaged approximate ly 127 IQ wh i l e the

controls averaged abou t 95 I Q po in t s . S imi l a r differences we re ev iden t in

other ve rba l and quant i ta t ive a c h i e v e m e n t s . A s they g rew older , the exper i -

menta l group c o n t i n u e d the i r " u p p e r midd le c l a s s " IQ b e h a v i o r w h i l e the i r

s ib l ings as wel l as the control group con t inued thei r " l o w e r s o c i o e c o n o m i c

class I Q " t rend.

Inev i tab ly , th is s tudy also b e c a m e controvers ia l even before the data were

pub l i shed . Page (21) con tends that the exper imenta l ch i ldren were coached ,

i . e . , told the answers to the I Q test . T h i s s e e m s to b e s imply chea t ing bu t , at

some poin t , the ch i ld m u s t b e e x p o s e d to the answers . H o w else can he

acqui re t h e m ? For example , it m a k e s no sense to keep secret all the words used

in the vocabu la ry sub tes t s , and then expec t a chi ld to define a word he has

never hea rd before . Hopeful ly , the exper imenta l g roup in the M i l w a u k e e

s tudy learned the specific IQ answers in a b road contex t of in format ion and


th is appears to b e the case . Bu t pr iv i leged ch i ld ren are a lways coached in

s o m e sense . Di rec t or ind i rec t exposure to the answer s o n an I Q test are m u c h

greater on the average for the chi ld w h o is reared in W e s t w o o d ' s academic

s u b u r b than for the chi ld reared in the C h i c a n o barr io o f Eas t Los Ange le s .

It wou ld b e a mi s t ake to a s s u m e that the " e n r i c h e d k i n d e r g a r t e n " th rough

some k ind of sensory s t imula t ion caused synapt ic t e rmina ls to proliferate in

the b ra in s of the exper imen ta l ch i ldren , and that the ghe t to h o m e actually

d i m i n i s h e d a s imi la r b io log ica l capac i ty in the controls . It is o b v i o u s that the

en r i ched k indergar ten w a s de s igned to t rain the mot iva t ions , the academic

ski l ls , and the specific l anguage of the social ly m o b i l e midd le class env i ron-

m e n t w h e r e a c h i e v e m e n t is m e a s u r e d b y in te l l igence tes ts . I f the ghe t to n i c h e

were ana lyzed in to i t ems and a c h i e v e m e n t quant i f ied , w e w o u l d p r o b a b l y

find that the intel lectual adapta t ions of the control ch i ldren to the ghe t to we re

no less r emarkab le . In fact, i f the exper imen ta l ch i ld ren are r e tu rned to the

ghet to , t hey m a y b e t ragical ly " d i s a d v a n t a g e d , " at least t emporar i ly , to cope

wi th that e n v i r o n m e n t .

T h e e n o r m o u s complex i ty of the he red i ty and e n v i r o n m e n t in te rac t ion is

b rough t h o m e w h e n o n e a t tempts to deal w i t h a concep t of in te l l igence in

te rms of gene t i c m e c h a n i s m s . A s s u m e that in te l l igence is d e t e r m i n e d b y a

finite set of po lygenes , th i s is he red i ty . All o ther inf luences inc lud ing cellular

ones are the env i ronmen t . E v e n the ac t ions of gene t i c modif iers u p o n expres-

s ion of those inte l lectual alleles can b e cons ide red e n v i r o n m e n t a l o n e s . T h e

bes t unde r s tood gene t i c effects u p o n intel lectual pe r fo rmance are ones in

w h i c h modif ie r g e n e s are apparent ly affecting the no rma l deve lopmen t of

hered i ta ry potent ia l . Fo r example , in pheny lke tonur i a (PKU) a recess ive

genet ic defect in m e t a b o l i s m of pheny la l an ine causes men ta l def ic iency and a

hos t of o the r p r o b l e m s . If P K U is ident i f ied in early in fancy , and the chi ld is

fed on a die t low in pheny la l an ine , t hen men ta l i m p a i r m e n t is r educed a long

wi th the o ther s y m p t o m s . In D o w n ' s s y n d r o m e , an extra c h r o m o s o m e of the

twenty-f irs t pa i r modif ies no rma l deve lopmen t of the hered i ta ry po ten t ia l

caus ing men ta l re tardat ion. In Kl inefe l ter ' s s y n d r o m e , extra sex c h r o m o s o m e s

increase the i n c i d e n c e of men ta l re tardat ion. In a s imi la r way , m o r e pure ly

e n v i r o n m e n t a l agen ts such as chemica l s , h igh t empera tu re , o r e v e n m e c h a n -

ical p ressure m a y m i m i c a gene t ic effect p roduc ing a p h e n o c o p y (23) . In the

face of these complex i t i e s it is i m p o s s i b l e to m a k e r easonab le s t a t emen t s abou t

the gene t ic b a s i s o f academic test pe r fo rmance n o mat te r h o w w i d e l y w e

sample cul tures and races of m a n .

BREEDING FAST HORSES AND SMART RATS

It is t emp t ing to specula te that the he red i ty ve r sus e n v i r o n m e n t i s sue m i g h t

b e set t led b y a eugen ic s p rog ram in w h i c h w e se lec t ive ly b r e d the parents in

436 John Garcia

order to p roduce in te l l igent offspring. B u t such is not the case , b e c a u s e in-

te l l igence defined as a genera l adapt ive factor quant i f ied in to a s ingle score

mus t a lways b e defined b y specific env i ronmen ta l tests des igna ted b y arb i -

trary value j u d g m e n t s .

Let us reluctant ly in t roduce ano ther ana logy in to a po lemic a l ready overbur -

dened b y ana log ies . If a b r eede r o f ho r ses w i s h e s to p roduce fast ho r ses , he

mus t opera t ional ly define " s p e e d " in fixed env i ronmen ta l t e rms . In thorough-

b red rac ing , fast is def ined as w i n n i n g a compe t i t i ve race over a flat dry

circular track of approx imate ly 1 mi le carrying a j o c k e y of approx imate ly 100

lb in we igh t . T h e second step is to ma te strictly in te rms of th i s cri teria.

Losers are e l imina ted from the b r e e d i n g stock and w i n n e r s are p laced in shel-

tered paddocks and served b y g rooms and med ica l exper ts . Se lec ted mates are

provided . T h i s fixed pro tec t ive e n v i r o n m e n t insures that the track records

exert the major inf luence upon survival and reduces the inf luence of other

select ive factors, such as ou twi t t ing predators , f inding food and shel ter , and

compe t ing for ma tes . T h e se lect ive pressure exer ted b y the horse b reede r is

s t r ingent and specif ic , l imi t ing the behav io ra l traits and the gene pool to

p roduce a horse spec ia l ized in speed . Bu t , does the t ho roughb red hor se have

the genera l capaci ty for speed? Wi l l it w i n a race aga ins t a M o n g o l i a n pony

across 100 mi le s of rocky deser t? Wi l l it w i n a race aga ins t a draft horse pul l ing

a loaded w a g o n ? Wi l l it speed to the lure of a ma te as it speeds to the s t ing of

the w h i p ? A n y sens ib le horse b reede r wou ld r e spond , " T h o s e ques t ions are

not relevant . S p e e d is w h a t m y speed test m e a s u r e s . " T h e hered i ta ry speed of

the t ho roughbred horse is t ied to the specific fixed e n v i r o n m e n t of the race-

track and the protec t ive paddock .

T h e s ame case can b e m a d e for b r e e d i n g for " in te l l ec tua l " pe r formance in

an imals . At the Un ive r s i ty of Cal i fornia , in 1929 , Professor R o b e r t C. T ryon set

out to b r eed a race of in te l l igent rats. Firs t he des igned the fixed env i ron-

menta l test , a large au tomat ic m a z e (28) . T h e correct cho ices in the m a z e led to

the o p e n path to the feed ing area, the w r o n g cho ice led to a b l ind alley

requ i r ing the an ima l to turn and retrace its s teps to the cho ice point . Floor

plates w i th electr ic swi t ches coun ted errors and c losed doors b e h i n d the

an imals to keep t h e m m o v i n g th rough the maze . " B r i g h t " rats w h i c h mas te red

the maze were ma ted to o ther b r igh t rats and "du l l " an imals w h i c h took

longer were ma ted to o ther dull an ima l s . T h e an imals we re protec ted and

cared for so that o ther se lect ive pressures were reduced . In abou t seven gener -

a t ions T ryon p roduced a relat ively b r igh t race and a relat ively dull race of rats.

Bu t T ryon w a s wel l aware the i r relat ive in te l l igence w a s spec ia l ized for that

m a z e , for i f the an imals we re tes ted w i th o ther apparatus on o ther p rob l ems ,

the b r igh t an imals we re no t any be t te r than dull an imals . In fact some re-

searchers felt that the b r igh t an imals w e r e super ior in the large au tomat ic

maze because they were i n sens i t i ve to the c lank ing doors and swi tches . At

th is po in t , T ryon did no t p roc la im the circular opera t ional a rgument , " i n -


te l l igence is w h a t m y m a z e m e a s u r e s , " and t hen p roceed to define w i t h corre-

la t ional m e a s u r e s all aspects o f the rat 's wor ld as intel lectual . T h e T r y o n m a z e

proved to b e an unsa t i s fac tory cr i ter ion for " i n t e l l i g e n c e " in rats b e c a u s e it d id

not select out a genera l adapt ive ab i l i ty to deal w i th a var ie ty of p r o b l e m s . To

select for genera l ab i l i ty it wou ld b e neces sa ry to dev i se a w i d e var ie ty of

tes t ing appara tus . T h e r e w a s s o m e ev idence that T ryon ' s b r igh t rats we re

more " food d r i v e n " than the dull an ima l s , therefore w e wou ld have to use a

var ie ty of depr iva t ions and rewards . The re w a s s o m e ev idence that the br igh t

an imals were less caut ious and less explora t ive , bu t t hese are not admi rab le

qua l i t i es in dangerous s i tua t ions so w e w o u l d have to set up s i tua t ions in

w h i c h so lu t ion d e p e n d e d on " p r u d e n t " j u d g m e n t and so on . If w e con t i nued

in th is v e i n , the end resul t wou ld b e a large g e n e pool y ie ld ing a he t e roge -

neous s train of rats differing from each o ther on a w i d e var ie ty of trai ts . In dif-

ferential e n v i r o n m e n t s h y b r i d s are apt to b e m o r e successful than purebreds .

S u p p o s e w e could agree on the fixed e n v i r o n m e n t w h i c h is b e s t for m a n

and set abou t se lec t ive ly b r e e d i n g h u m a n in te l l igence to fit spec ia l i zed n i c h e s

as in Aldous Hux ley ' s nove l " B r a v e N e w W o r l d " (14) . T h e resul ts o f such a

p rogram wou ld b e d i sappo in t ing to those peop le w h o value the p resen t racial

d i s t inc t ions of sk in color , ha i r form, and facial features , for it is no t ve ry l ikely

that the alleles w h i c h de t e rmine these superficial traits also de t e rmine the

b ra in m e c h a n i s m s gu id ing adapt ive behav io r . T h e p rog ram w o u l d call for

se lec t ing the ind iv idua l s e x h i b i t i n g super io r traits f rom w h a t e v e r racial g roup

and se lec t ively b r e e d i n g t h e m to p roduce superpersons . T h e end result w o u l d

surely b e surpr i s ing to those w h o proc la im racial super ior i ty today.

THE ANIMAL MODEL: GENERAL ADAPTATION VERSUS DIFFERENTIAL SPECIALIZATION

No one d id m o r e to e s t ab l i sh the no t i on of in te l l igence as a genera l adapt ive

factor in the b e h a v i o r of an ima l s than the p res t ig ious Karl Lash ley . H o w e v e r ,

h i s p r inc ip les of m a s s ac t ion of the cerebra l cor tex and equ ipo ten t i a l i ty of i ts

c o m p o n e n t parts w e r e m o r e ensu red b y h i s m e t h o d o l o g y than der ived from

h i s empi r i ca l research (18) . Lash ley ' s b a s i c m e t h o d w a s to l es ion the cortex

and to obse rve the effect of les ion m a g n i t u d e u p o n the an ima l ' s pe r fo rmance .

H i s behav io ra l tes ts were a se r ies of m a z e s of inc reas ing complex i ty wel l -

su i ted to rats w h i c h served as h i s sub jec t s . H o w e v e r , h i s m a z e tes ted a w i d e

var ie ty of funct ions w i t h o u t di f ferent ia t ing t h e m , h e n c e it could not cont radic t

the genera l p rocess h y p o t h e s i s .

Firs t the m a z e can b e cons ide red a po lysensory test, w h e r e pe r fo rmance is

gu ided b y a mul t i tude of cues m o n i t o r e d b y the m a n y sensory sys t ems (e .g . ,

olfactory, audi tory , cu t aneous , and k ines the t i c ) . S e c o n d , the m a z e per form-

ance is usual ly s u m m a r i z e d as a s ing le score ind ica t ing eff iciency in fo l lowing

438 John Garcia

the shor tes t pa th to the goal b o x . If a s enso ry sys t em is d a m a g e d e i ther cen-

trally o r per iphera l ly , the an ima l wil l t hen t raverse the m a z e wi th decreased

efficiency. If a s econd sensory sys tem is damaged then the dec remen t in m a z e

per formance is greater , i l lustrat ing the mass ac t ion pr inc ip le , i . e . , the greater

the d a m a g e the greater the dec remen t in per formance . G i v e n the s ingle m e a -

sure of m a z e per fo rmance , d a m a g e to o n e sensory sys t em produces a perform-

ance dec remen t w h i c h appears s imi lar to the dec remen t s caused b y damage to

o ther sys tems . T h e da m a ge d an imal t raverses the maze wi th wha teve r un-

damaged sensory inpu t that r ema ins , i l lustrat ing the equ ipo ten t i a l i ty pr inc i -

ple. L ike the IQ test , the Lash l ey mazes artificially l u m p e d a var ie ty of different

mot iva t iona l perceptua l , and cogn i t ive factors in to a s ingle genera l factor.

Lash ley ' s formula t ions of m a s s ac t ion and equ ipo ten t i a l i ty s e e m e d to imply

that differential analys is of central neuro logica l funct ions w a s not very l ikely

to y ie ld causal de te rminan t s of behav io r . Pavlov (22) also p roc la imed that all

s t imul i were equ iva len t s ignals as far as classic cond i t i on ing func t ions we re

conce rned . M o t o r r e sponses were also v i e w e d as equ ipo ten t i a l , that i s , an-

imals could subs t i tu te one set o f r e sponses for ano ther in order to reach their

goal. O b v i o u s l y i f (a) all afferent sys t ems serve equ iva len t ly as in format ion

channe l s , (b) all r e sponse sys t ems serve equ iva len t ly as m e a n s to the end , and

(c) central m e c h a n i s m s we re also equ ipo ten t i a l ; then the in ternal s t ructure of

the o rgan i sm was of small c o n s e q u e n c e to the behav io r i s t (10) . T h e b e h a v -

ioris t adapted to these p resuppos i t ions b e c o m i n g a strict env i ronmenta l i s t ,

s eek ing the causal de t e rminan t s o f b e h a v i o r in the s tat is t ical pa ramete rs of in-

tens i ty , f requency , and spa t io tempora l pa t te rn ing of per iphera l s t imulus and

r e sponse var iab les . T h e o r g a n i s m b e c a m e a b lack b o x con ta in ing a w e t in -

tegrat ive sys t em w h o s e genera l funct ional laws could b e d iv ined from the

per iphery . Hypo the t i ca l cons t ruc t s b e c a m e subs t i tu tes for de ta i led empi r ica l

research in to the centra l ana tomica l and p h y s i o c h e m i c a l s tructure of the orga-

n i sm . Paradoxical ly , J e n s e n and Her rns te in , no t ed for the i r strict env i ron-

menta l i s t t ra in ing and research , have a t t empted to expla in stat is t ical soc ioedu-

cat ional data in t e rms of gene t ic m e c h a n i s m s ; bu t the i r specula t ions are so

loose , that the empi r ica l sc ience of gene t ics canno t p rov ide even a vague

out l ine of the gene t ic control o f tes t pe r formance .

T h e formula t ion of in te l l igence in t e rms of a genera l p rocess is as archaic

and arbi t rary for an ima l s as it is for h u m a n sub jec t s . M o d e r n behav io r i s t s ,

r e spond ing to e thologica l s tud ies , n o w v i e w learn ing as differential ly specia l -

ized . Behav io ra l pa t te rns vary in accordance wi th specific detai ls of the test

s i tua t ions and the specific spec ies of an imal u s e d as the subjec t . S e l i g m a n and

Hager (24) have col lected a large array of s tudies to i l lustrate such specia l iza-

t ion. For example , all s t imul i are not equ iva len t as s ignals . Effect iveness m a y

depend upon spec ies . For the rat, taste of food or wate r is the mos t effective

w a r n i n g s ignal for p o i s o n , wh i l e v isua l s ignals are ex t remely inefficient . For a


quai l , the color of wate r is m o r e effective than its tas te for the same funct ional

purpose (30) .

For ano ther example , the ef fect iveness of reward and p u n i s h m e n t d e p e n d s

upon the r e sponse even w h e n w e are t ra in ing a s ingle spec ies . If w e w i s h to

increase or decrease a rat 's explora tory r e sponse to s o m e feature o f h i s cage

then reward and p u n i s h m e n t are effective. If w e w i s h to inc rease or decrease

h i s g r o o m i n g r e s p o n s e s toward h i s o w n b o d y , t hen the s a m e reward and

p u n i s h m e n t are ineffect ive (3) .

T h e ef fect iveness of a s ignal also d e p e n d s upon w h a t task w e i m p o s e u p o n

the an imal . Bo th dogs and m o n k e y s appear in te l l igent or s tupid d e p e n d i n g

u p o n h o w they are s ignal led . T h e y eas i ly learn to execute or w i thho ld a

r e sponse (go-s top) if they are s ignal led b y two different sounds c o m i n g from

the s ame loca t ion . W i t h these ident ica l s ignals they have ex t reme difficulty if

they are r equ i r ed to r e spond to o n e sound w i t h o n e p a w and to the o ther

sound wi th the al ternate p a w ( lef t - r ight) . If they are s ignal led b y the s a m e

sound c o m i n g from different loca t ions then the converse is true. T h e g o - s t o p

p r o b l e m is ve ry difficult and the lef t - r ight p r o b l e m is very easy (7) . M a n also

s eems to have the s a m e difficulties. Mi l i t a ry recrui ts have n o p r o b l e m re-

spond in g in te l l igent ly to " forward m a r c h " and " rea r m a r c h " e m a n a t i n g from

the s a m e drill se rgeant , bu t t hey appear s tupid w h e n he calls out " r i gh t flank,

m a r c h " or "left flank, m a r c h " often tu rn ing in the w r o n g d i rec t ion and run-

n i n g in to each o ther .

T h e s e data are comple te ly unpred ic tab le and i n c o m p r e h e n s i b l e from a gen-

eral p rocess theory . T h e y represen t evo lu t ionary spec ia l iza t ion w h e r e survival

d e p e n d e d on m o v i n g or f reezing, d e p e n d i n g u p o n the qual i ty o f audi tory

cues . T h e s ame cry s ignal l ing m o v e or freeze d e p e n d i n g on its loca t ion is

rarely encoun te red in nature . Evo lu t ionary specula t ion c o n c e r n i n g cepha lo -

caudal di f ferent ia t ion and bi la teral s y m m e t r y m a y b e heur i s t i c , bu t on ly

neuro logica l speci f ica t ion of the central m e c h a n i s m s wil l reveal w h y w e can

eas i ly hand le fo rward -backward s ignals wh i l e confus ing lef t - r ight s ignals .

T h e n e w differential spec ia l iza t ion approach to b e h a v i o r p r e s u p p o s e s w h a t

neu rophys io logy has a l ready demons t ra ted : that the b ra in is no t a s ingle en-

t i ty, b u t an aggregate of neura l o rgans h o u s e d in o n e b r a in case . T h e appro-

priate ana lys i s o f b e h a v i o r demons t ra tes that damage to o n e m e m b e r of the

aggregate m a y impa i r the l ea rn ing of o n e task and actually i m p r o v e l ea rn ing

in another . Examples taken from our research i l lustrate th is t ru i sm. Rats can

qu ick ly learn a n o i s e - s h o c k task w h e r e an audi tory cue s ignals the pa in of

shock . Defens ive suppres s ion of d r ink ing is the r e sponse measu re . U n d e r

s imi la r cond i t i ons , it is m u c h m o r e difficult for t h e m to learn a t a s te - shock

re la t ionsh ip for the s a m e defens ive purpose . Conver se ly , they qu ick ly learn to

i n h i b i t d r ink ing in a tas te- i l lness task , w h e r e a specific tas te cue i s fo l lowed

b y i l lness . Bu t they find it ex t remely difficult to learn a no i se - i l lness assoc ia -

440 John Garcia

t ion to defend aga ins t i l lness (9) . T h e evolu t ionary s ignif icance of th i s b e h a v -

ior has already b e e n d i scussed and n e e d only b e s u m m a r i z e d here . Tas te in-

format ion is vi tal i f the rat is to select nu t r ien t s effectively and avoid tox ins ,

thus m a i n t a i n i n g the in tegr i ty o f h i s in ternal homeos ta t i c env i ronmen t . H o w -

ever , in its natural h i s to ry the spec ies has neve r had to cope wi th audi tory

sounds that led to i l lness . O n the o ther h a n d , in the natural external env i ron-

men t , rats m a k e good use o f audi tory cues to avo id the painful at tacks of

predators u p o n the i r cu t aneous surface. B u t taste cues are no t part icularly in -

format ive w h e n it c o m e s to avo id ing predators (see F igure 4 ) .

L e s i o n s m a d e in the ventra l h i p p o c a m p u s or the lateral s ep tum disrupt the

n o i s e - s h o c k l ea rn ing m a k i n g the an ima l less in te l l igent in c o p i n g wi th the ex-

ternal wor ld , w h i c h is not surpr i s ing . It is surpr i s ing that the same les ions

facili tate f lavor-i l lness l ea rn ing m a k i n g the an ima l s more in te l l igent in cop ing

wi th in ternal p r o b l e m s (20) . If w e were to s u m m a t e the effects across bo th

tasks w e w o u l d conc lude that the l e s ions h a d n o effect u p o n genera l in-

te l l igence. It is the concep t of genera l in te l l igence that m a k e s th is type of anal-

ys i s ludicrous . W h e n e v e r w e damage an imals b y wha teve r m e t h o d , pro te in

s tarvat ion, v i t amin def ic iency, or drugs , w e are apt to b e confronted b y incon-

gruous resul ts i f w e v i e w adapt ive b e h a v i o r as a genera l funct ion. O u r treat-

m e n t s wil l have specific effects u p o n specific mo t ive sys t ems , on specific

in tegra t ive sys t ems , and on specific perceptual sys t ems w h i c h cannot b e

FIGURE 4. Rats quickly and appropriately adapt to noises followed by peripheral pain or to flavors followed by illness. Conversely these animals are relatively insensitive to noise-illness and flavor-pain contingencies. Moreover, specific brain lesions may disrupt one form of adapta-tion while enhancing another making a notion of general adaptation untenable. Survival appears to depend upon a variety of specialized input-output systems selected by differential evolutionary pressures and controlled by differential neurological and genetic mechanisms.


accoun ted for b y a s ing le factor. Behav io r , l ike o the r b o d y func t ions , mus t

b e dif ferent ia ted in to i ts c o m p o n e n t e l ements and related to specific neura l

s t ructures . Gene ra l in te l l igence is abou t as useful for th is pu rpose as elan

vital i s to d iagnos t ic m e d i c i n e .

R E F E R E N C E S

1. Anastasi, A. Psychological Testing. Macmillan, New York, 1954.

2. Anastasi, A. Differential Psychology. (3rd ed.) Macmillan, New York, 1958.

3. Bolles, R. , and Seelbach, S. Punishing and reinforcing effects of noise onset and termination for different responses. / . Comp. Physiol. Psychol., 1964, 58: 127-131.

4. Bridgman, P. W. The Logic of Modern Physics. Macmillan, New York, 1927. 5. Burt, C. The inheritance of intelligence Am. Psychol., 1958, 13: 1-15.

6. Cattell, J . M. Mental tests and measurements. Mind, 1890, 15: 373-381.

7. Dobrzecka, C , Szwejkowska, G., and Konorski, J. Qualitative versus directional cues in two forms of differentiation. Science, 1966, 153: 87-89.

8. Galton, F. Hereditary Genius. Macmillan, New York, 1869.

9. Garcia, J . , and Koelling, R. A. Relation of cue to consequence in avoidance learning. Psychon.

Sci., 1966, 4: 123-124.

10. Garcia, J . , McGowan, B . , and Green, K. Biological constraints on conditioning. In: Classical Conditioning: Current Research and Theory. (A. H. Black and W. F. Prokasy, Eds.). Appleton, New York, 1972: 3-27.

11. Heber, R., Garber, H., Harrington, S., and Hoffman, C. Rehabilitation of families at risk for mental retardation. Unpublished research report, University of Wisconsin, Milwaukee.

12. Herrnstein, R. IQ. The Atlantic, 1971, 228: 43-58. 13. Honzik, M. P., Macfarlane, J . W. , and Allen, L. The stability of mental test performance

between two and eighteen years. / . Exp. Educ, 1948, 17: 309-324. 14. Huxley, A. Brave New World. Harper, New York, 1932. 15. Jensen, A. R. How much can we boost IQ and scholastic achievement? In: Environment, Hered-

ity, and Intelligence. Harvard Review Reprint Ser. No. 2. Harvard Univ. Press, Cambridge, Massachusetts, 1969: 1-123.

16. Kamin, L. J. The Science and Politics of IQ. Earlbaum Associates, Potomac, Maryland, 1974; Heredity, Intelligence, Politics and Psychology. Invited address to the Eastern Psychological Association, Washington, D.C. , May, 1973.

17. Lannholm, G. V . , and Schrader, W. B. Predicting Graduate School Success: An Evaluation of the Effectiveness of the Graduate Record Examination. Educational Testing Service, Princeton, New Jersey, 1951.

18. Lashley, K. S. Brain Mechanisms and Intelligence. Univ. of Chicago Press, Chicago, Illinois, 1929.

19. McCall, R. B . , Appelbaum, M. I., and Hogarty, P. S. Developmental changes in mental per-formance. Monogr. Soc. Res. Child Dev., 1973, 38: 1-83.

20. McGowan, B. K., Hankins, W. G., and Garcia, J . Limbic lesions and control of the internal and external environment. Behav. Biol, 1972, 7: 841-852.

21. Page, E. B . Miracle in Milwaukee: Raising the IQ. Educ. Res., 1972, 1: 8-16. . 22. Pavlov, I. P. Lectures on Conditioned Reflexes. International Publishers, New York, 1928. 23. Rosenthal, D. Genetic Theory and Abnormal Behavior. McGraw-Hill, New York, 1970. 24. Seligman, M. E. P., and Hager, J . L. Biological Boundaries of Learning. Appleton, New York,

1972. 25. Terman, L. M., and Merrill, M. A. Stanford-Binet Intelligence Scale. (3rd ed.) Houghton,

Boston, Massachusetts, 1960.

442 John Garcia

26. Terman, L. M., and Oden, M. H. The Gifted Child Grows Up. Stanford Univ. Press, Stanford, California, 1947.

27. Tryon, R. C. A theory of psychological components—an alternative to "mathematical" factors. Psychol. Rev., 1935, 42: 425-454.

28. Tryon, R. C. Genetic differences in maze-learning ability in rats. 39th Yearb. Natl. Soc. Stud. Educ, 1940, Part I: 111-119.

29. Wechsler, D. The Measurement and Appraisal of Adult Intelligence. Williams & Wilkins, Bal-timore, Maryland, 1958.

30. Wilcoxon, H., Dragoin, W. , and Krai, P. Illness-induced aversions in the rat and quail: Rela-tive salience of visual and gustatory cues. Science, 1971, 171: 826-828.

16 Hepatic Phenylalanine Hydroxylase and PKU

SEYMOUR KAUFMAN

Laboratory of N euro chemistry, National Institute of Mental Health,

Bethesda, Maryland

As is we l l - known , pheny lke tonu r i a ( P K U ) , w a s d i scovered in 1934 b y a

N o r w e g i a n b i o c h e m i s t and phys i c i an , Foi l ing . A y o u n g m o t h e r b rough t her

two menta l ly re tarded ch i ld ren to h i m and po in ted out that from early infancy

a mus ty odor s e e m e d to c l ing to the chi ldren . Foi l ing e x a m i n e d ur ine samples

for ace toacet ic ac id u s ing the F e C l 3 test bu t ins tead of ge t t ing a r e d - b r o w n

color , the u r ine tu rned green . H e u l t imate ly isola ted from the i r u r ine the c o m -

p o u n d r e spons ib l e , pheny lpyruv ic acid . H e later found that t hese ch i ld ren

also h a d h igh b l o o d levels of pheny la l an ine .

W h i l e these f indings sugges ted that there w a s s o m e d e r a n g e m e n t of pheny l -

a lan ine m e t a b o l i s m in th is n e w d i sease , our knowledge of the in t e rmed ia ry

m e t a b o l i s m of pheny la l an ine and tyros ine w a s too p r imi t ive to local ize pre-

c ise ly the me tabo l i c error. In fact, it w a s 13 years before th is w a s done .

Foi l ing , h imsel f , pos tu la ted that in P K U there w a s an a b n o r m a l r acemiza -

t ion of pheny la l an ine , the D - a m i n o acid g iv ing r ise to the pheny lpyruv ic ac id .

A n o t h e r h y p o t h e s i s a s s u m e d that the me tabo l i c b lock w a s an inab i l i ty to

b reak d o w n pheny lpyruv ic ac id .

If Foi l ing w a s the father of P K U , the m i d w i f e w a s Je rv i s (8) . In 1947 he per-

formed a ser ies of expe r imen t s that led to the correct ident i f ica t ion of the

me tabo l i c defect. H e found that the admin i s t r a t ion of pheny la l an ine to an-

imals or h u m a n s led to a p rompt increase in Mi l lon- reac t ive subs t ances in the

b lood ( this test is a nonspec i f ic o n e for ty ros ine) . W h e n , on the o ther h a n d ,

pheny la l an ine w a s g iven to P K U pa t ien t s , there w a s abso lu te ly no increase in

tyros ine . H e conc luded that the b lock lay b e t w e e n pheny la l an ine and tyros ine ,

that i s , in the hydroxyla t ion of pheny la l an ine . In 1953 , Je rv i s (9) p resen ted

445

446 Seymour Kaufman

proof for th is hypo thes i s w i th the first in vitro demons t ra t ion that a l iver

sample from a P K U pat ient could no t conver t pheny la lan ine to tyros ine . T h i s

result no t on ly es tab l i shed the s tep in m e t a b o l i s m that was b locked in P K U ,

but also p rov ided impor tan t suppor t ing ev idence for the no t ion that w a s

ga in ing c redence at the t ime , name ly , that the conver s ion of pheny la l an ine to

ty ros ine is an ob l iga tory s tep in the comple te ox ida t ion of pheny la l an ine to

CO2 and water ; in m a m m a l s there are n o al ternat ive pa thways w h e r e b y th is

can b e accompl i shed . Pheny la l an ine can unde rgo a l imi ted n u m b e r of o ther

me tabo l i c t ransformat ions in the a b s e n c e of hydroxyla t ion , bu t these all in-

volve t ransformat ions of the a lan ine s ide cha in , e .g . , d e a m i n a t i o n and oxida-

t ive decarboxyla t ion , to g ive phenylpyruva te and phenylace ta te .

It shou ld also b e no ted that be s ide s b e i n g an ob l iga tory react ion in the

comple te c o m b u s t i o n of pheny la l an ine , the hydroxyla t ion s tep also provides

an e n d o g e n o u s source of ty ros ine . W h e n th is react ion occurs , tyros ine is a

nonessen t i a l amino acid , w h i c h m e a n s that w e are no t d e p e n d e n t on a dietary

source of it. O b v i o u s l y , in a P K U pat ient , ty ros ine is an essent ia l a m i n o acid

and m u s t b e p rov ided in the diet .

O n e migh t well ask w h a t m o r e r e m a i n e d to b e done abou t the nature of the

me tabo l i c defect in P K U after J e rv i s ' work . After all, he did locate correct ly the

site of the me tabo l i c les ion in the d i sease , and h e did demons t ra te that

pheny la lan ine hydroxylase is inac t ive in the l ivers of P K U pat ien ts .

If the sys tem that catalyzes the hydroxyla t ion of pheny la l an ine were c o m -

posed of a s ingle c o m p o n e n t , the hydroxylase , J e rv i s ' wo rk i ndeed w o u l d have

c losed the chapter on th is aspect of the P K U story. As s u b s e q u e n t work

s h o w e d , howeve r , the hydroxyla t ing sys tem is a complex o n e , c o m p o s e d of

m a n y essent ia l c o m p o n e n t s , b o t h pro te in and nonp ro t e in in na ture , the lack

of any of w h i c h could lead to a nonfunc t iona l hydroxyla t ing sys tem. T h u s , the

early conc lus ion that a defect in the abi l i ty to conver t pheny la l an ine to tyro-

s ine could b e equa ted w i t h a defect in the e n z y m e , pheny la l an ine hydroxyl-

ase , was premature .

W e have b e e n s tudy ing the pheny la lan ine -hydroxy la t ing sys tem of m a m -

mal ian l iver for m a n y years , one of our goals b e i n g to try to d issect the sys tem

into its ind iv idua l c o m p o n e n t s so that a prec ise de l inea t ion of the affected

c o m p o n e n t in P K U could b e made .

Under ly ing our approach has b e e n the conv ic t ion that the level of our un-

ders tanding of th is d i sease can b e no deeper than the level of our under -

s tand ing of th is complex e n z y m e sys tem. T o give on ly one specific example of

the close connec t i on b e t w e e n these two a reas—be tween the bas ic and the

c l in ical—it is apparent that future approaches to therapy of th is d i sease wou ld

b e profoundly affected b y w h e t h e r the nonfunc t iona l c o m p o n e n t of the hy-

droxylat ing sys tem were an e n z y m e or a nonpro t e in c o e n z y m e .

W h e n Je rv i s did h i s work , the conver s ion of pheny la l an ine to tyros ine was

though t to b e a s t ra ightforward, s ing le-s tep reac t ion , that could b e desc r ibed as

s h o w n in F igure 1.

16. Hepatic Phenylalanine Hydroxylase and PKU 447

OH

COOH COOH

FIGURE 1. Early scheme for the enzymatic conversion of phenylalanine to tyrosine.

Figure 2 s h o w s today ' s formula t ion of the reac t ion . A s can b e s een , the reac-t ion is complex and is cata lyzed b y a sys t em of several e n z y m e s and coen -z y m e s w o r k i n g i n a coord ina ted m a n n e r .

I wou ld n o w l ike to desc r ibe these var ious c o m p o n e n t s and the roles that they play in the hydroxyla t ion sys tem.

T h e ear l ies t ind ica t ion that the hydroxyla t ion reac t ion w a s m o r e compl i -cated t han the s c h e m e s h o w n in F igure 1 c a m e from the f inding b y us and b y others that at least two e n z y m e s we re s o m e h o w invo lved in the reac t ion (11 ,22) . For r easons w h i c h I wi l l no t go in to n o w , w e purif ied o n e of these en-z y m e s from rat l iver and the o the r from s h e e p l iver extracts . Before w e k n e w thei r funct ion, w e referred to these two c o m p o n e n t s as the rat and s h e e p l iver e n z y m e s .

T h e next impor tan t s tep in the analys is of the sys t em c a m e w i t h the d iscov-

ery b y us in 1958 that a n e w c o e n z y m e p layed an essen t ia l role in the reac t ion

(12) . I n 1963 , w e ident i f ied th i s c o e n z y m e as an uncon juga ted p ter in , cal led

b iop te r in , w h o s e s tructure is s h o w n in F igure 3 (16) .

Severa l years before w e had de t e rmined the final s t ructure of the c o e n z y m e ,

we k n e w that it w a s an uncon juga ted p te r in and h a d p repared cer ta in s imple

c o m p o u n d s of th i s c lass that h a d h i g h cofactor act ivi ty . O n e of t hese , the 6 ,7-

FIGURE 2. Modern formulation of the enzymatic conversion of phenylalanine to tyrosine.

448 Seymour Kaufman

H H

CHo - C - C - ^ n I I

OH OH

DIHYDROBIOPTERIN

FIGURE 3 . Structure of dihydrobiopterin.

dimethy l c o m p o u n d , has b e e n u s e d ex tens ive ly as an ana log for the natural ly

occurr ing cofactor in the pheny la l an ine hydroxyla t ing sys tem. In fact, it was

th rough the use of th is analog that m u c h of the fundamenta l work on the en-

z y m e sys t em w a s carr ied out .

T h e s c h e m e in F igure 4 shows the reac t ions that the rat and s h e e p l iver en-

z y m e s catalyze (14 ,17) . T h e rat l iver e n z y m e is pheny la l an ine hydroxylase . It

catalyzes a coupled reac t ion in w h i c h b o t h pheny la lan ine and the te t rahydrop-

ter in are ox id ized to ty ros ine and the d ihydrop te r in , respec t ive ly , b o t h a toms

of molecular oxygen b e i n g reduced to the level o f hydroxyl groups in the

process . T h e m i n i m u m requ i r emen t s necessa ry for the conve r s ion of phenyl -

a lanine to tyros ine are the hydroxylase and its subs t ra tes—a te t rahydropter in ,

pheny la l an ine , and oxygen . It shou ld b e no ted , h o w e v e r , that o n e canno t get

m u c h tyros ine in th i s way ; as you can see , one gets one mole of tyros ine for

each mo le of t e t rahydrobiop te r in . S i n c e the concen t ra t ion of the cof actor in

l iver is p robab ly on ly abou t 1 X 1 0 " 6 M , one m u s t have a sys t em for regenera t -

ing the te t rahydro form o f the cofactor to al low it to funct ion catalyt ical ly. T h e

e n z y m e that fulfills th is funct ion is the sheep l iver e n z y m e , w h i c h catalyzes

the D P N H - or T P N H - m e d i a t e d reduc t ion of the d ihdro- to the te t rahydrop-

ter in (14) . T h i s reac t ion comple te s the cycle . In the test t ube , w e s h o w e d years

ago that h igh concen t ra t ions of reduc ing agen ts , such as mercap tans and as-

corba te , can also b e used to regenera te the te t rahydro form of the cofactor (14) ,

bu t in the cell , the reduc tase p robab ly performs this funct ion.

7,8-;

FIGURE 4. Pterin transformations during the enzymatic conversion of phenylalanine to tyrosine.


T h e s e are the four abso lu te ly essen t ia l c o m p o n e n t s of the hydroxyla t ion

sys tem: the two e n z y m e s , hydroxylase and reduc tase , and the two c o e n z y m e s ,

the te t rahydropte r in and the r educed pyr id ine nuc leo t ide . Clear ly the lack of

any of these c o m p o u n d s will lead to a nonfunc t iona l hydroxyla t ing sys tem.

T h e d ihydrop te r in that is p roduced from the te t rahydro c o m p o u n d dur ing

the hydroxyla t ion reac t ion is an exceed ing ly uns tab le q u i n o n o i d der iva t ive

(17) . Un le s s it i s rapidly r educed b a c k to the te t rahydro level , it rear ranges b y

t au tomer iza t ion to a more s tab le i somer , the 7 ,8 -d ihydrop te r in (14 ,17) . D e -

p e n d i n g o n the s t ructure of the p te r in u sed , th i s i s o m e r is e i ther inac t ive in

the sys t em (e .g . , as wi th the 6 ,7 -d imethy l c o m p o u n d ) or it can b e b rough t

b a c k in to the sys t em b y still ano the r e n z y m e , dihydrofolate reduc tase , as in

the case w i t h 7 ,8 -d ihydrob iop te r in (18) . F igure 4 shows the s c h e m e w i t h th is

extra reac t ion inc luded . A l though dihydrofolate reductase is an essent ia l c o m -

p o n e n t of the sys t em w h e n o n e starts w i th 7 ,8 -d ihydrob iop te r in , w e do no t

k n o w wi th cer ta in ty if i t plays any role in the hydroxy la t ion reac t ion in vivo.

There are two reasons for suspec t ing that it does : (a) in all p robab i l i t y , the de

novo syn thes i s of b iop te r in p roceeds th rough the 7 ,8 -d ihydro der iva t ive so that

d ihydrofolate reduc tase wou ld b e obl iga tor i ly r equ i red to conver t th i s com-

p o u n d to the te t rahydro level ; (b) d ihydrofola te reductase p r o b a b l y serves to

salvage any 7 ,8 -d ihydrob iop te r in that m i g h t b e formed from the q u i n o n o i d

c o m p o u n d in any s i tua t ion w h e r e the d ihydrop te r id ine reductase-ca ta lyzed

reac t ion is no t fast e n o u g h to keep pace wi th the hydroxylase .

M o r e recent ly , w e have s h o w n that still ano ther c o m p o n e n t plays a role in

the hydroxyla t ion reac t ion . T h i s is a pro te in that w e have purif ied to h o m o g e -

ne i ty from rat l iver and w h i c h w e have cal led pheny la l an ine hydroxylase -

s t imula t ing pro te in or P H S (7 ,19) . W e have ev idence to suppor t the idea that

P H S is an e n z y m e that ca ta lyzes the b r e a k d o w n to products of an in t e rmed ia te

in the hydroxyla t ion react ion (6) . It is on ly r equ i red w h e n t e t rahydrob iop te r in

is u sed as the cofactor; w h e n the 6 ,7 -d imethy lp te r in is u sed , w e t h ink that the

ana logous in te rmed ia te b reaks d o w n rapid ly even in the a b s e n c e of P H S .

Final ly , there are c o m p o n e n t s that m a y b e invo lved in the sys t em in a less

direct way . W e have s h o w n wi th the purif ied e n z y m e that there is a marked

s t imula t ion of the rate of hydroxyla t ion b y catalase or pe rox idase (15) . T h e

reason for th is r e q u i r e m e n t is the sens i t iv i ty of the hydroxylase to H 2 0 2 .

W e have also s h o w n that cer ta in phospho l i p id s , such as lyso lec i th in , can

s t imula te the purif ied hydroxy lase 3 0 - to 50-fold. Jus t as w i t h the P H S requ i re -

men t , phospho l ip id s on ly s h o w a m a r k e d s t imula t ion w h e n te t rahydro-

b iop te r in is the cofactor (1) .

T h e last c o m p o n e n t to b e m e n t i o n e d is i ron . W e have recent ly s h o w n that

pheny la l an ine hydroxylase from rat l iver is an i r o n - e n z y m e (2) . A l t h o u g h the

e n z y m e from w h i c h i ts i ron is r e m o v e d is inac t ive , the meta l is no t readi ly

separa ted from the i r o n - e n z y m e .

To s u m m a r i z e , there are 4 factors that are direct ly invo lved in the react ion:

pheny la l an ine hydroxy lase , d ihydrop te r id ine reductase , t e t rahydrobiopte r in ,

450 Seymour Kaufman

and reduced pyr id ine nuc leo t ide . Pe rhaps p lay ing a less dynamic role is a fifth

factor, d ihydrofolate reductase . A n d finally, the three c o m p o n e n t s that s t imu-

late the hydroxyla t ion reac t ion on ly u n d e r specia l c i r c u m s t a n c e s — P H S , phos -

pho l ip id , and i ron.

As progress w a s m a d e in ana lyz ing the c o m p o n e n t s of the hydroxyla t ing

sys tem, there has b e e n parallel progress in def ining m o r e prec ise ly the af-

fected c o m p o n e n t in P K U .

T h e first s tep b e y o n d Je rv i s ' or ig inal work was accompl i shed after i t had

b e e n s h o w n that at least two e n z y m e s are invo lved in the hydroxyla t ion reac-

t ion. In 1957 , M i t o m a and M e i s t e r and thei r co-workers (23,24) s h o w e d that

one e n z y m e , the " s t a b l e " o n e , w a s presen t and the o ther , the " l a b i l e " one ,

was m i s s i n g in P K U l iver samples . Later , we s h o w e d that the " l a b i l e " e n z y m e

is the hydroxylase and the " s t a b l e " one is d ihydrop te r id ine reductase .

T h e next s tep in th i s ana lys is w a s m a d e poss ib l e b y our demons t ra t ion that

a p ter in c o e n z y m e is an essen t ia l c o m p o n e n t of the sys tem. In 1958 , we

s h o w e d that th is c o e n z y m e is p resen t in no rmal a m o u n t s in a ser ies of l iver

b i o p s y samples from P K U pat ien ts (13) . T h i s f inding appeared at last to put on

a firm b a s i s the conc lus ion that the hydroxylase is the m i s s i n g c o m p o n e n t in

P K U .

It is wor th e x a m i n i n g jus t h o w firmly this fact has b e e n es tab l i shed . Unt i l

qu i te recent ly , there w a s still a small degree of uncer ta in ty abou t the conclu-

s ion . T h e reason for th i s doub t s t ems from the type of ev idence that w a s avail-

able . In prac t ice , the type of expe r imen t that one does i s , first, to s h o w that

the t i ssue extract from the P K U pat ien t is inac t ive , and second , to supp lemen t

the extract w i th the ind iv idua l c o m p o n e n t s o f the sys tem—idea l ly , one at a

t ime—unt i l the act ivi ty is res tored. W h a t actually w a s done in all of the earl ier

expe r imen t s was to res tore act ivi ty w i th the addi t ion of fairly c rude prepara-

t ions of the hydroxylase . It is ev iden t that i f s u b s e q u e n t work had led to a

resolut ion of that crude hydroxylase in to two or m o r e c o m p o n e n t s , some un-

cer ta in ty wou ld have b e e n in t roduced into the or ig ina l conc lus ion .

In 1970 , w e purif ied the hydroxylase from rat l iver to a s tage w h e r e it is

abou t 9 5 % pure (20) . In our m o s t recent expe r imen t s on P K U l iver samples ,

w e were able to res tore act ivi ty w i th the add i t ion of this essent ia l ly pure en-

z y m e . T h e addi t ion of n o n e of the o ther c o m p o u n d s that I have desc r ibed

could subs t i tu te for the hydroxylase . Fur the rmore , direct assays s h o w e d that

P H S , the mos t recent ly ident i f ied pro te in c o m p o n e n t o f the sys t em, is present

in no rmal amoun t s in the P K U liver sample (7) . F r o m these resul ts , w e can

conc lude that the hydroxylase is firmly e s t ab l i shed as the on ly affected c o m p o -

nen t of the hydroxylase sys t em in P K U .

W e have conf i rmed and further s t r eng thened the above conc lus ion wi th i m -

munolog ica l m e t h o d s . W e have prepared a specific an t i se rum, in sheep , to

pure rat l iver pheny la l an ine hydroxylase (5) . U s i n g the doub le immunod i f -

fusion t e c h n i q u e , w e have s h o w n that this an t i se rum wil l g ive on ly a s ingle


prec ip i t in l ine w h e n tes ted agains t unfrac t ionated rat l iver extracts . T h e an-

t i se rum cross reacts w i t h normal h u m a n pheny la l an ine hydroxylase (5) . U n d e r

cond i t i ons w h e r e 5 to 1 0 % of the n o r m a l h u m a n l iver e n z y m e cou ld b e de -

tec ted , n o p rec ip i t in l ine w a s seen w i t h an extract from a P K U l iver sample (4) .

A s a l ready m e n t i o n e d , these resul ts demons t ra t e conc lus ive ly that the hydrox-

ylase is the affected c o m p o n e n t in P K U .

T h e next ques t i on that w e addressed ourse lves to w a s the na ture of the

gene t ic defect that leads to a loss o f the ac t ive hydroxylase . T h e resul ts of our

i m m u n o l o g i c a l s tud ies ruled out on ly a s ing le poss ib i l i ty : no rmal a m o u n t s of a

mutan t form of the hydroxylase wi th all of i ts an t igen ic de t e rminan t s intact ,

bu t devo id of hydroxylase act ivi ty , we re no t p resen t in P K U liver . O u r results

left o p e n the poss ib i l i ty that P K U w a s caused b y a de le t ion mu ta t i on of the

gene that codes for the hydroxylase .

W i t h the ident i f ica t ion of wha t w e h o p e are all of the essent ia l c o m p o n e n t s

of the hydroxyla t ing sys t em, w e felt that i t m i g h t b e poss ib l e to dev ise an

assay for the hydroxylase that w o u l d b e cons ide rab ly m o r e sens i t ive than

those that w e , or a n y b o d y e lse , h a d u sed in earl ier s tudies on P K U liver . In

part icular , w e w a n t e d to try to exploi t two character is t ics o f the e n z y m e : (a)

the dramat ical ly lower KM o f the e n z y m e for pheny la l an ine in the p resence of

t e t r ahydrob iop te r in as c o m p a r e d to i ts KM in the p resence of, for example , the

6 ,7 -d imethy l te t rahydrop te r in (19) , and (b) the s t imula t ion of the hydroxylase

b y lyso lec i th in i n t he p r e sence of t e t r ahydrob iop te r in (1) . W e an t ic ipa ted , of

course , that c rude prepara t ions of the hydroxylase wou ld no t b e l ikely to s h o w

the s a m e dramat ic s t imula t ion b y the p h o s p h o l i p i d as that of the purif ied en-

zyme .

In 1 9 7 3 , w e o b t a i n e d a b i o p s y l iver sample from a pa t ien t w i t h classic P K U

and assayed it for hydroxylase act iv i ty u s ing the m o r e sens i t ive assay cond i -

t ions w h i c h have jus t b e e n ou t l ined . W e de tec ted a small a m o u n t o f e n z y m e -

dependen t conve r s ion of 1 4 C - l a b e l e d pheny la l an ine to 1 4 C - l a b e l e d ty ros ine (3) .

T h e hydroxylase ac t iv i ty (per mi l l ig ram pro te in) was equa l to abou t 0 . 2 7 % of

that p resen t in a l iver extract from a n o n - P K U control . I should po in t ou t that

th is va lue is wel l b e l o w (by a factor of 20 to 40) the l imi t s of de tec t ion of the

i m m u n o l o g i c a l tests that w e h a d used previous ly . T h e hydroxyla ted product

was unequ ivoca l ly ident i f ied as ty ros ine b y ch romatograph ic and enzymat i c

m e t h o d s (3) .

B e c a u s e w e are dea l ing w i t h m i n u t e a m o u n t s of act ivi ty , and b e c a u s e there

is s o m e cross-speci f ic i ty a m o n g the var ious p t e r in -dependen t a romat ic a m i n o

acid hydroxylases—for example , adrenal and b ra in ty ros ine hydroxylase and

p inea l t ryp tophan hydroxylase can conver t pheny la l an ine to t y ros ine—we tr ied

to charac ter ize the hydroxyla t ing e n z y m e in the P K U l iver sample . For tu-

na te ly , ra ther specific i n h i b i t o r s of the th ree p t e r in -dependen t hydroxylases

are avai lable . T h e resul ts of our ana lys is are s h o w n in T a b l e I. Firs t , it can b e

seen that ty ros ine format ion is comple te ly dependen t on added te t rahydro-

452 Seymour Kaufman

Table I Characteristics of the Enzyme from the Liver of a Patient with

PKU that Converts 1 4C-Phenylalanine to 1 4C-Tyrosine

Additions (+) or Omissions (—) Activity (pmole tyrosine/hour/mg of protein)

Experiment A" Control 27

— Lysolecithin 12.5

—Tetrahydrobiopterin 0.15

Experiment Bb

Control 30 + 0 . 1 mM 3-iodotyrosine 26

Experiment C

Control 29 + 1 m M p-chlorophenylalanine 0.0

+ 1 mM 6-fluorotryptophan 27

+Adrenal tyrosine hydroxylase 13,600 +Adrenal tyrosine hydroxylase

+ 0 . 1 mM 3-iodotyrosine 250

" Total volume of reaction mixture was 0.05 ml. The extract was treated with ammonium sulfate to 75% of saturation, and the precipitate was dissolved in 0.12 M KC1 buffered with 0.01 MTris-HCl (pH 7.0). Four hundred micrograms of this ammonium sulfate fraction was added to each assay. Phenylalanine concentration was 0.5 m M (300,000 cpm/tube), and tetrahydrobiopterin concentra-tion was 0.05 m M . Each value has been corrected for any radioactivity in the tyrosine area (equiva-lent to 24 pmole tyrosine/hour/milligram of protein) after incubation with a boiled enzyme control. b Total volume was 0.1 ml; 0.05 ml of extract, containing 1 mg of protein, was added to each tube. Phenylalanine concentration was 0.11 m M (750,000 cpm), and tetrahydrobiopterin concentration was 0.025 mM. The radioactivity in the tyrosine area after incubation with a boiled enzyme control was equivalent to 31 pmole tyrosine/hour/milligram of protein. c Total volume was 0.05 ml; 0.015 ml (0.3 mg of protein) of extract was added to each tube. Phenyl-alanine concentration was 0.036 mM (375,000 cpm), and tetrahydrobiopterin concentration was 0.03 mM. The radioactivity in the tyrosine area after incubation with a boiled enzyme control was equivalent to 14 pmole tyrosine/hour/milligram of protein. The tyrosine hydroxylase used had been purified through the substituted Sepharose step from bovine adrenal glands. Thirty micro-grams of this fraction was added to each reaction tube assayed. The tyrosine concentration was 0.05 mM (400,000 cpm/tube), and the tetrahydrobiopterin concentration was 0.1 mM.

b iop te r in and is s t imula ted more than 2-fold b y lysolec i th in . S i n c e l iver has

detec table amoun t s of ty ros ine hydroxylase , p r e sumab ly local ized to the nerve

end ings in that o rgan , it w a s poss ib l e that w e were mere ly de tec t ing th is en-

z y m e in our P K U l iver sample . T h e resul ts of expe r imen t B (Table I) rule out

this poss ib i l i ty . T h e po ten t and specific ty ros ine hydroxylase inh ib i to r , 3-

iodo tyros ine , i n h i b i t e d tyros ine format ion b y only 1 4 % . S i n c e rat l iver phenyl -

a lanine hydroxylase is not i n h i b i t e d at all b y th is concen t ra t ion of iodo-

tyros ine , w e can conc lude that no m o r e than 1 4 % of the hydroxyla t ion that we

obse rved could b e due to ty ros ine hydroxylase . It can also b e calculated (from

the repor ted act iv i ty o f ty ros ine hydroxylase in l iver) that our obse rved spe-


cific act ivi ty of pheny la l an ine hydroxylase in the P K U l iver sample is abou t 50

t imes greater than the repor ted value for ty ros ine hydroxylase . T h e resul ts of

expe r imen t C (Table I) s h o w that the act ivi ty is sens i t ive to the nonspec i f ic

a m i n o acid hydroxy lase i nh ib i to r , p -ch lo ropheny la lan ine , w h e r e a s it is i n sen -

si t ive to the ra ther specific i n h i b i t o r of t ryp tophan hydroxylase , 6-f luorotryp-

tophan . T h e results of these s tudies s t rongly ind ica te that at least 8 6 % of

pheny la lan ine -hydroxy la t ing act iv i ty in th i s s ample is due to a form of pheny l -

a lan ine hydroxylase .

T h e de tec t ion of s o m e pheny la lan ine hydroxylase in P K U l iver obv ious ly

rules out the poss ib i l i ty that there is a de le t ion of the s tructural gene for the

hydroxylase as the gene t ic ba s i s for th is d i sease . T h e resul ts are compa t ib l e

w i th the idea that the hydroxylase act ivi ty in classical P K U is due to the pres-

ence of e i ther no rma l hydroxylase in ex t remely l o w concen t ra t ion or a s tructur-

ally al tered hydroxylase w i t h l o w catalytic act ivi ty.

In an a t tempt to d i s t ingu i sh b e t w e e n these two a l ternat ives , w e c o m p a r e d

s o m e of the k ine t i c p roper t ies o f the hydroxylase in no rma l and i n P K U liver .

T h e first difference is in the degree of s t imula t ion b y lyso lec i th in . As al ready

po in ted out ( expe r imen t A , Tab le I ) , the e n z y m e in P K U l iver is s t imula ted

abou t 2-fold b y lyso lec i th in . W e have prev ious ly s h o w n that t he no rma l

h u m a n l iver e n z y m e is s t imula ted 3 - to 4-fold. It is pos s ib l e , bu t not l ikely,

that th is represen ts a difference in e n d o g e n o u s p h o s p h o l i p i d con ten t b e t w e e n

the two l ivers , ra ther t han a difference in proper t ies b e t w e e n the two en -

z y m e s .

A more c o n v i n c i n g difference in p roper t i es w a s uncove red w h e n w e deter-

m i n e d the apparent KM va lue for pheny la l an ine . A l though the KM va lues , t hem-

selves , m a y no t b e s ignif icant ly different, the resul ts s h o w n in F igures 5 A

and B ind ica te that the e n z y m e from P K U liver is no t i n h i b i t e d b y excess

pheny la l an ine up to 0 .2 m M . B y contrast , the n o r m a l h u m a n e n z y m e i s 5 0 %

i n h i b i t e d at 0 .14 m M pheny la l an ine (3) .

F inal ly , the capac i ty of the an t i se rum to rat l iver hydroxylase to i n h i b i t the

no rma l and P K U l iver hydroxylases w a s s tudied . It was found that the max-

i m u m i n h i b i t i o n that could b e obse rved was 6 4 % wi th the no rma l and 1 8 %

wi th the P K U liver hydroxylases (3) .

All our resul ts , s u m m a r i z e d in T a b l e I I , ind ica te that i n th i s P K U pat ien t

there is a s tructural ly al tered hydroxylase w i th ve ry low act ivi ty ra ther than a

very low a m o u n t of no rma l e n z y m e , i . e . , th is P K U pat ien t has a mu ta t ion in

the g e n e cod ing for the s t ructure of pheny la l an ine hydroxylase .

To expla in w h y no c ross - reac t ing pro te in could b e de tec ted in P K U l iver

wi th our specific an t i s e rum to pheny la l an ine hydroxylase , w e can p ropose

several a l ternat ive mode l s : (a) the muta t ion in pheny la l an ine hydroxylase no t

only affects the e n z y m e ' s catalytic p rop te t ies , bu t also i ts an t igen ic deter-

m i n a n t s in such a w a y that it no longer forms a prec ip i ta t ing c o m p l e x w i t h the

an t ibod ie s . A c c o r d i n g to th is mode l , P K U liver m a y con ta in a n o r m a l a m o u n t

454 Seymour Kaufman

1 C

8 0 .06 -

V> I 0 . 0 4

^ Normal Liver E • Exp I

5 00J> * Exp n ILL

1 1 1 1 1 1 1 I

- 4 0 - 2 0 0 2 0 4 0 6 0 8 0 100 120

I / L - P H E N Y L A L A N I N E , m M " 1

FIGURE 5 A and B. A comparison of the reciprocal plots of phenylalanine concentration against

specific activity for the normal and the PKU liver enzyme.

Table II Properties of a PKU and a Normal Human Liver Phenylalanine Hydroxylase

Property Normal PKU

KM for phenylalanine" 0.028-0.039 m M 0.037 mM Substrate inhibition above

0.1 mM phenylalanine Present None Specific activity''

(nmole tyrosine/hour/milligram protein) 56 0.150 Stimulation by lysolecithin (%) 300-400 100 Inhibition by antiserum (%) 63 18

a In the presence of tetrahydrobiopterin. b V m a x with tetrahydrobiopterin and lysolecithin.

I / L - P H E N Y L A L A N I N E , m M " 1

ro

\ E

/v (

pm

ole

s

Ty

ros

ine

/hr/

mg

)"1

° \

- 4 C


of the structurally al tered hydroxylase b u t the m e t h o d used canno t detect it;

and (b) the al tered hydroxylase is syn thes i zed at a no rma l rate bu t is degraded

more rapid ly than i s the n o r m a l e n z y m e , pe rhaps as a c o n s e q u e n c e of i ts al-

tered structure. Accord ing to th is mode l , P K U l iver con ta ins less than the

normal a m o u n t of the structurally al tered hydroxylase . O t h e r mode l s could

also b e dev i sed that could account for our resul ts .

S i n c e there is n o reason to suspec t that th is P K U pat ien t is any th ing bu t a

typical o n e , w e be l i eve that the above conc lus ion wil l apply to mos t , i f no t all,

pa t ien ts w i t h classic P K U . Jus t h o w genera l the conc lus ion is can only b e deter-

m i n e d b y future s tud ies on the hydroxylase in o ther P K U pat ien ts .

T h e de l inea t ion of the ind iv idua l c o m p o n e n t s of the hydroxyla t ing sys t em

also p rov ides a pe rspec t ive for a cons ide ra t ion of poss ib l e var ian ts of the

d i sease . Firs t , there are the muta t ions that lead to a structural modi f ica t ion of

the hydroxylase that are different from the o n e seen in classic P K U . It i s l ike ly

that h y p e r p h e n y l a l a n i n e m i a falls in to th is ca tegory . Cl in ica l ly , th i s cond i t ion

is a mi ld form of P K U w h i c h does not appear to lead to men ta l re tardat ion. W e

s h o w e d that these pa t ien ts have abou t 5 % of the no rma l a m o u n t of pheny l -

a lan ine hydroxylase (10 ,21) . The re are ind ica t ions that th i s low act iv i ty is due

not to the p re sence of 5 % of the no rma l e n z y m e , b u t ra ther to the p re sence of

an al tered hydroxylase w i t h k ine t ic proper t ies that d i s t ingu i sh it from b o t h

the normal e n z y m e and the e n z y m e presen t in classical P K U . T h e m a i n dif-

ference is in the KM va lue for pheny la l an ine m e a s u r e d i n the p re sence of the

d ime thy lpter in. In three different h y p e r p h e n y l a l a n i n e m i a pa t i en t s , the KM val-

ues w e r e abou t 6 0 % of those found for the e n z y m e from three different control

pa t ien ts (4) .

Comple t e ly different types of var iants of P K U are theore t ica l ly poss ib l e in

w h i c h one of the o ther essen t ia l c o m p o n e n t s o f the hydroxyla t ing sys t em are

lacking , e .g . , the p ter in cofactor , or d ihydrop te r id ine reductase .

S i n c e ty ros ine and t ryp tophan hydroxy lases , the e n z y m e s invo lved in the

b i o s y n t h e s i s of n o r e p i n e p h r i n e (and ep ineph r ine ) and se ro ton in , respec t ive ly ,

also r equ i re these two c o m p o n e n t s , these two hypothe t i ca l var ian ts o f P K U

should b e charac te r ized b y addi t ional b i o c h e m i c a l abnorma l i t i e s , and should

therefore b e d i s t i ngu i shab le from classic P K U .

T w o o ther c o m p o n e n t s of the sys t em that m a y play an essen t ia l role in

pheny la l an ine hydroxyla t ion in vivo are d ihydrofola te reductase and P H S , the

recent ly i so la ted s t imula t ing pro te in . B e c a u s e of i ts cri t ical i nvo lvemen t in

o ther areas o f i n t e rmed ia ry m e t a b o l i s m , a comple te def ic iency of d ihydrofo-

late reductase w o u l d a lmost cer ta in ly b e a lethal mu ta t ion that w o u l d neve r b e

encoun te red in pract ice . A muta t ion that led to a loss o f P H S w o u l d p r o b a b l y

result in a mi ld form of P K U .

I shou ld l ike to e m p h a s i z e that a l though the a m o u n t of hydroxylase act ivi ty

that w e have found in the P K U l iver sample is ve ry low, it is p rofoundly dif-

ferent from zero act ivi ty . T h e difference can b e l ikened to that b e t w e e n a w e a k

456 Seymour Kaufman

heart bea t and no hear t bea t . No t on ly do these resul ts p rove that w e are not

dea l ing wi th a de le t ion muta t ion , they also m a y provide a ba s i s for al ternate

therapy in P K U . For the first t ime it b e c o m e s ra t ional to try to find w a y s in

w h i c h th i s small level of ac t iv i ty m i g h t b e ra ised in pa t ien ts . Pr ior to our

resul ts , the on ly w a y in w h i c h the hydroxylase act ivi ty migh t have b e e n

increased in such pa t ien ts wou ld have b e e n b y supply ing e i ther the e n z y m e

itself, o r the structural g e n e that codes for it.

At first g lance it m i g h t s e e m that there wou ld b e little chance of ever ra is ing

th is low level of act ivi ty to a therapeut ica l ly s ignif icant value . To ach ieve

no rma l levels , after all, an e n h a n c e m e n t factor o f a lmost 400 wou ld b e

requ i red . W e k n o w , howeve r , that na ture has p rov ided normal m a n wi th a

very comfor table excess o f the e n z y m e . W e k n o w th is because of an exper i -

m e n t that na ture has per formed for us , i . e . , the d i sease hyperpheny la lan in -

emia . A s already m e n t i o n e d , these pa t ien t s do no t appear to suffer from

menta l re tardat ion in spi te of the fact that , as w e have s h o w n , they possess

only 5 % of the normal levels of pheny la l an ine hydroxylase . T h e e n h a n c e m e n t

factor that is r equ i red to ach ieve normal b ra in funct ion is therefore abou t 18 ,

ra ther than 4 0 0 . Indeed , th is value is an upper l imi t ; w e do no t k n o w h o w low

a level of pheny la lan ine hydroxylase is r equ i red to afford comple te pro tec t ion

to the deve lop ing bra in—al l w e k n o w is that it l ies s o m e w h e r e b e t w e e n 0.27

and 5 % of the normal value . B a s e d on these cons ide ra t ions , w e have b e e n en-

couraged to try to explore w a y s in w h i c h the hydroxylase act ivi ty can b e

e n h a n c e d in vivo. O n e of the m o r e p r o m i s i n g poss ib i l i t i e s is that the hydroxyl-

ase act ivi ty m i g h t b e inc reased b y admin is t ra t ion of e i ther the p ter in cofactor,

itself, or an analog of the cofactor that has even more favorable proper t ies .

S U M M A R Y

T h e conver s ion of pheny la l an ine to ty ros ine in m a m m a l i a n t i s sues is cata-

lyzed b y a complex e n z y m e sys t em c o m p o s e d of several essen t ia l e n z y m e s and

cof actors . All of these c o m p o n e n t s have b e e n assayed in l iver b i o p s y samples

from pa t ien ts w i th the classic form of P K U . T h e y are all p resent except for

pheny la l an ine hydroxylase , thus e s t ab l i sh ing th is e n z y m e as the m i s s i n g

c o m p o n e n t . T h i s conc lus ion has b e e n conf i rmed in immuno t i t r a t i on exper i -

m e n t s w i t h a specific an t i se rum to pheny la l an ine hydroxylase .

W i t h the use of a h i g h l y sens i t ive assay for the hydroxylase , 0 . 2 7 % of the

normal act ivi ty of pheny la l an ine hydroxylase has b e e n detec ted in a l iver

sample from a pa t ien t w i t h classic P K U . The re is s o m e ev idence that th is low

level of catalytic act ivi ty is due to the p re sence of a mu tan t form of the e n z y m e

rather than to very low levels of the normal e n z y m e . T h e s e results rule out the

poss ib i l i ty that classic P K U is caused b y a de le t ion muta t ion .

T h e f inding that the proper t ies of the e n z y m e are different from the normal


e n z y m e also sugges t s that the low hydroxylase ac t iv i ty in P K U is not caused

b y a regulatory gene muta t ion , bu t ra ther b y a mu ta t ion in the gene that codes

for the structure of the hydroxylase .

R E F E R E N C E S

1. Fisher, D. B . , and Kaufman, S. The stimulation of rat liver phenylalanine hydroxylase by lysolecithin and a-chymotrypsin. / . Biol. Chem., 1973, 248: 4 3 4 5 ^ 3 5 3 .

2. Fisher, D. B . , Kirkwood, R., and Kaufman, S. Rat liver phenylalanine hydroxylase, an iron en-zyme. /. Biol. Chem., 1972, 247: 5161-5167.

3. Friedman, P. A., Fisher, D. B . , Kang, E. S., and Kaufman, S. Detection of hepatic 4-phenylalanine hydroxylase in classical phenylketonuria. Proc. Natl. Acad. Sci. USA, 1973, 70: 552-556.

4. Friedman, P. A., Kaufman, S., and Kang, E. S. Nature of the molecular defect in PKU and hyperphenylalaninaemia. Nature (London), 1972, 240: 157-159.

5. Friedman, P. A., Lloyd, T., and Kaufman, S. Production of antibodies to rat liver phenyl-alanine hydroxylase: Cross-reactivity with other pterin-dependent hydroxylases. Mol. Phar-macol, 1972, 8: 501-510.

6. Huang, C. Y . , and Kaufman, S. Studies on the mechanisms of action of phenylalanine hydrox-ylase and its protein stimulator. / . Biol Chem., 1973, 248: 4242-^251.

7. Huang, C. Y . , Max, E. E . , and Kaufman, S. Purification and characterization of phenylalanine hydroxylase stimulating protein from rat liver. / . Biol Chem., 1973, 248: 4235-4241.

8. Jervis, G. A. Studies on phenylpyruvic oligophrenia. The position of the metabolic error. / . Biol Chem., 1947, 169: 651-656.

9. Jervis, G. A. Phenylpyruvic oligophrenia deficiency of phenylalanine-oxidizing system. Proc. Soc. Exp. Biol Med., 1953, 82: 514-515.

10. Kang, E. S., Kaufman, S., and Gerald, P. S. Clinical and biochemical observations of patients with atypical phenylketonuria. Pediatrics, 1970, 45: 83-92.

11. Kaufman, S. The enzymatic conversion of phenylalanine to tyrosine. / . Biol Chem., 1957, 226: 511-524.

12. Kaufman, S. A new cofactor required for the enzymatic conversion of phenylalanine to tyro-sine. / . Biol. Chem., 1958, 230: 931-939.

13. Kaufman, S. Phenylalanine hydroxylation cofactor in phenylketonuria. Science, 1 9 5 8 ,128:1 5 0 6 . 14. Kaufman, S. Studies on the mechanism of the enzymatic conversion of phenylalanine to tyro-

sine. / . Biol Chem., 1959, 234: 2677-2682. 15. Kaufman, S. Aromatic hydroxylation. In Oxygenases. (O. Hayaishi, Ed.). Academic Press, New

York, 1962: 129-179.

16. Kaufman, S. The structure of phenylalanine hydroxylation cofactor. Proc. Nat. Acad. Sci. USA, 1963, 50 : 1085-1093.

17. Kaufman, S. Further studies on the structure of the primary oxidation formed from tetrahy-dropteridines during phenylalanine hydroxylation. / . Biol. Chem., 1964, 239: 332-338.

18. Kaufman, S. Metabolism of the phenylalanine hydroxylation cofactor. / . Biol. Chem., 1967, 242: 3934-3943.

19. Kaufman, S. A protein that stimulates rat liver phenylalanine hydroxylase. /. Biol. Chem., 1970, 245: 4751-4759.

20. Kaufman, S., and Fisher, D. B . Purification and some physical properties of phenylalanine hydroxylase from rat liver. / . Biol Chem., 1970, 245: 4 7 4 5 ^ 7 5 0 .

21. Kaufman, S., and Max, E. E. Studies on the phenylalanine hydroxylating system in human liver and their relationship to pathogenesis of PKU and hyperphenylalaninemia. In Phenylke-

458 Seymour Kaufman

tonuria and Some Other Inborn Errors of Amino Acid Metabolism. (H. Bickel, F. P. Hudson, and L. I. Woolf, Eds.). Thieme, Stuttgart, 1971: 13-19.

22. Mitoma, C. Studies on partially purified phenylalanine hydroxylase. Arch. Biochem. Biophys., 1956, 60: 476-^84.

23. Mitoma, C , Auld, R. M., and Udenfriend, S. On the nature of enzymatic defect in phenyl-pyruvic oligophrenia. Proc. Soc. Exp. Biol. Med., 1957, 94: 634-635.

24. Wallace, H. W. , Moldave, K., and Meister, A. Studies on conversion of phenylalanine to tyrosine in phenylpyruvic oligophrenia. Proc. Soc. Exp. Biol. Med., 1957, 94: 632-633.

16A Discussion: Phenylalanine Hydroxylase in

Human Kidney; Relevance to Phenylketonuria

JUNE E. AYLING Mental Retardation Research Center and Department of Biological Chemistry,



Pheny lke tonu r i a w a s or ig ina l ly desc r ibed as he red i t a ry men ta l re tardat ion

assoc ia ted w i t h pheny lpyruv ic ac id in the u r ine and a h i g h p l a sma pheny l -

a lan ine level . F r o m l iver b i o p s i e s it w a s s u b s e q u e n t l y s h o w n that th i s d isorder

resul ted from a defect ive hepa t i c pheny la l an ine hydroxylase (10 ,18) . Severa l

forms of th is d i sease have n o w b e e n desc r ibed . C a s e s of c lass ic pheny lke -

tonur ia h a v e b e e n repor ted that pos se s s n o e n z y m e act iv i ty in l iver b i o p s i e s

(8 ,9 ) , w h i l e h y p e r p h e n y l a l a n i n e m i a has b e e n assoc ia ted w i t h part ia l act ivi ty

(8 ,9 ,12 ) . A mi ld form of pheny lke tonu r i a has b e e n n o t e d in w h i c h the pa t ien t ,

t hough lack ing l iver pheny la l an ine hydroxylase , d isplays on ly modera te ly

h igh p l a sma pheny la l an ine levels . It h a s b e e n sugges ted that ind iv idua l s w i t h

the " m i l d " form m a y h a v e al ternate m e c h a n i s m s for m e t a b o l i z i n g or d is -

pos ing of pheny l a l an ine (9) .

Unt i l recent ly pheny la l an ine hydroxy lase w a s cons ide red to b e a l iver-

specific e n z y m e , and in pa t i en t s w i t h pheny lke tonu r i a on ly the l iver has b e e n

assayed for e n z y m e act ivi ty . In the last few years several inves t iga tors have

demons t ra t ed that the k i d n e y s of m o u s e ( 5 , 1 4 , 1 7 ) , rat (5 ,14 ) , and gu inea p ig (5)

possess a pheny la l an ine -hydroxy la t ing sys t em. Recen t ly w e have repor ted the

p re sence of th is e n z y m e act iv i ty in h u m a n k i d n e y (3) . T h i s chap te r s u m m a -

r izes our c o m p a r i s o n o f h u m a n l iver and k i d n e y levels and a t t empts to dif-

ferent iate b e t w e e n the two e n z y m e s .

H u m a n pheny la l an ine hydroxylase is ex t remely lab i le , and t i s sues o b t a i n e d

unde r no rma l au topsy cond i t i ons re ta in ve ry l i t t le, i f any , pheny la l an ine

459

460 June E. Ay ling

hydroxylase act ivi ty . There fore , levels of act ivi ty w h i c h w e have m e a s u r e d in

h u m a n k i d n e y and l iver are from b i o p s i e s from d i seased organs taken at o p e n

surgery. In the cases s tud ied there appears to b e n o effect of the d i sease on

the pheny la l an ine hydroxylase act ivi ty (Tables I and II) .

Pheny la l an ine hydroxylase levels we re m e a s u r e d in 15 surgical ly r e m o v e d

h u m a n k idneys . K i d n e y s we re genera l ly pu t s t raight on to i c e , and ext rac t ion

of the e n z y m e w a s b e g u n w i t h i n 2 0 m i n u t e s of removal . Ac t iv i ty is on ly

presen t in the cor tex of the k idney . T h e r e appears to b e n o corre la t ion

b e t w e e n the d i sease and the pheny la l an ine hydroxylase act ivi ty (Table I ) .

Therefore w e a s s u m e that the d i sease does no t affect the pheny la lan ine hy-

droxylase level and that t hese va lues represen t n o r m a l levels . T h e average

value is 4 7 . 2 m U of e n z y m e pe r gram of cor tex unde r s tandard assay condi -

t ions .

Va lues in the l i terature for pheny la l an ine hydroxylase levels measu red in

Table I Phenylalanine Hydroxylase Activity in Surgically Removed Human Kidney Cortex and Liver

Age (years) Sex Disorder "mU/gm Tissue

A. Kidney cortex 6 M Hydronephrosis 41.8 62 M Hydronephrosis 30.3 56 M Hydronephrosis 27.4 44 F Hydronephrosis 38.0 59 M Cancer 45.2 60 M Cancer 54.5 61 M Cancer 48.1 - F Aneurism 43.0 27 F Aneurism 50.3 56 F Renal vascular

hypertension 27.0

23 F Renal vascular hypertension

44.8


35.0


57.0


61.5

24 M Kidney stone 52.0 47.2 ± 11.2

B. Liver 25 M Hodgkin's disease 200 62 M Bile duct tumor 234

" A mU is defined as the amount of enzyme required to produce 1 nmole of product in 1 minute at pH 7.4 and 27°C in the presence of 1 mM phenylalanine and 0.2 mM Me 2 H 4 -pterin (6,7-dimethyl-tetrahydropterin).

16 A. Phenylalanine Hydroxylase in Human Kidney 461

Table II Levels of Phenylalanine Hydroxylase Activity Found in Human Liver

[Me2H4- IxMolesIgm nMoles/gm Temp. [Phe] pterin] Supernatant No. of Protein/ Tissue/

Ref. (°C) pH (mM) (mM) patients hour min"

13 25 7.0 1.33 0.94 — 2 — 171 9 25 7.0 1.33 0.153 16,000 (15 min) 6 57 ± 10 119

12 25 6.8 2.0 0.3 6,000 (20 min) 5 74 ± 15 154 7 25 6.8 1.33 0.4 20,000 (10 min) 18 61.5 ± 9 . 2 128

15 ? 6.8 2.0 0.6 6,000 (20 min) 5 130.6 ± 22.8 276 Table I 27 7.4 1.0 0.2 - 2 - 217

" Calculated on the basis of 125 mg protein/gm tissue.

b i o p s i e s of hea l thy h u m a n l ivers are s u m m a r i z e d in T a b l e II. In sp i te of the

differences in p H , in concen t ra t ion of subs t ra te and cofactor u sed in the assay

procedures , and also differences in the m e t h o d s of ex t rac t ing the e n z y m e , the

act ivi t ies repor ted b y the different groups are in fairly c lose ag reement . T h e

act ivi t ies w h i c h w e have m e a s u r e d in b i o p s i e s o f two d i seased l ivers , o n e

from a pa t ien t w i th H o d g k i n s d i sease and the o ther w i t h a b i l e duct t umor are

200 and 234 m U of e n z y m e per gram of l iver , and thus fall in the s a m e genera l

range of ac t iv i t ies repor ted b y o thers .

A s s u m i n g our l iver va lues are represen ta t ive , the level in k i d n e y is 4 - 5

t imes less pe r g ram of t i ssue than found in l iver . If the w e i g h t of the l iver of

an average adult is t aken to b e 1500 g m , and each k i d n e y w e i g h s 150 g m , half

of w h i c h is cor tex , there is 10 t imes less k i d n e y t i s sue than l iver , w i t h a level

of ac t iv i ty that is 5 t imes less than that of l iver . T h e levels repor ted he re in -

dicate that on ly abou t 2 % of the total b o d y pheny la l an ine hydroxylase is in

the k idneys of no rma l ind iv idua l s (Table III) .

W h e t h e r or not the k i d n e y e n z y m e is affected in pheny lke tonur i a w o u l d

depend o n w h a t type of mu ta t i on had occurred and w h e t h e r the k i d n e y and

l iver e n z y m e are coded for b y the s ame structural gene . A regulatory mu ta t ion

m i g h t cause a d i m i n i s h e d level o f e n z y m e in the l iver w h i l e that in the k idney

r e m a i n e d normal . Al te rna t ive ly , if pheny lke tonur i a were due to a structural

muta t ion in the pheny la l an ine hydroxylase g e n e , such as appears to b e the

case in o n e pa t ien t repor ted (11) , the k i d n e y e n z y m e wou ld also b e affected, i f

the k i d n e y e n z y m e is coded for b y the s ame structural g e n e as the l iver en-

Table HI Distribution of Phenylalanine Hydroxylase in Man

Organ Weight (gm) Units/gm Tissue Total Units

Liver 1500 0.217 325 Kidney cortex 75 x 2 = 150 0.047 7

462 June E. Ayling

zyme . Final ly , if the k i d n e y con ta ins a different i s o z y m e from the l iver , then

the k i d n e y e n z y m e m a y r e m a i n unaffected in pheny lke tonur i a .

In an a t tempt to de t e rmine if m a m m a l i a n k i d n e y e n z y m e is the s a m e or dif-

ferent f rom that of the l iver , the phys ica l , catalyt ic , and i m m u n o l o g i c a l proper-

t ies of the k i d n e y e n z y m e were c o m p a r e d w i t h the proper t ies of the e n z y m e

from l iver (4) .

T h e phys ica l p roper t ies o f k i d n e y and l iver pheny la l an ine hydroxylase from

rat were c o m p a r e d b y (a) gel filtration and (b) sucrose gradient centr i fugat ion.

(a) W h e n rat l iver pheny l a l an ine hydroxylase is pas sed th rough a B i o - G e l A-5

co lumn, two b a n d s of act ivi ty are e luted. If e i ther b a n d is run again through

the co lumn the same e lu t ion profile resul ts . T h i s ind ica tes that the e n z y m e

exis ts in two different molecu la r w e i g h t forms w h i c h are in te rconver t ib le . Rat

k idney e n z y m e , w h e n p a s s e d over the s ame co lumn produces a ve ry s imi lar

e lut ion pat tern. T h i s s h o w s that the k i d n e y also has two different molecular

we igh t forms, the molecu la r w e i g h t of each b e i n g s imi lar to those of the l iver

enzyme , (b) Sucrose gradients ( 5 - 2 0 % ) were run in the a b s e n c e or p resence of

5 m M pheny la l an ine . E n z y m e was p re incuba ted for 20 m i n u t e s , e i ther w i th

buffer a lone or w i th buffer con t a in ing pheny la l an ine , before b e i n g appl ied to

the appropr ia te gradient . It has b e e n repor ted that pheny la l an ine increases the

s ed imen ta t ion ve loc i ty o f rat l iver pheny la l an ine hydroxylase (16) . In the

gradients w i thou t pheny la l an ine the rates of s ed imen ta t i on of the k i d n e y and

l iver e n z y m e s were the s a m e . In the p re sence of pheny la l an ine the peak of

act ivi ty s e d i m e n t e d m o r e rapidly , l eav ing a t ra i l ing shoulder . T h i s effect is

also obse rved w i t h the k i d n e y e n z y m e to the same extent as w i t h the l iver en-

zyme .

T h e catalytic and i m m u n o l o g i c a l proper t ies were de t e rmined for k i d n e y and

l iver pheny la lan ine hydroxylase from b o t h m a n and rat. T h e c o m p a r i s o n of

spec ies served as a measu re of the sens i t iv i ty o f the m e t h o d s for d is t in-

gu i sh ing b e t w e e n e n z y m e s from different o rgans of the same spec ies .

T h e apparent M i c h a e l i s cons tan t s (KM) (6) we re m e a s u r e d for pheny la l an ine ,

the pseudocofac tor , 6 ,7 -d imethy l te t rahydrop te r in , and the natural cofactor,

t e t rahydrob iop te r in . All of the M i c h a e l i s cons tan t s were de t e rmined at least in

t r ipl icate (Table IV) . In each case the l iver and k i d n e y va lues agree w i t h i n

exper imen ta l error. H o w e v e r , the K M ' s for pheny la l an ine and 6 ,7 -d imethy l -

te t rahydropter in also s h o w no spec ies d e p e n d e n c e . O n the o ther h a n d , the KM

for t e t rahydrob iop te r in s h o w s a 2 -3- fo ld difference b e t w e e n spec ies , bu t has

ident ica l va lues for the KM w i th l iver and k i d n e y e n z y m e of the s ame spec ies .

T h i s is of specia l in te res t s ince the d ihydroxypropyl group of t e t r ahydrob iop-

ter in , w h i c h the pseudocofac tor lacks , is r e spons ib l e no t on ly for the t ight

b i n d i n g of the natural cofactor , b u t is also invo lved in allosteric regula t ion of

the e n z y m e (1 ,2 ) . It is no t surpr i s ing therefore that spec ies differences are

more readi ly de tec ted w i t h the natural cofactor. T h e r e is also a spec ies dif-

ference in the rat ios of m a x i m u m ve loc i ty w i th natural cofactor to the max-

16A. Phenylalanine Hydroxylase in Human Kidney 463

Table IV Kinetic and Immunological Properties of Phenylalanine Hydroxylase from Liver and Kidney of Man and Rat

Rat Human

Property Liver Kidney Liver Kidney

KM Phenylalanine (mM)" KM Me 2 H 4 -pterin (mM)'' KM H 4-biopterin (mM)h

VM H 4 -biopterin r

VM Me 2 H 4 -pterin Milliliter antiserum/mU enzyme

50% inhibition' '

0.83 ± 0.05 0.95 ± 0 . 1 6 0.101 ± 0 . 0 1 4 0.107 ± 0 . 0 0 5 0.023 ± 0.002 0.021 ± 0.003

4.5 4.5

for 0.008 0.008

0.62 ± 0.06 0.84 ± 0 . 1 6 0.154 ± 0.018 0.144 ± 0 . 0 4 3 0.057 ± 0.014 0.057 ± 0.014

2.0 2.0

0.2 0.2

" Apparent Michaelis constants were determined at pH 7.4 and 27°C. The KM for phenylalanine was measured at 0.2 mM Me 2H 4-pterin, with phenylalanine concentration varied from 0.2 to 4.0 mM. '' The KM's for Me 2 H 4 -pterin and H 4-biopterin (tetrahydrobiopterin) were measured at 1 mM phenylalanine, with Me 2 H 4 -pterin varied from 0.02 to 0.5 mM, and H 4-biopterin varied from 0.01 to 0.2 mM.

Ratio of the apparent maximum velocity with H 4-biopterin as cofactor to the apparent maximum velocity with Me 2 H 4 -pterin as cofactor. ( l See legend to Figure 1 for experimental details.

i m u m ve loc i ty w i t h pseudocofac tor , bu t for the different t i s sues of the s a m e spec ies the va lues are the s a m e .

T h e i m m u n o l o g i c a l p roper t ies of the k i d n e y and l iver e n z y m e s we re c o m -

pared on the b a s i s o f the i r react iv i ty w i th an t i bod i e s to rat l iver pheny la l an ine

hydroxylase . E n z y m e w a s i n c u b a t e d w i t h inc reas ing a m o u n t s of an t i se rum.

The prec ip i ta te fo rmed w a s r e m o v e d b y cent r i fugat ion, and the superna tan t

was assayed for r e m a i n i n g act ivi ty . T h e a m o u n t o f an t i s e rum requ i r ed to g ive

5 0 % i n h i b i t i o n of the rat k idney e n z y m e w a s ident ica l to that r equ i r ed for the

rat l iver . T h e e n z y m e from the two h u m a n t i s sues w a s t hen t i t rated w i th rat

l iver e n z y m e an t i se rum. Bo th h u m a n l iver and h u m a n k i d n e y r equ i r ed 25

t imes m o r e an t i s e rum than d id the rat e n z y m e s , to g ive 5 0 % i n h i b i t i o n (Fig-

ure 1, T a b l e I V ) . T h u s , a l though d i s t i ngu i sh ing b e t w e e n spec ie s , t hese i m -

muno log ica l s tud ies were unab l e to d i s t ingu i sh b e t w e e n e n z y m e s from

k idney and l iver of the s a m e spec ies .

B y all of the above cr i ter ia w e have no t b e e n ab le to different iate the k i d n e y

from the l iver e n z y m e , e i ther from m a n or from rat. W e are at p resen t devel-

op ing m e t h o d s to a l low a c o m p a r i s o n of the e lec t rophore t ic p roper t i es of the

k i d n e y and l iver e n z y m e s . T h e u l t imate m e t h o d of c o m p a r i s o n wi l l b e the

a m i n o acid s e q u e n c e of the e n z y m e s from the two t i s sues .

S o m e of the mi lde r forms of pheny lke tonur i a m i g h t b e exp la ined b y re ten-

t ion of pheny la l an ine hydroxylase act iv i ty in the k idney . A l though in normal

ind iv idua ls it appears that on ly 2 % of the total b o d y pheny la l an ine hydroxyl -

464 June E. Ayling

0 0.001 0.01 0-1

ml antiserum per milliunit enzyme

FIGURE 1. Precipitation of phenylalanine hydroxylase from rat liver ( A ) , rat kidney ( • ) , human liver ( x ) , and human kidney ( O ) , with rat liver phenylalanine hydroxylase antibodies. Enzyme was incubated with varying concentrations of antiserum for 30 minutes at 25°C and 3 hours at 0°C. Enzyme-antibody complex was then precipitated by 3 3 % saturation with ammonium sulfate, and the enzyme activity remaining in the supernatant was assayed after precipitation with 5 0 % am-monium sulfate. Each point on the graph is the average of four measurements.

ase is in the k idney , it i s pos s ib l e that w h e n the l iver e n z y m e is defect ive the

k i d n e y e n z y m e m a y b e m o r e ac t ive . If there are forms of pheny lke tonur i a in

w h i c h the k i d n e y e n z y m e r e m a i n s ac t ive , ac t ivat ion or induc t ion of th i s en-

z y m e m i g h t p rov ide a m e a n s of t rea tment .


This work was supported in part by USPHS Grant Nos. HD-05061 and HD-04612.

R E F E R E N C E S

1. Ayling, J . E. , Boehm, G. R., Textor, S. C., and Pirson, R. A. Kinetics of phenylalanine hydroxylase with analogs of tetrahydrobiopterin. Biochemistry, 1973, 12: 2045-2051.

Act

ivit

y, %

16A. Phenylalanine Hydroxylase in Human Kidney 465

2. Ayling, J . E . , and Helfand, G. D. Active sites of phenylalanine hydroxylase. Fed. Proc, Fed. Am. Soc. Exp. Biol, 1974, 33: 1243.

3. Ayling, J . E . , Helfand, G. D., and Pirson, W. D. Phenylalanine hydroxylase from human kidney. Enzyme, 1975, 20: 6-19.

4. Ayling, J . E . , Pirson, W. D. , Al-Janabi, J . M. , and Helfand, G. D. Kidney phenylalanine hydroxylase from man and rat: Comparison with the liver enzyme. Biochemistry, 1974, 13: 78-85.

5. Berry, H. K., Cripps, R., Nichols, K., McCandless, D. , and Harper, C. Development of phenylalanine hydroxylase activity in guinea pig liver. Biochim. Biophys. Acta, 1972, 261: 315-320.

6. Dixon, M., and Webb , E. C. Enzymes. New York: Academic Press, 1964. 7. Grimm, U. , Knapp, A. , and Teichmann, W. Phenylalaninhydroxylase-Aktivitatsbes-

timmungen in Leberhomogenat gesunder probanden. Acta Biol. Med. Ger., 1971, 27: 443-446.

8. Grimm, U., Knapp, A., Tischer, W. , and Schlenzka, K. Phenylalanine hydroxylase activity in hyperphenylalaninemia and classical phenylketonuria. Acta Biol. Med. Ger., 1972, 28: 549-552.

9. Justice, P., O'Flynn, M. E . , and Hsia, D. Y. Phenylalanine hydroxylase activity in hyperphen-ylalaninemia. Lancet, 1967, 1: 928-929.

10. Kaufman, S. Phenylalanine hydroxylating cofactor in phenylketonuria. Science, 1958, 128: 1506-1508.

11. Kaufman, S. Hepatic phenylalanine hydroxylase and phenylketonuria. This volume, Chapter 16.

12. Kaufman, S., and Max, E. Studies on the phenylalanine hydroxylating system in human liver and their relationship to pathogenesis of PKU and hyperphenylalaninemia. In: Phenylke-tonuria. (H. Bickel, F. P. Hudson, and L. I. Woolf, Eds.). Thieme, Stuttgart, 1971: 13-19.

13. La Du, B. N. , and Zannoni, V. G. Inhibition of phenylalanine hydroxylase in liver. In: Phenylketonuria and Allied Metabolic Diseases. (J. A. Anderson, and K. F. Swaiman, Eds.). US Govt. Printing Office, Washington, D. C , 1967: 193-202.

14. McGee, M. M., Greengard, O., and Knox, W. E. The quantitative determination of phenyl-alanine hydroxylase in rat tissues. Biochem. J . , 1972, 127: 669-672.

15. Raiha, N. C. R. Phenylalanine hydroxylase in human liver during development. Pediat. Res., 1973, 7: 1-4.

16. Tourian, A. Activiation of phenylalanine hydroxylase by phenylalanine. Biochem. Biophys. Acta, 1971, 242: 345-354.

17. Tourian, A., Goddard, J . , and Puck, T. T. Phenylalanine hydroxylase activity in mammalian cells. / . Cell. Physiol., 1969, 13: 159-170.

18. Wallace, H. W. , Moldave, K., and Meister, A. Studies on conversion of phenylalanine to tyrosine in phenylpyruvic oligophrenia. Proc. Soc. Exp. Biol. Med., 1957, 94: 632-633.

17 Studies on the Molecular Defect

in Galactosemia

THOMAS A. TEDESCO* Department of Human Genetics, University of Pennsylvania

School of Medicine, Philadelphia, Pennsylvania

I N T R O D U C T I O N

Ga lac tosemia is an i n b o r n error in m e t a b o l i s m first de sc r ibed as a c l inical

abnormal i ty b y v o n R e u s s in 1908 (19) . S i n c e th i s obse rva t ion n u m e r o u s in-

ves t iga tors have con t r ibu ted to our unde r s t and ing of the enzymology ,

gene t i c s , and pa tho logy assoc ia ted w i th mutan t s of galactose m e t a b o l i s m in

m a n (6) . W e have advanced cons ide rab ly in our unde r s t and ing of the galac-

tose me tabo l i c p a t h w a y and have deve loped soph is t i ca ted quan t i t a t ive and

qual i ta t ive t e c h n i q u e s for iden t i fy ing mu tan t genes as wel l as s o m e of the i r

phys io log ica l effects in va r ious cell types . A l t h o u g h it i s n o w wel l r ecogn ized

that classic ga lac tosemia is a c o n s e q u e n c e of a def ic iency of ga lac tose -1 -

phospha t e ur idyl t ransferase act ivi ty , little is k n o w n abou t the type or n u m b e r

of different mu ta t i ons that can p roduce th is d i sease , or w h a t the different

molecu la r defects are that can cause catalyt ic mal funct ion of th i s e n z y m e . T h e

research to b e p re sen ted he re is p r imar i ly conce rned w i t h a n s w e r i n g two

q u e s t i o n s abou t g a l a c t o s e - l - p h o s p h a t e ur idyl t ransferase def ic iency in man : (a)

is there gene t ic he t e rogene i t y a m o n g t ransferase def ic iency ga lac tosemics and

(b) does the m e c h a n i s m of act ion of no rma l h u m a n t ransferase sugges t that w e

can d i s t i ngu i sh at a molecu la r level the bas i s o f defect ive catalyt ic funct ion in

mutan t e n z y m e s ?

* Present address: Department of Pediatrics, University of South Florida, College of Medicine, Tampa, Florida.

467

468 Thomas A. Tedesco

T H E G A L A C T O S E P A T H W A Y

There are two e n z y m e s in the galactose me tabo l i c pa thway , ga lac tokinase

(k inase) and g a l a c t o s e - l - P ur idyl t ransferase ( t ransferase) , that are deficient in

two dis t inct forms of ga lac tosemia , bo th o f w h i c h are au tosomal recess ive

d i seases in m a n . K i n a s e def ic iency results in the inab i l i ty to conver t galactose

(gal) to g a l - l - P . T h i s e n z y m e def ic iency first de sc r ibed b y G i t z e l m a n n (4)

p roduces ga lac tosemia ( i . e . , e levated b lood ga lac tose) , and cataracts appear to

b e i ts p r inc ip le c l in ical c o n s e q u e n c e . Transferase def ic iency results in the

classic form of ga lac tosemia . S i n c e the conve r s ion of g a l - l - P to g l u - l - P does

not occur normal ly , b o t h g a l - l - P and gal accumula te in the t i s sues of these pa-

t ients (6) . T h e cl inical abnormal i t i e s assoc ia ted w i th t ransferase def iciency

inc lude failure to thr ive , often mani fes t w i t h i n the first few w e e k s of l ife, cata-

racts , and l iver and neuro log ica l dysfunct ion , the last f requent ly leading to

menta l re tardat ion.

F igure 1 s h o w s the ga lac tose me tabo l i c p a t h w a y and i ts re la t ionsh ip to sev-

eral areas o f m e t a b o l i s m . T h e r e is good ev idence that the conve r s ion of galac-

tose to galacti tol b y a ldose reductase in lens t i s sue p roduces osmot i c effects

w h i c h lead to cataract format ion (6) c o m m o n in b o t h k inase and transferase

Gal-l-P

Glycogen Glycol ipids Glycoproteins

r GT

UDPGIu

Glu-l -P • UDPGal

GE jUDPHex PooFI

UDPGIu

Glu-6 Glycolytic Pathway

FIGURE 1. The galactose metabolic pathway and its relationship to several areas of metabolism. AR, aldose reductase; GD, galactose dehydrogenase; AP, nonspecific phosphatase; GK, galacto-kinase; GT, galactose-l-P uridyltransferase; GE, UDPgalactose-4-epimerase; PGM, phosphogluco-mutase; and PP, UDPglucose pyrophosphorylase.

17. Studies on the Molecular Defect in Galactosemia 469

def ic iency, and m o r e recen t ev idence desc r ibes a specif ic i n h i b i t o r of a ldose

reductase that p reven t s th i s effect (3) .

T h e o b v i o u s c l in ical and b i o c h e m i c a l d i f ferences b e t w e e n k inase and trans-

ferase def ic iency sugges t that the accumula t ion of intracel lular g a l - l - P is re-

lated to the more severe c l in ical c o n s e q u e n c e s found in t ransferase def ic iency.

G a l - l - P has b e e n repor ted to accumula te in red b lood cells (2) and sk in

f ibroblast cell cul tures der ived from ga lac tosemic pa t ien ts (10) . T h e toxic ef-

fects of g a l - l - P as a me tabo l i c i n h i b i t o r have b e e n suspec ted , ye t no good evi -

dence has b e e n p roduced a long these l ines . Severa l e n z y m e s have b e e n tes ted

and found to b e unaffected b y g a l - l - P (15) . W i t h concen t ra t ions u p to 2 m M

w e have found that g a l - l - P has little or n o effect on the ac t iv i ty o f U D P g a l - 4 -

ep imerase or U D P g l u py rophosphory la se unde r s tandard assay cond i t ions .

P h o s p h o g l u c o m u t a s e ( P G M ) , an impor tan t l ink in ga lac tose , g lucose , and

g lycogen m e t a b o l i s m , has b e e n repor ted to b e i n h i b i t e d b y g a l - l - P , b u t on ly

in the a b s e n c e of the c o e n z y m e g l u - l , 6 - d i P (15) . W e have conf i rmed th i s find-

ing wi th P G M extrac ted from h u m a n d ip lo id f ibroblast cell cul tures . T h e r e

w a s n o de tec tab le i n h i b i t i o n w h e n g l u - l , 6 - d i P w a s added to the reac t ion m i x -

ture and on ly abou t 5 0 % i n h i b i t i o n w i t h a g a l - l - P / g l u - l - P rat io of 100/1 w h e n

g l u - l , 6 - d i P w a s de le ted from the reac t ion . T h e levels of P G M found in extracts

of h u m a n f ibroblas ts are 4 to 5 fold h i g h e r w h e n cells are cul tured in g lucose

m e d i u m than w h e n g rown in galactose m e d i u m . T h i s di f ference, h o w e v e r ,

m a y b e due to dec reased levels of the c o e n z y m e g l u - l , 6 - d i P in the ga lac tose-

g rown cells s imi la r to the f inding of dec reased levels of th i s me tabo l i t e in the

t i s sues of galac tose-fed rats (15) .

A v ic ious cycle of p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n has b e e n pos tu la ted

b y s o m e inves t iga tors as a toxic m e c h a n i s m in ga lac tosemia ( 7 , 8 , 1 0 ) . T h e ac-

t ion of nonspec i f ic p h o s p h a t a s e s on g a l - l - P w o u l d cause repea ted phosphory l -

a t ion of galac tose at the e x p e n s e of A T P . H o w e v e r , in the l ivers o f rats fed a

5 0 % galactose diet , A M P levels dec reased , A D P levels were u n c h a n g e d or

on ly s l ight ly i nc reased , and A T P levels inc reased (15) sugges t ing s t imula t ion

of A T P - g e n e r a t i n g sys t ems ra ther than exhaus t ion of A T P b y cyclic p h o s p h o -

ryla t ion of ga lac tose . S i n c e no true an ima l m o d e l exis ts for t ransferase defi-

c i ency ga lac tosemia , it is difficult to compare the data from s tudies in w h i c h

normal an ima l s are fed d ie ts o f 20 to 5 0 % galactose w i t h those o b t a i n e d from

ga lac tosemic pa t ien t s or cul tured cells de r ived from them. O u r p re l imina ry

data s h o w that t ransferase deficient cells in cul ture accumula te g a l - l - P w h e n

g rown in galac tose m e d i u m to levels severalfold that found w h e n these cells

are g rown in g lucose m e d i u m ; and unde r the s a m e cond i t i ons , no rma l cells

con ta in bare ly de tec tab le levels of g a l - l - P . T h e s e resul ts agree w i t h those

recent ly repor ted b y M a y e s and Mi l le r (10) w h o demons t r a t ed that g a l - l - P

accumula tes in ga lac tosemic cells . In cells de r ived from he te rozygo tes for

t ransferase def ic iency they found g a l - l - P accumula t ions in t e rmed ia t e b e t w e e n


those found in no rma l and ga lac tosemic f ibroblas ts . T h e s e resul ts correlate

wel l w i th the re la t ive ac t iv i t ies o f t ransferase and k inase act ivi ty desc r ibed b y

us in normal , he te rozygo te , and ga lac tosemic f ibroblasts (17) . N o r m a l cell

extracts con ta in rat ios o f t ransferase/kinase greater than 1.0 and he te rozygous

cells have rat ios less than 1.0. Ga lac tok inase appears to b e rate l imi t ing in

normal cel ls , bu t w h e n t ransferase act ivi ty is decreased or absen t as in he te ro-

zygous and ga lac tosemic cel ls , t ransferase b e c o m e s rate l imi t ing as reflected

in the accumula t ion of g a l - l - P . If the accumula t ion o f intracel lular g a l - l - P leads

to the pa tho logy of ga lac tosemia , s tudies wi th f ibroblast cells in culture that are

truly deficient in t ransferase act iv i ty should e lucidate its m e c h a n i s m , bo th in

cellular and molecular t e rms .

T H E G E N E T I C N A T U R E O F T H E D E F E C T

O n e first approach toward de t e rmin ing w h a t k i n d o f mu ta t ion produces

t ransferase def ic iency ga lac tosemia w a s to p repare a r a b b i t an t i se rum to t rans-

ferase purified to e lec t rophore t ic h o m o g e n e i t y from h u m a n l iver (16) . B y

doub le immunod i f fus ion t e c h n i q u e s it w a s found that red b lood cell extracts

from ga lac tosemic pa t ien ts c o n t a i n e d a c ross - reac t ing mater ia l ( C R M ) i m -

munolog ica l ly i nd i s t i ngu i shab l e from the t ransferase found in normal red

b lood cells (18) . Transferase C R M has n o w b e e n ident i f ied in 16 different

ga lac tosemic fami l ies ; w e have no t ye t found a t ransferase-def ic ient pa t ien t

w h o lacks C R M , sugges t ing that th is d i sease is due to a structural gene muta -

t ion. P roo f that the a n t i b o d y w e were us ing reacted specifically w i th galac-

t o s e m i a C R M w a s demons t r a t ed b y quant i t a t ive i m m u n o p r e c i p i t a t i o n tech-

n i q u e s . Norma l t ransferase was t i t rated w i th an t i s e rum to de t e rmine the

a m o u n t n e e d e d to neut ra l ize an a l iquot of no rmal e n z y m e act ivi ty. T h i s vol-

u m e of an t i se rum w a s then a b s o r b e d wi th different amoun t s of part ial ly

purif ied red b lood cell extract from t ransferase 'def ic ient pa t ien t s , and the

amoun t of t ransferase-specif ic a n t i b o d y r e m a i n i n g w a s measu red . T h e results

clearly s h o w e d that these ga lac tosemic red cells con ta ined an an t igen that

reacted w i t h t ransferase an t ibody , and the amoun t s of an t igen were s imi la r to

those found in no rma l red b lood cells (18) . T h e lack of t ransferase act ivi ty in

ga lac tosemia thus appears to b e due to a mutan t p ro te in p resen t in apparent ly

normal quan t i t i e s in the red cell that is defect ive in i ts catalytic funct ion.

Normal t ransferase and ga lac tosemic C R M are also s imi lar in molecu la r s ize as

de t e rmined b y a s tandard ized S e p h a d e x G-200 gel filtration co lumn. T h e elu-

t ion vo lume of n o r m a l t ransferase d e t e r m i n e d b y specific e n z y m e act ivi ty w a s

ident ica l to that o f ga lac tosemic C R M , ident i f ied b y i ts immunoreac t i v i t y in

doub le immunod i f fus ion aga ins t t ransferase-specif ic an t i se rum (16) . T h e s e

data indica te that the t ransferase C R M found in ga lac tosemia has the same

molecular s ize and an t igen ic de te rminan t s as the no rma l e n z y m e .


GENETIC HETEROGENEITY AMONG GALACTOSEMICS (1)

Al though there is a t ransferase C R M in 16 different ga lac tosemic fami l ies ,

immuno log ica l ly i n d i s t i n g u i s h a b l e from normal t ransferase , w e have cons id -

ered w h e t h e r there is molecu la r he t e rogene i ty w i t h i n th is g roup . Nad le r et al.

(12) s h o w e d that c o m p l e m e n t i n g h y b r i d s w i t h t ransferase act ivi ty could b e

p roduced b y fusing cer ta in pairs o f ga lac tosemic f ibroblast cell cul ture l ines .

F r o m th i s result , the p red ic t ions that can b e r ea sonab ly m a d e are (a) there are

funct ionless p ro te ins in the parenta l cel ls , (b) t ransferase is c o m p o s e d of

subun i t s , and (c) there is he t e rogene i ty a m o n g var ious ga lac tosemic muta -

t ions . T h e p re sence of C R M in ga lac tosemia w h e r e molecu la r s ize is s imi lar to

normal t ransferase has a l ready b e e n d i scussed and thus the first p red ic t ion is

satisfied. H u m a n t ransferase purif ied from l iver as wel l as red b lood cells

w h e n e lec t rophoresed on S D S polyacry lamide gels , y ie lds a s ing le spec i e s

wi th an apparen t molecu la r w e i g h t of 3 0 , 0 0 0 (16) . T h e s e data satisfy the sec-

ond p red ic t ion s ince the normal e n z y m e and ga lac tosemic C R M have an

apparent molecu la r w e i g h t in the range of 9 0 , 0 0 0 . T h e thi rd p red ic t ion w a s sa-

tisfied b y o b t a i n i n g ev idence for molecu la r he t e rogene i ty a m o n g galac-

to semics b y quan t i t a t ive and qual i ta t ive i m m u n o c h e m i c a l t e c h n i q u e s . T h e

con ten t of C R M in red b lood cell extracts from different ga lac tosemic pa t ien t s

were ana lyzed b y e lec t ro immunodi f fus ion in an agarose layer con ta in ing

h u m a n t ransferase-specif ic an t i se rum (9) .

Resu l t s for five different pa t ien ts are l i s ted in T a b l e I. O n each plate a di lu-

t ion ser ies w a s run for each ga lac tosemic sample a long wi th an ident ica l ly

t reated no rma l control . I m m u n o p r e c i p i t i n peaks of s imi lar s ize w e r e c h o s e n

from each d i lu t ion ser ies and the pro te in con ten t of that par t icular sample

calculated. O n each plate the peak he igh t in mi l l ime te r s /mic rogram pro te in

w a s de t e rmined for the control and each ga lac tosemic sample . T h e a m o u n t of

C R M in each ga lac tosemic sample is expres sed as the rat io of CRM/cont ro l . As

seen in T a b l e I , two samples have a ratio of 0 .96 ind ica t ing that these two pa-

t ien ts have C R M levels in the i r red cells s imi la r to the controls . O n e pa t ien t

Table I Quantitative Variation of Human Transferase CRM from the Red Blood Cells of Different Galactosemic Patients"

Galactosemic Patient

Peak Height (mm)

CRM Control Galactosemic

Patient Peak Height

(mm) Protein (/mg) Peak Height/Protein Peak Height/Protein CRM/Control

DT 16.5 11.36 1.450 1.51 0.96 LW 12.5 7.20 1.740 1.81 0.96 FR 15.0 7.45 2.010 1.63 1.23 c w 12.5 16.04 0.779 1.81 0.43 AM 10.0 16.25 0.615 1.92 0.32

" Based on electroimmunodiffusion in agar containing antibody to human transferase.


Table II Variation in Electrophoretic Mobility of Transferase CRM in Red Blood Cells from Different Galactosemic Patients Identified by Electroimmunodiffusion

Electrophoretic Mobility

Galactosemic Patient CRM Rf (mm) Control Rf (mm) CRM/Control

D T 43 43 1.00

M W 52 5 4 0.96

W W 55 5 4 1.01

F R 56 5 2 1.07

A M 37 43 0.86

L L 44 5 2 0.84

has 2 0 % m o r e C R M than no rma l (FR) , and two have on ly 32 and 4 3 % of the

control sample ( A M and C W , respec t ive ly) . T h i s quan t i t a t ive var ia t ion in red

cell con ten t of ga lac tosemic C R M m a y ind ica te that different mutan t t rans-

ferases have different s tabi l i t ies or that the structural a l terat ions in these pro-

te ins affect the i r rates o f syn thes i s or degradat ion . Var ia t ion in e lec t rophore t ic

mob i l i t y w a s also found for ga lac tosemic C R M extracted from red cells o f dif-

ferent pa t ien ts . B y e m p l o y i n g a t w o - d i m e n s i o n a l sy s t em in w h i c h the samples

are separa ted b y e lec t rophores i s in the first d i m e n s i o n , fo l lowed b y e lec t ro im-

munodi f fus ion in the s econd d i m e n s i o n (11) , in to an agarose gel con ta in ing

t ransferase-specif ic an t i se rum, the e lec t rophore t ic m o b i l i t y of C R M can b e de-

t e rmined . The resul ts o b t a i n e d from six different ga lac tosemic pa t ien t s are

l is ted in Tab le II. Norma l and mutan t t ransferases we re run on the s ame plate,

and the e lec t rophore t ic mig ra t ion (Ry) of each ga lac tosemic C R M is expressed

as a ratio of C R M Ry/control Rf. Four pa t ien ts have t ransferase C R M wi th the

same e lec t rophoret ic m o b i l i t y as the control sample . Pa t ien ts M W and W W are

bro thers w i th t ransferase def ic iency. T w o o ther pa t ien ts from different fami-

l ies have t ransferase C R M w h i c h migra tes at a s ignif icant ly s lower rate than

normal ( A M and L L ) .

THE MECHANISM OF ACTION OF HUMAN TRANSFERASE (20)

To under s t and the structural ba s i s o f defect ive catalyt ic funct ion in mu tan t

e n z y m e s , the m e c h a n i s m of ac t ion of the no rma l e n z y m e needs to b e

thoroughly charac ter ized . W e have ana lyzed the k ine t i cs of no rmal h u m a n red

cell t ransferase purif ied to e lec t rophore t ic h o m o g e n e i t y ; in i t ia l ve loc i t ies as a

funct ion of subs t ra te concen t ra t ion , in b o t h forward and reverse d i rec t ions ,

have b e e n de t e rmined . F r o m these s tud ies w e have conc luded that h u m a n

transferase funct ions b y the P i n g - P o n g m e c h a n i s m for a two-subs t ra te e n z y m e


UDPGIu Glu-I-P Gal-l-P UDPGal

Gt« GtUDPGIu GtUMP GtUDPGal

FIGURE 2. Scheme showing the Ping-Pong mechanism of action for human galactose-l-P uridyl-

(18) . S u c h a m e c h a n i s m i m p l i e s that b o t h subs t ra tes are no t s imul t aneous ly

complexed wi th the e n z y m e . T h e e n z y m e r ecogn izes the first subs t ra te and

p roceeds t h rough a hal f - react ion to p roduce the first product and a react ive

in te rmedia te . T h e react ive in t e rmed ia t e then complexes w i t h the s econd s u b -

strate and p roceeds th rough the s econd ha l f of the reac t ion to y ie ld the final

product and free e n z y m e .

T h e reac t ion s e q u e n c e sugges ted for the t ransferase e n z y m e is i l lustrated in

F igure 2 w i t h an e n z y m e - U M P complex as the reac t ive in t e rmed ia te . Produc t

i n h i b i t i o n pa t te rns for b o t h forward and reverse reac t ions reveal that g l u - l - P

and g a l - l - P c o m p e t e w i th each o ther as do U D P g l u and U D P g a l , b u t U D P -

hexose and h e x o s e - l - P (e i ther glu or gal) do no t c o m p e t e w i t h o n e another .

Therefore th i s e n z y m e m u s t have separa te b i n d i n g s i tes for U D P h e x o s e and

h e x o s e - l - P . T h e e n z y m e s h o w s n o apparen t preferent ia l affinity for glu or gal

as the h e x o s e i n these subs t ra tes in e i ther the forward or reverse d i rec t ion .

T h i s can b e suppor ted b y i so tope exchange reac t ions w i t h e i ther glu or gal as

i l lustrated in F igu re 3 . T h e abi l i ty of h u m a n t ransferase to exchange i so tope

b e t w e e n U D P g a l and g a l - l - P was p rev ious ly desc r ibed b y Sega l et al. (14) . T h e

nonspec i f ic i ty of h u m a n transferase for e i the r glu or gal ra i ses a genera l q u e s -

t ion of subs t ra te specif ic i ty . W e found that h u m a n t ransferase is h igh ly spe-

cific for u r id ine as the nuc leo t ide subs t ra te in the i so tope e x c h a n g e reac t ion

wi th 1 4 C - g l u - l - P (Table III) and for g l u - l - P and g a l - l - P as the h e x o s e - l - P s u b -

strate. Severa l o the r h e x o s e s , inc lud ing g l u - l , 6 - d i P , were found to b e unreac-

t ive in the i so tope exchange reac t ion wi th U D P g l u - 1 4 C (Table III) . Transferase

therefore is a molecu le that is h igh ly specif ic for u r id ine d iphospha t e h e x o s e ,

and for h e x o s e - l - P , bu t glu and gal readi ly subs t i tu te for o n e ano the r as the

hexose in these subs t ra tes .

A l though a reac t ive in t e rmed ia t e has no t b e e n i so la ted , the P i n g - P o n g

m e c h a n i s m predic ts a t r ans fe rase -UMP complex . T h i s has b e e n suppor ted b y

expe r imen t s in w h i c h inc reas ing concen t ra t ions of g l u - l - P w e r e found to c o m -

pete w i th g a l - l - P for the react ive in te rmed ia te , and b y demons t r a t ing that

transferase.

Glu-l-P + UDPGIu( , 4C) Glu-l-P( , 4C) + UDPGIu

Gal-l-P + UDPGalft) + •> G a l - h P ^ ) + UDPGal

FIGURE 3. Isotope exchange reactions catalyzed by human galactose-l-P uridyltransferase with glucose and galactose substrates.


Table III Relative Activities of Human Transferase with Various Nucleoside and Hexose Substrates 0

Nucleoside-DPglu % Activity Hexose % Activity

UDPglu TDPglu CDPglu ADPglu GDPglu

100 5.9 2.9 1.9 1.9

Glucose-l,6-diP Glucose

Glucose-l-P Mannose-l-P Glucose-6-P

100 0 0 0 0

" The reaction mixture for testing various nucleoside diphosphate glucose substrates contained in a final volume of 75 /xl; 0.06 /xmoles of the substrate to be tested; 20 /xmoles Tris, pH 8.5; 0.52 /xmoles dithiothreitol; 0.2 mg of partially purified red cell transferase protein (specific activity, 0.03 units/ mg); and 0.16 1 4 C-glu-l-P (specific activity, 0.6 ^tCi//xmole). The same reaction mixture was used to test the various hexose substrates except that 0.03 /nmoles UDPglu- 1 4 C (specific activity, 3.3 /xCi/ /umole) replaced 1 4 C-glu-l-P. After 1 hour incubation at 37°C an aliquot of the reaction mixture was chromatographed on DEAE-cellulose paper with 0.05 M NaCl to separate hexose-1-P from UDP-hexose which were then counted in a liquid scintillation spectrometer.

g l u - l - 3 2 P reacts w i th t ransferase to p roduce U - P - 3 2 P g l u . A s c h e m e for the

h e x o s e - l - P compe t i t i on e x p e r i m e n t s is i l lustrated in F igure 4 . Inc reas ing con-

cent ra t ions of g l u - l - P we re added to the i so tope exchange reac t ion mix ture

con t a in ing U D P g a l and 1 4 C - g a l - l - P . U D P g a l - 1 4 C format ion w a s m e a s u r e d b y

scint i l la t ion spec t romet ry after separa t ing U D P h e x o s e from the reac t ion m i x -

ture w i t h DEAE-ce l lu lose pape r ch roma tog raphy (13) . T h e format ion of

U D P g l u i n the reac t ion mix tu re w a s m e a s u r e d w i t h U D P g l u dehydrogenase .

T h e resul ts p lot ted in F igure 5 clearly s h o w that U D P g l u format ion increases

as a funct ion of g l u - l - P concen t ra t ion , and that U D P g a l - 1 4 C format ion de-

creases p ropor t iona te ly as U D P g l u increases . W h e n g l u - l - 3 2 P is u sed as a s u b -

strate in th i s reac t ion the label is t ransferred to U - P - 3 2 P g l u , further ev idence

that an e n z y m e - U M P complex is the react ive in t e rmed ia t e in addi t ion to in-

dicat ing w h i c h ester b o n d is invo lved in the reac t ion . O b v i o u s l y one of the

FIGURE 4. Scheme describing the hexose- l -P competition for the reactive intermediate in the transferase reaction.

Glu-l-P

Gal - l -P ( , 4 C)


0 0.4 0.8 1.2 1.6 2.0 Glu-l-P (mM)

FIGURE 5. The results of hexose-l-P competition for the reactive intermediate in the transferase reaction, as illustrated in Figure 4. Increasing concentrations of glucose-l-P were incubated in a reaction mixture which contained in a final volume of 85 fil; 10 jiimoles Tris, pH 8.5; 0.52 /nmoles dithiothreitol; 2 mg of partially purified red cell transferase protein (specific activity, 0.03 units/mg); 0.19 /xmoles UDPgalactose; and 0.14 /mnoles 1 4 C-galactose-l-P. (Specific activity, 0.7 /xCi/per /imole.) UDPgal- 1 4 C was measured by liquid scintillation spectrometry after separation from the reaction mixture by DEAE-cellulose paper chromatography (13). UDPglucose was measured with UDPglucose dehydrogenase.

b o n d s in the es ter l inkage b e t w e e n the two p h o s p h a t e s in the U D P h e x o s e

subs t ra te is b e i n g b r o k e n and re formed in the course of th i s reac t ion . Appro -

priate expe r imen t s w i t h specifically l abe led l s O - U D P h e x o s e ( U - P - l s O - P g l u )

should n o w ind ica te exact ly w h i c h covalent b o n d is invo lved .

T h e in terpre ta t ion of these data is s u m m a r i z e d in F igure 6 w h i c h i l lustrates

the act iv i ty of h u m a n transferase b r o k e n d o w n in to s ix ind iv idua l s teps , each

of w h i c h is revers ib le . W e are n o w a t t empt ing to detect ha l f - react ions b y

m e a s u r i n g h e x o s e - l - P format ion from U D P h e x o s e in the a b s e n c e of a h e x o s e -

l - P subs t ra te , as wel l as o ther ind iv idua l parts of th i s s equen t i a l reac t ion . Ar-

tificial subs t ra tes m a y p rov ide s o m e of th is capabi l i ty . For example , U M P f -

n i t ropheno l complex , an assay for the first s tep in the reac t ion (Figure 6) wil l

no t react w i th the e n z y m e to p roduce e n z y m e - U M P and free /?-ni t rophenol . If

w e can successful ly separa te and quant i ta t ive ly m e a s u r e the enzyme-UMP-/? -

n i t ropheno l complex , an assay for the first s tep in the reac t ion (F igure 6) will

b e avai lable for tes t ing mutan t t ransferases to de t e rmine if they can perform

th is part o f the reac t ion . W i t h the use of addi t iona l artificial subs t ra tes we

wou ld u l t imate ly h o p e to deve lop the ab i l i ty to a s say each of the ind iv idua l

s teps in the total reac t ion . T h i s w o u l d al low the de t e rmina t ion of the specific

UD

PG

aftK

(cp

m x

I0

3 )

UD

PG

Iu (

n mo

les)


U-P-P-Gal U-P-P-Glu

GT:U-P-P-Gal GT:U-P-P-Glu

GT:U-P rP-Gal GT-U-P.P-Glu

P-Gal

GT=U-P < FIGURE 6. The reaction sequence proposed for human galactose-l-P uridyltransferase. Activity moving clockwise represents the forward reaction.

catalytic defect in different ga lac tosemic t ransferases . P r e s u m a b l y those n o n -

funct ional parental e n z y m e s that are complemen ta ry in cell hybr id iza t ion

expe r imen t s wi l l have different types of funct ional defects .

T h e galactose me tabo l i c p a t h w a y and s o m e of the c o n s e q u e n c e s of deficient

ga lac tokinase or g a l - l - P ur idyl t ransferase act ivi ty have b e e n d i scussed . T h e

ex i s tence of C R M in t ransferase def ic iency ga lac tosemia is p resen ted as ev i -

dence that th is d i sease is the resul t of a structural g e n e muta t ion . T h e f inding

of bo th quan t i t a t ive and qual i ta t ive var ia t ion in t ransferase C R M a m o n g dif-

ferent ga lac tosemic pa t ien ts argues that gene t ic he t e rogene i ty exis ts w i th in

this group. Da ta suppor t ing a P i n g - P o n g m e c h a n i s m of ac t ion for h u m a n

transferase have b e e n p re sen ted , and a s e q u e n c e of s ix ind iv idua l s teps in the

t ransferase reac t ion is p roposed .

I gratefully acknowledge Dr. William J. Mellman, Chairman, Department of Human Genetics, University of Pennsylvania School of Medicine, for his critical evaluation and direction throughout the course of this work; Dr. Roland G. Kallen, Associate Professor of Biochemistry, for consultation and advice in the studies on the mechanism of action of human transferase; Karen Miller, Jr. Research Specialist, for her work on all phases of this project; Dr. Joseph W-T Wu, Post Doctoral Fellow, for his work on the molecular studies; Francie Boches, graduate student in bio-chemistry, for her work on genetic heterogeneity of transferase CRM; and last but not least, Bar-bara Needleman and Constance Page for their cell culture work.

This work was supported by NIH Grant Nos. HD-00588 and HD-04861.

S U M M A R Y



R E F E R E N C E S

1. Boches, F . , Tedesco, T. A., Miller, K., and Mellman, W. J. Unpublished observations. 2. Donnell, G. N. , Koch, R., and Bergren, W. R. Observations on results of management of

galactosemic patients. In: Galactosemia. (D. Y. Y. Hsia, Ed.). Chapter 31. Thomas, Spring-field, Illinois, 1969: 247-268.

3. Dvornik, D. , Simard-Duquesne, N., Krami, M., Sestauj, K., Gabbay, K. H., Kinoshita, J . H., Varma, D. S., and Merola, L. O. Polyol accumulation in galactosemic and diabetic rats: Con-trol by an aldose reductase inhibitor. Science, 1973, 182: 1146-1148.

4. Gitzelman, R. Hereditary galactokinase deficiency, a newly recognized cause of juvenile cata-racts. Pediat. Res., 1967, 1 : 14-23.

5. Hersh, L. B . , and Jencks, W. P. Coenzyme A transferase; kinetics and exchange reactions. / . Biol. Chem., 1967, 242: 3468-3480.

6. Kalckar, H. M. , Kinoshita, J . H., and Donnell, G. N. Galactosemia: Biochemistry, genetics, pathophysiology, and developmental aspects. In: Biology of Brain Dysfunction. (G. E. Gaull, Ed.). Chapter 2. Plenum, New York, 1973: 31-88 .

7. Kozak, L. P. , and Wells, W. W. Effect of galactose on energy and phospholipid metabolism in the chick brain. Arch. Biochem. Biophys., 1969, 135: 371-377.

8. Kozak, L. P., and Wells, W. W. Studies on the metabolic determinants of D-galactose induced neurotoxicity in the chick. / . Neurochem., 1971, 18: 2217-2228.

9. Laurell, C. B . Quantitative estimation of proteins by electrophoresis in agarose gels con-taining antibodies. Anal. Biochem., 1966, 15: 45-52.

10. Mayes, J. S., and Miller, L. R. The metabolism of galactose by galactosemic fibroblasts in vitro. Biochim. Biophys. Acta, 1973, 313: 9-16.

11. Minchin Clark, H. G., and Freeman, T. Quantitative immunoelectrophoresis of human serum proteins. Clin. Sci., 1968, 35: 4 0 3 ^ 1 3 .

12. Nadler, H. L. , Chacko, C. M., and Rachmeler, M. Interallelic complementation in hybrid cells derived from human diploid strains deficient in galactose-l-phosphate uridyltransferase activ-ity. Proc. Natl. Acad. Sci. USA, 1970, 67: 967-982.

13. Ng, W. G., Bergren, W. R. , and Donnell, G. N. Assay of hemolysate gal-l-P uridyltransferase activity by radioactivity. In: Galactosemia. (D. Y. Y. Hsia, Ed.). Chapter 13. Thomas, Spring-field, Illinois, 1969: 98-104.

14. Segal, S., Rogers, S., and Holtzapple, P. G. Liver galactose-l-phosphate uridyltransferase: activity in normal and galactosemic subjects. / . Clin. Invest., 1971, 50: 500-506.

15. Sidbury, J . B . , Jr. Investigations and speculations on the pathogenesis of galactosemia. In: Galactosemia. (D. Y. Y. Hsia, Ed.). Chapter 2. Thomas, Springfield, Illinois, 1969: 13-29.

16. Tedesco, T. A. Human galactose-l-phosphate uridyltransferase: Purification, antibody pro-duction, and comparison of the wild type, Duarte variant, and galactosemic gene products. / . Biol. Chem., 1972, 247: 6631-6636.

17. Tedesco, T. A., and Mellman, W. J . Galatose-l-phosphate uridyltransferase and galactokinase activity in cultured human diploid fibroblasts and peripheral blood leukocytes. / . Clin. Invest., 1969, 48: 2390-2397.

18. Tedesco, T. A., and Mellman, W. J. Galactosemia; evidence for a structural gene mutation. Science, 1971, 172: 727-728.

19. von Reuss, A. Zuckerauwscheidung in Sauglinsalter. Wien. Med. Wochenschr., 1908, 58: 799. 20. Wu, J . , Tedesco, T. A. , and Kallen, R. G. Human galactose-l-phosphate uridyltransferase;

kinetic mechanism, substrate specificity, and exchange reactions. (Submitted to / . Biol. Chem.)

18 The Enzymology of Tay-Sachs Disease and

Its Variant Forms

JOHN F. TALLMAN Developmental and Metabolic Neurology Branch,

National Institutes of Health,

Bethesda, Maryland

A. INTRODUCTION: THE CATABOLISM OF GANGLIOSIDES

T h e s tudy of cer ta in progress ive ly degenera t ive neuro log ica l d isorders

k n o w n as the l ip idoses ( 1 - 3 ) has necess i t a t ed an in t ens ive inves t iga t ion of

reac t ions invo lved in the ca t abo l i sm of gang l ios ides and re la ted neutra l gly-

cosph ingo l ip ids . T h e s e c o m p o u n d s w h i c h are der iva t ives o f s p h i n g o s i n e ,

C H 3 ( C H 2 ) 1 2 — C H = C H — C H ( O H ) — C H ( N H 2 ) — C H 2 O H , con ta in a long cha in

fatty ac id b o u n d in an a m i d e l inkage to the n i t rogen a tom on C-2 . T h i s N-

acy l sph ingos ine complex is called ce ramide . Add i t iona l cons t i tuen t s , such as

m o n o - or o l igosacchar ides , m a y b e l inked th rough the p r imary hydroxyl

group of s p h i n g o s i n e to y ie ld the g lycosph ingo l ip ids . T h e gang l io s ides are

g lycosph ingo l ip ids w h i c h are charac ter ized b y the p resence of o n e or more

molecu les of sial ic ac id (N-acetyl or N-g lyco ly lneuramin ic acid) (F igure 1) .

It is p resen t ly be l i eved that the ca t abo l i sm of a gang l ios ide p roceeds s tep-

w i s e from the n o n r e d u c i n g t e rminus of the ca rbohydra te por t ion of the mo le -

cule toward the c e r a m i d e m o i e t y (Figure 2) (35) . T h e e n z y m e s ca ta lyz ing these

reac t ions are located in the l y sosomes and e x h i b i t p roper t i es character is t ic of

such e n z y m e s . O f the n u m e r o u s e n z y m e s invo lved in gang l ios ide b r e a k d o w n ,

cl inical d isorders have b e e n assoc ia ted w i th def ic ienc ies in each of the reac-

t ions (wi th the excep t ion of the n e u r a m i n i d a s e ) . A d i scus s ion of these d is-

orders i s avai lable ( 1 - 3 , 3 6 , 4 0 ) . T h i s r e v i e w wil l b e c o n c e r n e d w i t h T a y - S a c h s

d i sease and i ts var ian ts ( known col lect ively as the G M 2 - g a n g l i o s i d o s e s ) .

479

480 John F. Tollman

Ceramide-glucose-galactose-iV-acetylgalactosamine-galactose

W-aeetyl- ^ -ace ty l -neuraminic acid neuraminic acid

Major disialoganglioside ( G n i a )

Ceramide-glucose-galactose-^-acetylgalactosamine-galactose

A/-acetyl-neuraminic acid

Major Monosialoganglioside (GMI)

Ceramide -glucose -galacto se-N-acetylgalactosamine

N-acetyl -neuraminic acid

Tay-Sachs Ganglioside (GM2)

FIGURE 1. The structure of important gangliosides.

B. TYPE I GM2-GANGLIOSIDOSIS (TAY-SACHS DISEASE)

1. Clinical Signs

T h e or ig ina l desc r ip t ions of th is d i sorder date from the last cen tury and

were m a d e separa te ly b y W a r r e n Tay (41) and Berna rd S a c h s (24) in ch i ld ren of

Ashkenaz i c J e w i s h descent . S i n c e these p r imary desc r ip t ions , n u m e r o u s cases

have b e e n repor ted . It is th i s form of G M 2 - g a n g l i o s i d o s i s w h i c h is r ight ly

cal led T a y - S a c h s d i sease . M o t o r w e a k n e s s i s an early s ign and i s usual ly

mani fes t b e t w e e n the third and s ixth postnata l m o n t h . T h i s w e a k n e s s pro-

gresses and after 1 year of age , inc reas ing men ta l re tardat ion b e c o m e s appar-

ent. T h e pa t ien t dec l ines w i t h the further appearance of b l i n d n e s s , deafness ,

and convu l s ions . Affected ch i ld ren c o m m o n l y d ie b e t w e e n age 3 and 4 .

2. Pathology

Pat ien ts wi th T a y - S a c h s d i sease are charac ter ized b y the p re sence of a

cher ry- red spot in the macula r r eg ion of the eye (41) . Mac rocepha ly occurs

after 1 yea r bu t there are no viscera l changes such as hepa to sp l enomega ly o r

b o n y changes in T a y - S a c h s pa t ien ts .

Mic roscop i c s igns o f an acute l ip id s torage d i sease are found in neurona l

cells in var ious o rgans . T h e cy top lasm of these cells is d i s t ended and con ta ins

n u m e r o u s granules w h i c h on e lect ron mic roscop ic examina t ion appear as

spira l l amina ted s tructures w i t h a cross sec t ion o f 50 A (25) (Figure 3 ) . T h r o u g h

18. The Enzymology of Tay-Sachs Disease 481

/3-Galactosidase

Glc-Cer

0-Glucosidase

Ceramide

FIGURE 2. Enzymatic hydrolysis of gangliosides.

the use of b o t h con t i nuous and d i s con t inuous sucrose grad ien t cent r i fugat ion ,

it has b e e n p o s s i b l e to isolate these b o d i e s and s h o w that t hey con ta in a large

quan t i ty of the gang l ios ide G M 2 (F igure 1) (25) . Enzymat i c ana lys i s ind ica tes

that these i so la ted m e m b r a n o u s cy top lasmic b o d i e s con ta in h i g h levels of

var ious e n z y m e s w h i c h are character is t ic o f the l y s o s o m e ; th is f inding po in t s

out the i r p r o b a b l e lysosomal or ig in (38) . D e g e n e r a t i o n of the axons , fol lowed

b y demye l ina t i on , m a y also occur in T a y - S a c h s pa t ien t s (42) .

3. C h e m i s t r y o f the S to rage Ma te r i a l

A n al terat ion in the c h e m i c a l c o m p o s i t i o n of the b ra in of pa t ien t s w i t h Tay -

Sachs d i sease w a s first de tec ted b y K l e n k (11) w h o s h o w e d an inc rease in the

482 John F. Tollman

FIGURE 3. Typical isolated MCB from a patient with Tay-Sachs disease magnification x34,000.

Courtesy Dr. Suzuki.

acidic g lycosph ingo l ip id (gangl ios ide) con ten t of such pa t ien ts . Conf i rmat ion

of th is repor t fol lowed and the par t icular gang l ios ide presen t in e levated

quan t i t i e s was ident i f ied as G M 2 b y S v e n n e r h o l m (33) . Its s t ructure w a s later

de t e rmined (16) and th is c o m p o u n d c a m e to b e called T a y - S a c h s gang l ios ide

(Figure 1) . T h e total gang l ios ide con ten t of T a y - S a c h s b ra in is e leva ted to 4 to 5

t imes normal and the greates t p ropor t ion of th is is G M 2 (about 8 0 % ) . T h e asialo

der ivat ive of G M 2 / N-ace ty lga lac tosaminy lga lac tosy lg lucosy lce ramide(G A 2 ) is

also presen t in the b r a in s of these pa t ien ts (about 1 mo le GA2IS mo le s G M 2 ) and

is e levated approx imate ly 20 to 50 t imes normal (29) .

4. Nature o f the M e t a b o l i c D e f e c t

B a s e d on prev ious s tud ies conce rn ing the nature of enzymat ic defects in

related l ip idoses , such as G a u c h e r ' s d i sease and N i e m a n n - P i c k ' s d i sease (1) , a

logical si te for a defect in T a y - S a c h s d i sease was in the ca t abo l i sm of the ac-

cumula t ing gang l ios ide ( G M 2 ) and asialo der ivat ive ( G A 2 ) - S i n c e G M 2 i s

b r a n c h e d at its n o n r e d u c i n g t e rminus , ca t abo l i sm of G M 2 could conce ivab ly

p roceed e i ther t h rough the removal o f N-ace ty lneu ramin ic ac id (neu ramin i -

dase) to y ie ld G A 2 or N-ace ty lga lac tosamine (hexosamin idase ) to y ie ld G M 3

(Figure 2 ) . U s i n g artificial c h r o m o g e n i c subs t ra tes , ini t ia l s tudies on h u m a n

h e x o s a m i n i d a s e s ind ica ted that there w a s a h i g h e r total h e x o s a m i n i d a s e act iv-


Table I Subcellular Distribution of Cortical G M 2 -Hydrolases a

Fraction Gm-Sialidase

(pmoles/mg protein/hr) GM2-Hexosaminidase

(p moles/mg protein/hr)

Homogenate 62 31 Myelin-rich 0 0 Nerve endings, mitochondria,

and some lysosomes 79 56 Lysosome-rich'' 198 140 Cytosol 26 10

" Data from Tallman and Brady (34). h 64% of total G M 2 -sial idase activity; 81% of total G M 2 -hexosaminidase activity.

i ty in b r a in t i s sue from pa t ien ts w i t h T a y - S a c h s d i sease than in cont ro ls (28) .

T h e poss ib i l i ty h a d to b e cons ide red that a G M 2 - n e u r a m i n i d a s e def ic iency w a s

r e spons ib l e for the gang l ios ide accumula t ion and that the s torage of the asialo

c o m p o u n d was secondary to G M 2 accumula t ion . T o inves t iga te th i s a l ternat ive ,

G M 2 w a s b iosyn the t i ca l ly labe led in the N-ace ty lneu raminosy l por t ion of the

molecu le f rom a specific sial ic ac id precursor , N - a c e t y l m a n n o s a m i n e (13) . A

n e u r a m i n i d a s e w h i c h pos se s sed act ivi ty toward 3 H - A c n e u - G M 2 w a s d e m o n -

strated in m a m m a l i a n t i s sue (15 ,34) and pos se s sed a ly sosomal subcel lu lar d is -

t r ibu t ion (Table I ) . T h e act ivi ty of th is n e u r a m i n i d a s e w a s de t e rmined in

skeletal musc le (14) and b ra in t i ssue (39) from no rma l and T a y - S a c h s pa t ien ts .

T h e specific act ivi ty o f th is n e u r a m i n i d a s e was s imi la r in t i ssue s p e c i m e n s

from no rma l h u m a n s and pa t i en t s w i t h T a y - S a c h s d i sease (Table I I ) .

Table II Ganglioside Catabolism by Human Brain Lysosomes"

Enzyme Activities

Hexosaminidase Neuraminidase,

Source of Tissue GM2-l4C-Gangliosideh Artificial Substrate'' GM2-'-

iH-Gangliosideh

Control series (n = 7) G M 2 -Gangliosidosis patients

1. V.D. (Type I) 2. J.K. (Type I) 3. D.T. (Type II)

Mixed experiment Control + D.T.

140 ± 53

0 0 6

138 (Theory = 98)

134 ± 11

513 1416

3

Not determined

219 ± 3 3

225

Not determined 232

210 (Theory = 224)

" Data from Tallman et al. (39). b Picomoles G M 2 hydrolyzed/mg protein/hour. c Nanomoles 4-methylumbelliferyl-N-acetyl-D-glucosaminide hydrolyzed/mg protein/hour.

484 John F. Tollman

Reso lu t ion of the d i sc repancy b e t w e e n these resul ts and the ex i s t ence of G M 2

storage (and T a y - S a c h s d isease) d e p e n d e d on a n u m b e r of obse rva t ions . T w o

h e x o s a m i n i d a s e i s o z y m e s (A a n d B) h a d b e e n demons t r a t ed in h u m a n t i s sues

(23) . U s i n g b o t h hea t dena tura t ion (act ivi ty of h e x o s a m i n i d a s e A is labi le to

hea t ing at 50°C for 4 hours at p H 4 .4 ) and e lec t rophore t ic t e c h n i q u e s (A is a

more acidic p ro te in ) , O k a d a and O ' B r i e n (20) were able to s h o w a def ic iency

in h e x o s a m i n i d a s e A in t i s sues from pa t ien t s w i t h T a y - S a c h s d i sease . At that

t ime , the act ivi ty of the h e x o s a m i n i d a s e s w i th regard to the i r abi l i ty to b reak

d o w n G M 2 h a d no t b e e n thorough ly inves t iga ted . T h e re lease of N-acety lga lac-

to samine from doub ly labe led G M 2 w a s d i m i n i s h e d in skeletal musc le prepara-

t ions from pa t ien t s w i t h T a y - S a c h s d i sease (14) ; h o w e v e r , the poss ib i l i ty of a

two-s tep reac t ion in w h i c h N-ace ty lneu ramin i c ac id w a s first r e m o v e d could

no t b e ruled out. U s i n g G M 2 / specifically l abe led in the N-ace ty lga lac tosaminyl

por t ion of the molecu le (22) , it w a s poss ib l e to s h o w that direct hydro lys is of

N-ace ty lga lac tosamine from G M 2 occur red in b ra in l y sosomes (Table I ) , that the

e n z y m e invo lved in th i s p rocess (a h e x o s a m i n i d a s e ) pos se s sed m a n y proper-

t ies different from the n e u r a m i n i d a s e [e .g . , different p H o p t i m u m (Figure

4) ] and , m o s t impor tan t ly , that th is G M 2 - h e x o s a m i n i d a s e act ivi ty w a s m i s s i n g

in the b ra in t i ssue of pa t ien t s w i t h T a y - S a c h s d i sease (Table II) . It is cr i t ical to

m e n t i o n at th is po in t that in these s tudies (14 ,34 ,39 ) n o a t tempt w a s m a d e to

separate h e x o s a m i n i d a s e A from B and that these resul ts ind ica ted that a

def ic iency in G M 2 hydro lys i s w a s the under ly ing cause o f T a y - S a c h s d i sease . It

s e e m e d l ikely at the t ime b y the corre la t ion of our f indings w i th those of

O k a d a and O ' B r i e n (20) that h e x o s a m i n i d a s e A a lone pos se s sed hydrolyt ic

act ivi ty toward G M 2 ; s u b s e q u e n t expe r imen t s w i t h purif ied e n z y m e s have

s h o w n th i s to b e false. (See E for a further d i scuss ion of th i s topic . )

3.5 4.0 4.5 5.0 5.5 6.0 6.5

pH

FIGURE 4. Effect of varying pH on brain lysosomal hydrolases. Data from Tallman and Brady (34).

pM

OL

ES

P

RO

DU

CT

IN

CU

BA

TIO

N

JSialidase with 6D| 0 Substrate

• Sialidase with GM 2 Substrate

^ Hexosaminidase with G M 2 Substrate


In order to o b t a i n a full unde r s t and ing of the pa tho log ica l b i o c h e m i s t r y of

T a y - S a c h s d i sease , several addi t ional cons ide ra t ions m u s t b e t aken in to ac-

count . A h e x o s a m i n i d a s e B- l ike p ro te in , w h i c h is p re sen t in the t i s sues of pa-

t ien ts w i t h G M 2 - g a n g h ° s i d o s i s T y p e s I and III , can catalyze the hydro lys i s o f

G A 2 p roduced b y G M 2 _ n e u r a m i n i d a s e w h i c h is still ac t ive in these pa t ien ts .

S i n c e the specific ac t iv i ty o f th i s B- l ike e n z y m e is e levated (29) , w h y does G M 2 accumula te at all?

T h e q u e s t i o n m a y b e part ly reso lved b y the fact that in T y p e II G M 2 -gang l i -

os idos i s (Sec t ion C) in w h i c h pa t ien t s lack b o t h h e x o s a m i n i d a s e A and B

i s o z y m e s , the abso lu te quan t i ty of G A 2 accumula t ing in b ra in is h i g h e r and the

rat io o f G A 2 to G M 2 i s larger (V3) t han in T a y - S a c h s pa t i en t s (Type I) (29) . T h i s

f inding sugges t s that the al ternate p a t h w a y does par t ic ipa te in GM2 b r e a k d o w n

bu t is insuff ic ient to hand le the load of G M 2 c aused b y rap id gang l ios ide turn-

over in ear ly life. T h e insuff ic iency o f th i s p a t h w a y m a y b e due to a n u m b e r of

factors inc lud ing : nonop t iona l spat ial a r r angemen t o f the T a y - S a c h s pa t i en t ' s

h e x o s a m i n i d a s e B- l ike e n z y m e to the n e u r a m i n i d a s e , i n h i b i t i o n o f G A 2 hydrol -

ys i s in later s tages of the d i sease b y G M 2 (26) , i n h i b i t i o n of the n e u r a m i n i d a s e

act iv i ty aga ins t G M 2 b y the di- and t r i s ia logangl ios ides s ince the s a m e e n z y m e

works o n all t hese subs t ra tes (35) , or n o n o p t i m a l p H c o n d i t i o n s for o n e or b o t h

of the a l ternate e n z y m e s (pH 3.8 o p t i m u m for G A 2 hydro lys i s and see F igure 4 ) .

T h e ex i s t ence of th i s a l ternate p a t h w a y m a y also account for the relat ively

ins igni f icant accumula t ion of G M 2 ou t s ide the centra l ne rvous sys t em.

5. Prenatal Diagnosis and Treatment

Screen ing for carr iers of T y p e I G M 2 -gang l io s idos i s ha s b e e n successful ly

under t aken u t i l i z ing the hea t dena tura t ion m e t h o d on s e rum h e x o s a m i n i -

dases . Large-sca le tes t ing p rograms for h i g h r isk popu la t ions are current ly

b e i n g i m p l e m e n t e d (6 ,10) and successful in t rau ter ine d i agnos i s o f T a y - S a c h s

d i sease h a s b e e n m a d e direct ly on amnio t i c fluid s amples (30) and cul tured

amnio t i c cells (19) . T h e r e is n o current therapy for T a y - S a c h s d i sease and the

success of e n z y m e r ep l acemen t trials s e e m s s l ight b e c a u s e of the bar r ie r (9) to

e n z y m e en t rance in to the b ra in .

C. TYPE II GM2-GANGLIOSIDOSIS (SANDHOFF'S DISEASE)

1. Clinical and Pathological Aspects

T h e s e pa t ien ts have neuro log ica l s igns s imi la r to those of pa t ien t s w i t h Type

I G M 2 - g a n g l i o s i d o s i s . Addi t iona l ly , there are c h a n g e s in v i scera l o rgans at ten-

dant on the accumula t ion of neutra l g lycosph ingo l ip id called g lobos ide : N - ace -

ty lga lac tosaminylga lac tosy lga lac tosy lg lucosy lceramide in b o t h l iver and sp leen

486 John F. Tallman

(28). T h e p rogress ion of th i s d i sease is s imi lar to T y p e I. The re is no pred i lec t ion

to J e w i s h ances t ry in these pa t ien t s .

Microscop ica l ly , there is l i t t le difference b e t w e e n the neurona l inc lus ions in

Type I and II G M 2 - g a n g l i o s i d o s i s . T h e p r e d o m i n a n t difference is the p re sence of

n u m e r o u s m e m b r a n o u s cy top lasmic b o d i e s in the l iver cells of Type II pa-

t ients . O n electron mic roscop ic examina t i on these b o d i e s appear qu i te s imi lar

to those desc r ibed in the b r a i n s of pa t ien ts w i t h T y p e I d i sease .

2. C h e m i s t r y o f the S to rage Ma te r i a l

Pa t ien ts w i th Type II G M 2 - g a n g l i o s i d o s i s are also charac ter ized b y the ac-

cumula t ion of G M 2 and i ts as ia lo der iva t ive i n neurona l cells. It i s in teres t ing to

note that the a m o u n t of G A 2 accumula t ion relat ive to G M 2 is greater than in

Type I (about 1 mo le G A 2 / 3 mo les G M 2 ) . T h i s m a y b e related to the na ture of the

enzymat i c defect and al ternate p a t h w a y d i scussed in Sec t ion B , 4 . In Type II pa-

t ien ts , there is also a s ignif icant ly h i g h e r accumula t ion of G M 2 in l iver com-

pared to T a y - S a c h s pa t ien ts w h e r e G M 2 accumula t ion is m i n i m a l (12) .

3. M e t a b o l i c D e f e c t , D i a g n o s i s and T r e a t m e n t

Pa t ien ts w i th T y p e II G M 2 - g a n g l i o s i d o s i s are charac te r ized b y a total lack of

h e x o s a m i n i d a s e act iv i ty w h e n m e a s u r e d w i t h artificial subs t ra tes (29) . T h e s e

pa t ien ts also lack the ab i l i ty to degrade G M 2 (Table II) (39) and neutra l gly-

cosph ingo l ip ids con ta in ing a t e rmina l molecu le o f N-ace ty lga lac tosamine

(12 ,29) . De t ec t i on of he te rozygo tes for T y p e II G M 2 - g a n g l i o s i d o s i s i s poss ib l e

and prenata l de tec t ion of th is d i sease has b e e n carr ied out (12) .

T rea tmen t of pa t ien t s w i th Type II G M 2 - g a n g l i o s i d o s i s has b e e n a t t empted

b y the in t ravenous in jec t ion o f h i g h l y purif ied h e x o s a m i n i d a s e A (9).

Al though s o m e lower ing of the se rum g lobos ide levels w a s o b t a i n e d , there

was no increase in h e x o s a m i n i d a s e ac t iv i ty in the b ra in . T h u s , th is therapeu-

tic effort m u s t b e regarded as unsuccessfu l .

D . O T H E R V A R I A N T F O R M S

4. T y p e I I I G M 2 - G a n g l i o s i d o s i s

A pa t ien t w i th T y p e III G M 2 - g a n g l i o s i d o s i s has b e e n desc r ibed w h o ap-

peared cl inical ly as a T y p e I pa t ien t and did no t s h o w any viscera l changes .

T h e amoun t of b o t h G M 2 and G A 2 accumula ted in the b r a in of th is pa t ien t were

h i g h e r than in Type I pa t ien t s , a l though the relat ive p ropor t ions are close to

those of T y p e I pa t ien t s (29) . W h e n artificial subs t ra tes are u sed to m o n i t o r act ivi ty , b o t h hex -


o s a m i n i d a s e s A and B appeared to b e p resen t a l though the abi l i ty of th i s pa-

t ient to b r e a k d o w n G M 2 i s d i m i n i s h e d (29) . T h e s e resul ts are in te rpre ted and

d i scussed in Sec t i on F .

2. H e x o s a m i n i d a s e A-Def i c i en t Adu l t s

V e r y recent ly , no rma l adults have b e e n desc r ibed w h o are totally lack ing in

h e x o s a m i n i d a s e A and yet do no t exh ib i t any of the s igns of T a y - S a c h s d i sease

(18 ,44) . In these cases , prenata l d i agnos i s is i m p o s s i b l e us ing current

m e t h o d s . A poss ib l e exp lana t ion of th i s s e e m i n g paradox is p re sen ted in S e c -

t ion F .

E . P R O B L E M S I N G M 2 H Y D R O L Y S I S

T h e p r o b l e m s in e s t ab l i sh ing G M 2 hydro lys i s w e r e a lmost i n s u r m o u n t a b l e . It

was i m p o s s i b l e to s h o w direct ly b y color imet r ic m e t h o d s the reac t ion products

from e i the r G M 2 - h e x o s a m i n i d a s e or G M 2 - n e u r a m i n i d a s e . For th i s r eason , the

use of G M 2 specif ical ly l abe led e i the r in the N-ace ty lga lac tosaminy l and N - a c e -

ty lneu raminosy l por t ion of the molecu le w a s in i t i a ted . W h e n the l abe led s u b -

strates are u sed , it is p o s s i b l e to s h o w the l ow levels o f ac t iv i ty repor ted in

T a b l e s I and II. B o t h e n z y m e s have specif ic ac t iv i t ies that are m u c h lower than

the o the r e n z y m e s of the gang l ios ide ca tabol ic p a t h w a y [e .g . , g lucoce rebros i -

dase is 100 t i m e s m o r e ac t ive in the same sys t em (37)] and m a k e it l ikely that

hydro lys i s o f G M 2 i s the ra te - l imi t ing s tep in gang l ios ide turnover .

T h e b ra in w a s c h o s e n for s tudy b e c a u s e the p r e d o m i n e n t effects in T a y -

Sachs d i sease are man i fes t in th is organ. T o m i m i c as c losely as pos s ib l e the

phys io log ica l s tate , a c rude ly sosomal sys t em w a s p repa red and great care w a s

taken to avoid rupture of the l y sosomes . M a n y a t tempts we re m a d e to op-

t imize the i n c u b a t i o n cond i t i ons . De te rgen t s and var ious ca t ions had n o s ig-

nif icant s t imula tory effect and in m a n y cases we re inh ib i to ry . F resh t i s sue

(never frozen) w a s u sed in all these s tud ies b e c a u s e f reezing inac t iva ted the

h e x o s a m i n i d a s e w i t h G M 2 as subs t ra te . S o n i c d i s rup t ion of the in tac t lyso-

s o m e s w h i c h p o s s e s s e d h e x o s a m i n i d a s e act iv i ty toward G M 2 resu l ted in prepa-

ra t ions w h i c h n o longer p o s s e s s e d G M 2 c leav ing act iv i ty ; ac t iv i ty t oward the

artificial subs t ra te , 4 -methy lumbel l i fe ry l -N-ace ty l -^-D -g lucosamin ide , w a s un-

affected b y th is t r ea tment and G M i - / 3 - g a l a c t o s i d a s e act iv i ty w a s also unaf-

fected. Mul t i s t ep hydro lys i s o f G a l N A c - 1 4 C - G M i w i th the re lease o f G a l N A c -1 4 C (22) , w h i c h w e w e r e ab le to demons t ra t e in th is sy s t em was also lost . It is

fa tuous to d is regard the a m o u n t of ac t iv i ty repor ted in these s tud ies b y

b l i the ly dec lar ing that op t ima l cond i t i ons have no t b e e n ach ieved . I ndeed , it

is l ikely that effect ive G M 2 hydro lys i s m a y d e p e n d on a m e m b r a n e - b o u n d h e x -

o s a m i n i d a s e or the assoc ia t ion of th is e n z y m e w i t h a cr i t ical l ip id or p ro te in

488 John F. Tallman

Table III Activity of Normal Lysosomes after the Addition of Various Purified Enzymes a

Addition Activity (pmoles GalNAc/incubation/hr)

Lysosomal extract 49 Urinary hexosaminidase A 0 Urinary hexosaminidase B 0

Lysosomal extract (i) + U Hex A 4- U. Hex B 23

" Data from Tallman et al. (39).

i ns ide the ly sosomal m e m b r a n e as w a s o b t a i n e d in in tac t l y sosomes . D i s rup-

t ion of th is sys t em or ul t rastructure leads to drast ic loss in G M 2 hydro lys i s b y

the h e x o s a m i n i d a s e . If th i s is t rue, it w o u l d also expla in our inab i l i ty to b r ing

abou t s t imula t ion of the G M 2 - c l e a v i n g ab i l i ty of e x o g e n o u s h e x o s a m i n i d a s e

added to the lysosomal sys t em (39) (Table III) .

V e r y recent ly , w e (37) have b e e n ab le to purify h e x o s a m i n i d a s e A and B ex-

tens ive ly from fresh h u m a n placenta . U s i n g these purif ied e n z y m e s w e found

that bo th normal h u m a n placental h e x o s a m i n i d a s e s A and B possess act ivi ty

toward G a l N A c - 1 4 C - G M 2 (Table I V ) . Hydro lys i s o f th is subs t ra te w a s ob t a ined

only in the p resence of the de te rgen t s o d i u m taurochola te . T h i s d e p e n d e n c e is

in contras t to the s tud ies in c rude b ra in l y sosomes w h e r e no de te rgent w a s

r equ i red for G M 2 hydro lys i s . A g a i n , in s tud ies in c rude t i s sue p repara t ions no

a t tempt w a s m a d e to separa te the ac t iv i t ies o f h e x o s a m i n i d a s e A and B . A

h i g h e r rat io of natural to artificial subst ra te hydro lys i s w a s also o b t a i n e d in

crude prepara t ions (1 :1000 versus 1 :10 6 for the purif ied e n z y m e ) . T h e lyso-

somal prepara t ions also s h o w e d a m o r e bas i c p H o p t i m u m than the h igh ly

purif ied e n z y m e s (compare F igures 4 and 5) and a classical k ine t i c s i tua t ion

(compare F igures 6 and 7) . W i t h the use of e i ther purif ied e n z y m e , a complex

r e sponse of the e n z y m e to inc reas ing subs t ra te concen t ra t ion w a s no ted and

an op t imal de te rgen t to subs t ra te rat io could b e e s t ab l i shed . H o w e v e r , the l ow

levels of ac t iv i ty o b t a i n e d in these expe r imen t s w i th the purif ied hex-

o s a m i n i d a s e s canno t b e cons ide red phys io log ica l ly s ignif icant .

Table IV Ratio of G M 2 Cleaving to Artificial Substrate Activity of Purified Hexodaminidases a

Artificial Activity GM2 Activity Ratio Natural/Artificial Enzyme (ixmoles/hr) (pmoles/hr) (pmole/ ixmole)

Hexosaminidase A 2.4 121 50.4 Hexosaminidase B 0.6 35 57.5

a Data from Tallman et al. (39).


375 r-

2.0 4.0 5.0 6.0 P H

FIGURE 5. Effect of varying pH on G M 2 hydrolysis by purified hexosaminidase A or B . Data from Tallman et al. (37).

Simi la r expe r imen t s (27) u s ing a purif ied prepara t ion of h e x o s a m i n i d a s e s

from h u m a n l iver demons t r a t ed the hydro lys i s of 3 H - G M 2 ( reduct ively tr i t iated

in the s p h i n g o s i n e mo ie ty ) u s ing long- te rm i n c u b a t i o n s (24 hours ) in the pres -

ence of s o d i u m taurochola te . O n l y h e x o s a m i n i d a s e A w a s capab le of th is

hydro lys i s . Purif ied B had no act ivi ty . H o w e v e r , because of the small

50 100 2 0 0 3 0 0

FIGURE 6. Effect of varying substrate concentration on G M 2 hydrolysis by brain lysosomal hydro-lases. Data from Tallman and Brady (34).

pM

OL

ES

N

-AC

ET

YL

GA

LA

CT

OS

AM

INE

/

INC

UB

AT

ION

jt/xm

ole

s P

RO

DU

CT

/hr

490 John F. Tallman

FIGURE 7. Effect of varying substrate concentration on G M 2 hydrolysis by purified hex-osaminidases A or B. Dark circles, hexosaminidase A . Open circles, hexosaminidase B . Data from Tallman et al (37).

amoun t of product ( G M 3 ) ( ~ 5 % ) fo rmed and the compl ica ted assay procedure

necess i t a ted b y the na ture of the l abe l ing , a s ignif icant decrease in the act ivi ty

of h e x o s a m i n i d a s e B dur ing the course of the i n c u b a t i o n wou ld m a k e it difficult

to detect act ivi ty p resen t on ly in the ini t ia l s tages of the reac t ion ( i . e . , a

smal ler a m o u n t of product ) . S u c h a decrease in act iv i ty w a s no ted i n our

exper imen t s (Figure 8) and m i g h t account for the inab i l i ty to demons t ra t e

act ivi ty wi th B .

1500

HOURS

FIGURE 8. Linearity of G M 2 hydrolysis with time by purified hexosaminidase A or B. Data from Tallman et al (37).

pmol

es N-ACETYLGALACTOSAMME

RELEASED/1NCUBATION / Hr

pMOLES

N-A

CETY

LGAL

ACTO

SAMI

NE

RELEASED

/ INC

UBAT

ION


T h e use of frozen t i s sue as the purif icat ion source m a y account for the dif-

ficulties of ano the r g roup (45) in demons t r a t i ng any G M 2 hydro lys i s . S i n c e I

have b e e n e m p h a s i z i n g the neces s i t y o f fresh t i s sue to s tudy adequa te ly the

gang l ios ide ca tabol ic e n z y m e s in l ight o f our expe r i ence , the i r inab i l i ty to

demons t ra te act iv i ty is no t surpr i s ing . H o w e v e r , in the i r expe r imen t s , the ad-

di t ion o f a bac ter ia l n e u r a m i n i d a s e led to a s ignif icant conve r s ion of G M 2 to

lac tosy lceramide . T h e p H of th i s i ncuba t ion w a s 3 . 8 , the o p t i m u m for the

hydro lys i s o f G A 2 and far from the o p t i m u m of the bacter ia l n e u r a m i n i d a s e . It

is no t clear from the i r data w h e t h e r th is is a two-s tep p rocess invo lv ing G M 2

hydro lys i s to G A 2 fo l lowed b y the hydro lys i s o f th i s c o m p o u n d to lactosyl-

ce ramide or an abs t ruse m e c h a n i s m u s i n g a " n e u r a m i n i d a s e - h e x o s a m i n i d a s e "

complex . O u r a t t empts to recons t ruc t th is e x p e r i m e n t w i th our op t ima l s u b -

strate/detergent rat io we re unsuccessfu l . W e s h o w e d a s l ight i n h i b i t i o n of our

act ive h e x o s a m i n i d a s e A at p H 3.8 b y the add i t ion of n e u r a m i n i d a s e ( S i g m a ,

Type V I ) . A t p H 5 .0 in our h a n d s , e i ther s tab i l i za t ion of the h e x o s a m i n i d a s e

or s o m e conve r s ion of G M 2 to G A 2 fo l lowed b y re lease of N-ace ty lga lac tosamine

from th is c o m p o u n d is o b t a i n e d . B a s e d on this ev idence and our p rev ious

nega t ive resul ts (Tab le III) in w h i c h w e s tud ied p o s s i b l e synerg i s t i c effects

after the add i t ion of purif ied m a m m a l i a n n e u r a m i n i d a s e to b o t h crude lyso-

somal p repara t ions f rom h u m a n b r a in and puri f ied h u m a n h e x o s a m i n i d a s e s ,

we feel that the pos tu la t ion of a " c o m p l e x of n e u r a m i n i d a s e and h e x -

o s a m i n i d a s e " is p rematu re and c louds the m e c h a n i s m of an o the rwise

" s i m p l e " hydro lase . It is also pos s ib l e that a c o n t a m i n a n t in the i r n e u r a m i n i -

dase, such as a l ip id , led to s t imula t ion of the i r h e x o s a m i n i d a s e in the same

w a y as the de te rgent , s o d i u m taurochola te .

Ve ry recent ly , L i and co-workers (17) have repor ted the p re sence of a heat -

s table factor from l iver superna tan t w h i c h , w h e n added to part ly purif ied h e x -

o s a m i n i d a s e s in the a b s e n c e of o ther de te rgen t s , s t imula ted the A form bu t

not B . A l though a p rovoca t ive f inding, and pe rhaps of phys io log ica l s ignif i -

cance , the i r fully s t imula ted h e x o s a m i n i d a s e was on ly one - t en th as ac t ive as

our purif ied A or B w a s w i t h s o d i u m taurochola te . T h u s , the i r hea t - s tab le

factor mere ly s t imula tes the i r e n z y m e s of n o act iv i ty in to l ow act ivi ty . T h e y do

not approach a phys io log ica l ly s ignif icant level b y several orders of m a g n i t u d e

and further w o r k is r equ i red to assess the s igni f icance of th i s f inding.

F. PHYSICAL PROPERTIES AND POSSIBLE RELATIONSHIP OF HEXOAMINIDASES

T h e apparen t molecu la r w e i g h t is iden t ica l for b o t h h e x o s a m i n i d a s e A and B

as de t e rmined b y gel filtration on S e p h a d e x G - 2 0 0 . O u r value of 127 ,000

dal tons is in a g r e e m e n t w i t h that repor ted ear l ier (29) for h u m a n l iver and

s l ight ly lower than the repor ted w e i g h t s of the b e e f sp leen e n z y m e s (43) .

492 John F. Tallman

W h e n the molecular w e i g h t s of h e x o s a m i n i d a s e s A and B were de t e rmined

us ing gel e lec t rophores i s in the p resence of urea , s o d i u m dodecylsulfa te , and

reduc ing agent at h i g h p H , a un i fo rm subun i t w i t h a m a s s of 3 3 , 0 0 0 dal tons is

ob t a ined . S u b u n i t s o f m a s s e s of 3 3 , 0 0 0 and 6 6 , 0 0 0 dal tons were s een after

p r e incuba t i on in the p re sence of these agents near neutra l i ty . T h e s e resul ts are

in the range of subun i t w e i g h t s o b t a i n e d w i t h the b e e f sp leen e n z y m e s (43)

and the p resence of on ly a s ing le b a n d on the h igh p H gels w h e n hex -

o s a m i n i d a s e s A and B are run s imul taneous ly ind ica tes that the subun i t s of

each e n z y m e are of ident ica l m a s s . In separate expe r imen t s , w h e n b o t h en-

z y m e s were added to the s a m e gel , no differences could b e de tec ted . A n inter-

es t ing difference w a s no t ed w h e n urea and r educ ing agents were no t inc luded

in the p re incuba t ion . In th is case , only trace amoun t s of the 3 3 , 0 0 0 molecular

w e i g h t un i t we re seen . H e x o s a m i n i d a s e A had a dark b a n d at the 6 5 , 0 0 0 -

dalton level and trace a m o u n t s of a set of h i g h e r (—120 ,000-140 ,000) molecular

w e i g h t p ro te ins . In contras t , h e x o s a m i n i d a s e B was ra ther res is tant to b reak-

d o w n in to i ts subun i t s and d isp layed a pat tern of p ro te ins of molecular we igh t

r ang ing from 120 ,000 to 140 ,000 dal tons (Figure 9 ) . T h i s pat tern was qual i ta-

FIGURE 9A. Polyacrylamide gel electrophoresis of purified hexosaminidases A (left gel) and B (right gel) after preincubation in the presence of 0 . 1 % SDS at pH 7.2. Tallman et al. (37).


FIGURE 9B. Polyacrylamide gel electrophoresis of purified hexosaminidases A (left gel) and B

(right gel) after preincubation in the presence of 0 . 1 % SDS, 1 0 % mercaptoethanol, 3 M urea, and

0 . 5 % dithiothreitol at pH 7.2. Tallman et al (37).

t ive ly l ike the trace a m o u n t s of h i g h molecu la r w e i g h t A . F r o m these data it

w o u l d s e e m that disulf ide b o n d s play an impor t an t part of the assoc ia t ion of

the two 6 5 , 0 0 0 un i t s in B fo rming the te t ramer . In h e x o s a m i n i d a s e A , th i s type

of b a n d does no t ye t exis t or is labi le unde r the cond i t i ons e m p l o y e d ; i n d e e d ,

it i s l ike ly that the un i t s are hyd rophob ica l ly assoc ia ted b u t no t covalent ly

l inked . T h e he t e rogene i ty of b o t h e n z y m e s on S D S gels at the h i g h molecu la r

w e i g h t r ange is in te res t ing b e c a u s e it m a y represen t the a s soc ia t ion of va ry ing

amoun t s of S D S w i t h the e n z y m e . P r e s u m a b l y , the e n z y m e s are qu i t e c lose to

the i r na t ive s tate and m a y con ta in vary ing a m o u n t s o f ca rbohydra t e (43) ; such

s ide cha ins c h a n g e the in te rac t ion of S D S w i t h p ro te ins (31) .

It i s p o s s i b l e to conve r t h e x o s a m i n i d a s e A in to B b y h e a t i n g at va r ious t e m -

pera tures in di lute p H 6.0 buffer (Figure 10 ) , and th i s conve r s ion approaches

1 0 0 % at 5 0 ° C . A m e c h a n i s m in w h i c h the c o m p l e m e n t a r y - S H groups are in

close appos i t i on after hea t ing and form o n e or more disulfide b r idges s e e m s

to b e i m p l i e d b y th i s expe r imen t . T h i s m a y b e due to an in i t ia l change in the

h y d r o p h o b i c in te rac t ions of the subun i t s . T h e inab i l i ty to conver t B b a c k in to

A to a degree greater than the error in the m o n i t o r i n g of each form is also

B

494 John F. Tallman

100

FIGURE 10. The conversion of hexosaminidase A to B . Purified hexosaminidase A was heated in pH 6.0 buffer for 2 hours at the temperatures indicated. Percentage B was determined as described in Tallman et al. (37).

exp la ined b y the p re sence of th is n e w disulfide b o n d . Inheren t ly , B wou ld

then appear to b e the m o r e s table pro te in and differences in the charge proper-

t ies of the two e n z y m e s m a y exis t b e c a u s e of the conformat iona l changes of

the pro te in e i ther p reced ing or a c c o m p a n y i n g the format ion of the n e w disul-

fide bond( s ) . In di lute so lu t ion , w h e r e c h a i n - c h a i n in te rac t ions are of great

impor t ance , s imi lar the rmos tab i l i ty profiles for bo th h e x o s a m i n i d a s e s are o b -

served (Figure 11) . A n in t e rmed ia te in the dena tura t ion of A in such so lu t ions

m i g h t b e a " B - l i k e " spec ie s . In s e r u m and crude t i s sues , o n e m i g h t no t expect

conver s ion of A to B , bu t ra ther unfo ld ing of the A pro te in on hea t ing under

acidic cond i t ions and in te rac t ion of the cri t ical sulfhydryl groups of A wi th

o ther c o m p o n e n t s of the pro te in- r ich mix tu re l ead ing to loss of enzymat ic

act ivi ty. H e x o s a m i n i d a s e B m i g h t not b e expec ted to s h o w th is dena tura t ion

in se rum.

O t h e r ev idence suppor ts th i s concep t of the close re la t ionsh ip of the hex-

osamin idases . In h u m a n placenta , the a m i n o acid conten t of b o t h en-

zymes w a s qui te s imi lar , and the on ly differences b e t w e e n the two spec ies

was in the i r sialic ac id and neut ra l ca rbohydra te content . Immuno log i ca l evi -

dence (desc r ibed b e l o w ) also suppor ts th is c lose re la t ionsh ip .

I wou ld l ike to s u b m i t a w o r k i n g mode l for the in te r re la t ionsh ip of hex-

o s a m i n i d a s e s w h i c h is b a s e d on our ev idence and that p u b l i s h e d b y o ther in-

ves t iga tors . It i s fully real ized that the concep t m a y have to b e modif ied or

even rejected as a c o n s e q u e n c e of further s tud ies , bu t for n o w it represents to

us the clearest exp lana t ion for the data o b t a i n e d for no rmal e n z y m e s and the

e n z y m e s from the var ious cl inical forms of T a y - S a c h s d i sease .

H e x o s a m i n i d a s e s A and B represen t different conformat iona l states of the

Q3r\IU

0J 9

%


HEAT STABILITY OF HEXOSAMINIDASE ACTIVITY (Hexosaminidase B )

2 HOURS

—•44° —•30° - - o 3 7 °

- - •53°

- -^61° 4

FIGURE 11. A and B. Thermostability of purified hexosaminidase A and B in pH 6.0 buffer.

From Tallman et al. (37).

same e n z y m e . E v i d e n c e for th i s theory is b a s e d on the a lmos t identical k ine t i c

pat terns o b t a i n e d w h e n the catalyt ic act ivi ty w i t h var ious subs t ra tes is i nves -

t iga ted , o n ident ica l the rmal dena tura t ion curves in di lute so lu t ion nea r n e u -

trali ty, on iden t ica l n u m b e r o f subun i t s pe r h o l o e n z y m e and ident ica l mo lecu -

lar we igh t s of these un i t s , on essent ia l ly ident ica l a m i n o acid c o m p o s i t i o n of

purified placental e n z y m e s , and on i m m u n o l o g i c a l ev idence (5,32) w h i c h

ind ica tes that A and B cross react ex tens ive ly . Adso rp t ion of a n t i - A b y hex -

o s a m i n i d a s e B has b e e n sa id to lead to the p roduc t ion of specific an t i -A an-

t i se rum (32), a l though the o the r groups (5) have d i spu ted th is f inding. T h e

specific an t i se rum A m a y resul t f rom an an t igen character is t ic of the hex -

osamin idase w i thou t the disulfide b o n d . R e d u c t i o n o f such b o n d s has b e e n

s h o w n to change the an t igen ic proper t ies o f o ther p ro te ins (4). It is s ignif icant ,

PE

RC

EN

T O

F

INIT

IAL

AC

TIV

ITY

A HOURS

PE

RC

EN

T O

F

INIT

IAL

AC

TIV

ITY

HEAT STABILITY OF HEXOSAMINIDASE ACTIVITY (Hexosaminidase A )

B 3

496 John F. Tallman

howeve r , that there are no specific an t i -B an t ibod ie s w h i c h migh t b e expec ted

if there were o n e c o m m o n and o n e u n i q u e subun i t for A and B .

S ince syn thes i s of p ro te ins p roceeds from the a m i n o te rmina l end of the

molecu le , the d is t inc t poss ib i l i t y exis ts that the early folding of the subun i t s is

fol lowed b y the i r assoc ia t ion to form a te t ramer w h i c h is a metas tab le A ,

despi te the fact that the t he rmodynamica l ly m o r e s table form m a y b e hex -

o s a m i n i d a s e B . In fact, an ene rgy bar r ie r b e t w e e n the two forms m a y exis t ,

and th is bar r ier is o v e r c o m e b y hea t ing . If ex t rus ion of n e w l y syn thes ized en-

z y m e after g lycosyla t ion in to the extracellular m e d i u m is fol lowed b y uptake

to form ly sosomes (8) , a h i g h propor t ion of the A i s o e n z y m e m i g h t be ex-

pec ted in t i ssue culture m e d i u m and extracellular fluids such as fresh p lasma

w h e r e A is found a lmos t exclus ive ly (21) .

In pa t ien ts w i t h classic T a y - S a c h s d i sease , the ini t ia l folding of the e n z y m e

in the A conformat ion is less s tab le than usual or the ene rgy bar r ie r b e t w e e n

the two forms is lower b e c a u s e of a m i s s e n s e muta t ion w h i c h results in an

amino acid subs t i tu t ion at a po in t cri t ical for the folding of the subun i t s and

the i r assoc ia t ion (hyd rophob ic r eg ion) . In this case , the disulfide b o n d n e c e s -

sary for the format ion of the s table B is readi ly formed. T h u s , h e x o s a m i n i d a s e

B is fo rmed a lmost i m m e d i a t e l y at the modera te t empera tures o f the b o d y . T h e

inc reased ins tab i l i ty of the A form of the e n z y m e accounts for the m i s s i n g A

in T a y - S a c h s pa t ien ts and the i r lack of the A-speci f ic an t igen (32) . T h e s e pa-

t ients do have a very small amoun t of res idual (newly syn thes ized?) A act ivi ty

and e levated " B - l i k e " e n z y m e . T h e y do no t have a full c o m p l e m e n t of inact ive

A pro te in . It is in te res t ing to no te that the " B - l i k e " e n z y m e w h i c h i s fo rmed in

these pa t ien ts is no t the no rma l B w h i c h wou ld b e necessa ry i f the c o m m o n

and u n i q u e subun i t theory he ld , bu t an e n z y m e w h i c h m o v e s m o r e s lowly

toward the anode in neutra l p H e lec t rophore t ic s c h e m e s ; the res idual A m o v e s

s imilar ly (5) . Concur ren t w i t h th is muta t ion , bu t no t as a result of the loss of

the A conformat ion a b o v e , there is a loss o f the abi l i ty o f e i the r form (A or B)

to catalyze the b r e a k d o w n of G M 2 . T h i s loss o f G M 2 - c l e a v i n g abi l i ty is the real

deficit in the G M 2 - g a n g l i o s i d o s e s .

In the " O " var ian t (Sandhoff ' s d i sease) bo th h e x o s a m i n i d a s e A and B cross-

react ing pro te ins are sa id to b e present (32) , a l though th is has not b e e n totally

e s tab l i shed (5) . T h e muta t ion , different from that in classic T a y - S a c h s d i sease ,

wou ld b e at a si te w h i c h is impor tan t in the actual m e c h a n i s m of hydro lys i s of

N-ace ty lhexosamine from any of the subs t ra tes . T h u s , a mechan i s t i ca l ly i m -

portant a m i n o acid is impl ica ted in th is d i sease and no t necessa r i ly an amino

acid invo lved in conformat iona l aspec ts .

In the " A B " var ian t whe re b o t h i s o z y m e s are present , G M 2 degrada t ion i s

decreased . Here w e have a muta t ion w h i c h involves G M 2 hydro lys i s bu t does

no t affect the conformat iona l s tab i l i ty of the A form; accord ingly , A is p resent .

O n e m i g h t predict , on theore t ica l g rounds , that s ince b o t h A and B have


act ivi ty aga ins t G M 2 / there m a y exis t a b e n i g n cond i t ion in w h i c h pa t ien ts m i g h t

lack the A i s o z y m e yet the i r B m a y re ta in G M 2 - h y d r o l y z i n g abi l i ty . T h e s e pa t ien ts

w o u l d p resen t no c l in ical s igns and w o u l d on ly b e no t i ced as a resul t o f the

mass - sc r een ing p rograms presen t ly b a s e d on differential hea t dena tura t ion of

h e x o s a m i n i d a s e A . Recen t ly , such pat ients have b e e n desc r ibed b y N a v o n (18)

and Vidgof f (44) .

R E F E R E N C E S

1. Brady, R. O. The Sphingolipidoses. N. Engl J . Med., 1966, 275: 312. 2. Brady, R. O. Lipidoses. Biochimie, 1972, 54: 723.

3. Brady, R. O. Hereditary diseases—causes, cures, and problems. Angew Chem. [Engl], 1973, 12: 1.

4. Brown, R. Studies on the antigenic structure of ribonuclease. / . Biol Chem., 1962, 237: 1162.

5. Carroll, M. , and Robinson, D. Immunological properties of N-acetyl-/3-D-glucosaminidase of normal human liver and of G M2-gangliosidosis liver. Biochem. ] . , 1973, 131: 91.

6. Graves, R. , Manunes, P., and Bakerman, S. Screening for Tay-Sachs disease carriers. Fed. Proc, 1973, 32: 866.

7. Handa, S., and Yamakama, T. Biochemical studies in cat and human gangliosidosis. / . Neurochem., 1971, 18: 1275.

8. Hickman, S., and Neufeld, E. A hypothesis for I-cell disease: Defective hydrolases that do not enter lysosomes. Biochem. Biophys. Res. Commun., 1972, 49: 992.

9. Johnson, W. G. , et al. Intravenous injection of purified hexosaminidase into a patient with Tay-Sachs disease. Birth Defects, Orig. Art. Ser., 1973, 9: 120-125.

10. Kaback, M. M., and Zeiger, R. Heterozygote detection in Tay-Sachs disease: A prototype community screening program for the prevention of recessive genetic disorders. In: Sphingolipids, Sphingolipidoses and Allied Diseases. (B. Volk, and S. Aronson, Eds.). Plenum, New York, 1972: 613-632.

11. Klenk, E. Uber die ganglioside des gehirns bei der infantlen amaurotischen Idiotie von Typus Tay-Sachs. Ber. Deut. Chem. Ges., 1942, 75: 1632.

12. Kolodny, E. H. Sandhoff's disease: Studies on the enzyme defect in homozygotes and detec-tion of heterozygotes. In: Sphingolipids, Sphingolipidoses and Allied Diseases. (B. Volk and S. Aronson, Eds.). Plenum, New York, 1972: 221-341.

13. Kolodny, E. H., Brady, R. O., Quirk, J . M., and Kanfer, J . N. Preparation of radioactive Tay-Sachs ganglioside labeled in the sialic acid moiety. / . Lipid Res., 1970, 11: 144.

14. Kolodny, E. H., Brady, R. O., and Volk, B. W. Demonstration of an alteration of ganglioside metabolism in Tay-Sachs disease. Biochem. Biophys. Res. Commun., 1969, 37: 526.

15. Kolodny, E. H., Kanfer, J . N. , Quirk, J . M. , and Brady, R. O. Properties of a particle-bound enzyme from rat intestine that cleaves sialic acid from Tay-Sachs ganglioside. / . Biol. Chem., 1971, 246: 1426.

16. Ledeen, R. , and Salsman, K. Structure of the Tay-Sachs ganglioside. Biochemistry, 1965, 4: 2225.

17. Li, Y. T., Mazzotta, M., Wan, C., Orth, R., and Li, S. C. Hydrolysis of Tay-Sachs ganglioside by /3-hexosaminidase A of human liver and urine. / . Biol. Chem., 1973, 248: 7512.

18. Navon, R., Padeh, B . , and Adam, A. Apparent deficiency of hexosaminidase A in healthy members of a family with Tay-Sachs disease. Am. J . Hum. Genet., 1973, 25: 287.

19. O'Brien, J . S., Okada, S., Fillerup, D. L. , Veath, M. L. , Adornato, B . , Brenner, P., and Leroy, J. Tay-Sachs disease—prenatal diagnosis. Science, 1971, 172: 61.

498 John F. Tallman

20. Okada, S., and O'Brien, J . S. Tay-Sachs disease: Generalized absence of a /3-D-N-acetylhex-osaminidase component. Science, 1969, 165: 698.

21. Price, R. , and Dance, N. The demonstration of multiple heat stable forms of N-acetyl-/3-glucosaminidase in normal human serum. Biochim. Biophys. Acta, 1972, 2 7 1 : 145.

22. Quirk, J . M., Tallman, J . F . , and Brady, R. O. Preparation of trihexosyl- and tetrahexosyl-gangliosides specifically labelled in the N-acetylgalactosamine moiety. / . Label. Compounds,

1972, 8: 484.

23. Robinson, D. , and Stirling, J . L. N-Acetyl-/3-gulcosaminidases in human spleen. Biochem. J . ,

1968, 107: 321.

24. Sachs, B. On arrested cerebral development with special references to its cortical pathology. /. New. Ment. Dis., 1887, 14: 541.

25. Samuels, S., Korey, S., Gonatas, J . , Terry, R., and Weiss, M. Studies on Tay-Sachs disease. IV. Membranous cytoplasmic bodies. / . Neuropathol. Exp. Neurol, 1963, 22 : 81 .

26. Sandhoff, K. Auftrennung der Sanger N-Acetyl-^-Hexosaminidase in multiple formen durch elektrofokussierung. Hoppe-Seyler's Z. Physiol Chem., 1968, 349: 1095.

27. Sandhoff, K. The hydrolysis of Tay-Sachs ganglioside by hexosaminidase A. FEBS Lett., 1970, 1 1 : 342.

28. Sandhoff, K. , Andrea, U. , and Jatzkewitz, H. Deficient hexosaminidase activity in an excep-tional use of Tay-Sachs disease with additional storage in three variants of Tay-Sachs disease. /. Neurochem., 1968, 18: 2469.

29. Sandhoff, K., and Wassle, W. Anreicherung and Charakterisierung zweier Formen der Menschlichen N-acetyl-/3-hexosaminidase. Hoppe-Seyler's Z. Physiol Chem., 1971, 352: 1119.

30. Schneck, L . , Adachi, M., and Volk, B . Chemical pathology of Tay-Sachs disease in the fetus. In: Sphingolipids, Sphingolipidoses and Allied Diseases. (B. Volk, and S. Aronson, Eds.). Plenum, New York, 1972: 385-394.

31. Segrest, J . , and Jackson, R. Molecular weight determination of glycoproteins by polyacryl-amine gel electrophoresis in sodium dodecyl sulfate. In: Methods in Enzymology. Vol. 28, Part B . (V. Ginsberg, Ed.). Academic Press, New York, 1973: 54.

32. Srivastava, S., and Beutler, E. Hexosaminidase A and Hexosaminidase B Tay-Sachs and Sandhoff s disease. Nature (Lond.), 1973, 2 4 1 : 463.

33. Svennerholm, L. The chemical structure of normal human brain and Tay-Sachs gangliosides. Biochem. Biophys. Res. Commun., 1962, 9: 436.

34. Tallman, J . F . , and Brady, R. O. The catabolism of Tay-Sachs ganglioside in rat brain lyso-somes. / . Biol. Chem., 1972, 247: 7570.

35. Tallman, J . F . , and Brady, R. O. The purification and properties of a mammalian neuroamini-dase (sialidase). Biochim. Biophys. Acta, 1973, 293: 434.

36. Tallman, J . F . , and Brady, R. O. Disorders of ganglioside catabolism. In: The Sialic Acids. (C. Schelgrund and A. Rosenberg, Eds.). 1975: in press.

37. Tallman, J. F . , Brady, R. O., Quirk, J. M. , Villalba, M. , and Gal, A. E. Isolation and relationship of human hexosaminidases. / . Biol. Chem.. 1974, 249: 3489.

38. Tallman, J . F . , Brady, R. O. and Suzuki, K. Enzymatic activities associated with membranous cytoplasmic bodies and isolated brain lysosomes. / . Neurochem., 1971, 18: 1775.

39. Tallman, J . F . , Johnson, W. G., and Brady, R. O. The metabolism of Tay-Sachs ganglioside: Catabolic studies with lysosomal enzymes from normal and Tay-Sachs brain tissue. / . Clin. In-vest., 1972, 5 1 : 2339.

40. Tallman, J . F . , Pentchev, P. G., and Brady, R. O. An enzymological approach to the lipidoses. Enzymes, 1974, 18: 136.

41. Tay, W. Symmetrical changes in the region of the yellow spot in each eye of an infant. / . Ophthalmol. Soc. U. K., 1881, 1 : 55.

42. Terry, R. D. , and Weiss, R. Studies in Tay-Sachs disease. II. Ultrastructure of the cerebrum. / . Neuropathol. Exp. Neurol, 1963, 22 : 18.


43. Verpoorte, J . Purification of two /3-N-acetyl-D-glucosaminidases. / . Biol. Chem., 1972, 247: 4787.

44. Vidgoff, J . , Buist, N. , and O'Brien, J . S. Absence of the /3-N-acetyl-D-hexosaminidase A activ-ity in a healthy woman. Am. J. Hum. Genet., 1973, 25: 372.

45. Wenger, D. , Okada, S., and O'Brien, J . Studies on the substrate specificity of hexosaminidase A and B from liver. Arch. Biochem. Biophys., 1972, 153: 116.

18A Discussion: Metachromatic Leukodystrophy,

An Unusual Case with a Subtle Cerebroside Sulfatase Defect

HAYATO KIHARA The Neuropsychiatry Institute, Pacific State Hospital,

Research Group, Pomona, California

Metach roma t i c l eukodys t rophy ( M L D ) is o n e of the genet ica l ly d e t e r m i n e d

sph ingo l ip idoses in w h i c h there is a def ic iency of the lysosomal e n z y m e aryl-

sulfatase A . A s a c o n s e q u e n c e , ce rebros ide sulfates (sulfatides) accumula te in

t i s sues , part icular ly in the central and per iphera l ne rvous sys t ems . T h e r e are

demye l ina t ion and p rogress ive neuro logica l degene ra t ion (4) .

M L D has b e e n arbi t rar i ly d iv ided in to three forms , late infant i le , j uven i l e ,

and adult , b a s e d on the age of onse t o f cl inical s y m p t o m s . Each of the forms

appears to b e an i n d e p e n d e n t au tosomal recess ive d isorder w i t h on ly one

form occur r ing in any k i n s h i p .

In several o ther l i p idoses , the degree of e n z y m e def ic iency h a s b e e n dec id-

edly less severe in var ian ts w i t h de layed age of onse t than in the infant i le

type (5) , for example , an e n z y m e level o f 10 to 2 0 % of no rma l c o m p a r e d to less

than 5 % of no rma l in the infant i le type . In contras t , arylsulfatase A act iv i ty in

u r ine , l eukocy tes , or cul tured f ibroblasts from all forms of M L D is un i formly

p resen t at a level less than 5 % of that found in normal samples . It has b e e n

difficult, therefore , to ra t ional ize h o w s o m e pa t ien ts reach adul thood wi thou t

c l inical s y m p t o m s .

Cul tured f ibroblas ts der ived from pa t ien ts w i t h M L D are deficient in aryl-

sulfatase A , bu t unde r no rma l cond i t i ons they do no t have me tach roma t i c

inc lus ions . T h e y can , h o w e v e r , b e i n d u c e d to b e c o m e me tach roma t i c w h e n

sulfatides are p rov ided in the cul ture m e d i u m (7) . T h i s depos i t i on of the

501

502 Hayato Kihara

sph ingo l ip id can b e quant i f ied b y the use of 3 5 S - l a b e l e d sulfat ides. Cont ro l

f ibroblas ts also incorpora te e x o g e n o u s sulfat ides , bu t the i r no rma l comple -

m e n t of arylsulfatase A enab l e s t h e m to degrade the inges t ed sulfol ipid and

re lease radioac t ive inorgan ic sulfate in to the g rowth m e d i u m .

T h i s in tact cell c e reb ros ide sulfate sul fohydrolase sys t em has p rov ided the

first ind ica t ion that latent forms of M L D do pos se s s a t tenuated bu t finite levels

of ce rebros ide sulfatase wh i l e the infant i le form does no t appear to have any

funct ional e n z y m e (Figure 1) (2 ,8 ) . T h e r e i s a direct corre la t ion b e t w e e n age of

onse t o f c l inical s y m p t o m s and intact cell e n z y m e act ivi ty. Adul t M L D fibro-

blas ts appeared to d i spose of the intracellularly accumula ted sulfat ides about

one -ha l f as rapidly as no rma l cells unde r th is test cond i t i on , ye t the arylsulfa-

tase A level in extracts o f t hese cells was b e l o w our l imi t s o f re l iable quan-

tif ication (less than 5 % of normal ) . Va r ious approaches failed to provide ex-

tracts w i th re l iably assayable levels of e n z y m e , so 100 t imes the usual n u m b e r

of cells w e r e cul tured , extracts p repared , and f ract ionated wi th a m m o n i u m

sulfate. In th is w a y w e were able to s h o w hydro lys i s of an exper imenta l ly s ig-

nif icant p ropor t ion of the sulfatide in the assay mix tu re , e l imina t ing any un-

cer ta in ty abou t its val idi ty . T h i s r ep resen ted 1 % of the sulfat idase act ivi ty in

control f ibroblasts . T i t ra t ion w i t h ant i -arylsulfatase A se rum s h o w e d that

adult M L D fibroblasts con ta ined abou t one- t en th as m u c h a n t i b o d y react ing

mater ia l as no rmal cells (2) . T h u s , the mutan t arylsulfatase A in adult M L D

fibroblasts appeared to b e catalyt ical ly less efficient as wel l as p resen t in

d i m i n i s h e d quant i ty . W e have p r e s u m e d that the latter was due to inc reased

0 4 8 12 16 0 4 8 12 16

D a y s D a y s

FIGURE 1. Correlation of intracellular cerebroside sulfate sulfohydrolase activities with age of

onset of clinical symptoms. Petri dishes (60 mm) were plated with fibroblasts (300,000/dish) in

3 ml medium containing 3 5 S-cerebroside sulfate (23 nmoles/ml; 3.6 x 1 0 3 cpm/nmole). On the days

indicated each cell strain was analyzed for intracellular 3 5S-sulfatide and extracellular 3 5S-sulfate.

The M L D cells were derived from patients whose age of onset of clinical symptoms are indicated

by the numbers opposite the plots. From Kihara et al. (2,p.22).

Extracellular 3 5 S - S u l f a t e Intracellular 3 5 S-Su l fa t ide

nm

ole

s-m

g"1

Prot

ein

18A. Metachromatic Leukodystrophy 503

Table I Leukocyte and Urinary Arylsulfatase A Activities

Specific Activity (nmole/hr/mg)

Subject Leukocyte Urine

Proband 5 30

MLD patients 7 0

Control 208 ± 97 558 ± 97

labi l i ty of the mu tan t e n z y m e . W h i l e w e w e r e in te res ted in charac te r iz ing the

defect in the adult M L D mutan t e n z y m e , i ts ex t remely l ow catalytic act iv i ty

has se rved as a deterrent .

W e have d i scovered wha t appears to b e a n e w var ian t of M L D . A menta l ly

re tarded pa t ien t w i t h neu ropa thy of u n d e t e r m i n e d e t io logy s h o w e d a defi-

c i ency of arylsulfatase A in leukocytes typical of M L D (Table I ) . Ur ina ry en-

zyme level w a s also great ly depres sed and the u r ine con ta ined me tach roma t i c

granules w h i c h y ie lded l ip ids coch roma tog raph ing wi th ce reb ros ide sulfate.

T h e pa t i en t p re sen ted w i t h the b i o c h e m i c a l b e n c h m a r k s of sulfat ide l ip idos i s ,

a l though there we re c l in ical i n c o n s i s t e n c i e s . N e u r o p a t h y and m y o p a t h y h a d

b e e n ev iden t from infancy . D e v e l o p m e n t w a s s low and ch i ldhood years we re

s to rmy b e c a u s e of emo t iona l d i s tu rbances . H o w e v e r , she has r e m a i n e d un-

changed neuro log ica l ly for m a n y years and there has b e e n no ev idence of

p rogress ive degenera t ion .

A f ibroblast cul ture w a s in i t ia ted from a sk in b i o p s y . Surpr i s ing ly , f ibro-

blas ts con t a ined abou t 1 0 % of no rmal arylsulfatase A act iv i ty b y the assay w i t h

the synthe t ic subs t ra te , 4 -n i t roca techol sulfate (Table II) . H o w e v e r , no e n z y m e

act iv i ty could b e demons t r a t ed toward the phys io log ica l subs t ra te , ce reb ros ide

sulfate. F ib rob la s t s cul tured in the p re sence of e x o g e n o u s sulfat ides failed to

exh ib i t m e t a c h r o m a s i a , sugges t ing the p re sence of funct ional ce reb ros ide sul-

fatase ac t iv i ty in in tac t cells . T h e 3 5 S-su l fa t ide load ing t e c h n i q u e w a s appl ied ,

and the resul ts i nd ica ted that these cells w e r e able to d i spose of sulfat ides at a

level i n t e rmed ia t e b e t w e e n adul t M L D and no rma l cells (F igure 2 ) .

A large ba t ch of these cells w a s cul tured and p rocessed in the s ame m a n n e r

as the adult M L D cel ls . A g a i n , ample act iv i ty toward the syn the t i c subs t ra te

Table II Fibroblast Arylsulfatase A Activity

Subject Specific Activity (nmole/hr/mg)

Proband 131

MLD patients 72

Control 1335 ± 652

504 Hay at o Kihara

Extracellular 3 5S-Sulfate Intracellular 3 5 S - Sulfatide

DAYS DAYS

FIGURE 2. Subnormal intracellular cerebroside sulfate sulfohydrolase activity of fibroblasts from

the proband. Conditions were similar to those described for Figure 1.

80

Concentration, mg/ml

FIGURE 3. The effect of varying concentrations of sodium taurodeoxycholate or sodium cholate on cerebroside sulfate sulfohydrolase activity. Assay conditions were as described previously (6) with bile salt as indicated. The enzyme was human urinary arylsulfatase purified to a specific activity of 2400 /xmoles of 4-nitrocatechol sulfate hydrolyzed per hour per milligram of protein.

cpm

x

IQ"4 •

m

g 1

Pro

tein

[35S

| su

lfat

e (

jjm

ole

s •

h~'

- m

g-1)

18 A. Metachromatic Leukodystrophy 505

Table III Cerebroside Sulfate Sulfohydrolase Activities

Subject Sodium Cholate (mg/ml) Activity (nmole/hr/ml)

Proband 1.25 0

2.50 0

5.00 189

Control 1.25 2850

2.50 770

5.00 4730

was presen t , bu t w e could no t demons t ra t e any act iv i ty toward the phys io log-

ical subs t ra te . F rac t iona t ion p rocedures , inc lud ing a m m o n i u m sulfate p rec ip i -

ta t ion and DEAE-ce l lu lose ch romatography , s h o w e d that syn the t ic subs t ra te

act ivi ty f ract ionated l ike e n z y m e from control f ibroblas ts . A c o m p a r i s o n of the

var iant and normal e n z y m e b y such parameters as mig ra t ion proper t ies on

po lyacry lamide gel e lec t rophores i s , p rec ip i t in reac t ion w i t h an t i bod i e s , rate of

heat inac t iva t ion , and i n h i b i t i o n b y A g + failed to reveal any difference

b e t w e e n the e n z y m e from b o t h sources .

T h e in vitro c e r eb ros ide sulfate sul fohydrolase reac t ion has an obl iga tory

b i le salt r e q u i r e m e n t w h i c h can b e m e t b y e i ther s o d i u m taurodeoxychola te or

s o d i u m chola te (3 ,6 ) . W e normal ly use taurodeoxychola te b e c a u s e resul ts have

b e e n m o r e r ep roduc ib l e than w i t h chola te . O n the o ther h a n d , w e have found

that the chola te sys tem prov ides a greater degree of ac t iva t ion in an unusua l

b i m o d a l pat tern (Figure 3 ) . T h u s , w h e n the f ract ionated var iant e n z y m e w a s

e x a m i n e d in the taurodeoxychola te sys t em and ce reb ros ide sulfatase act ivi ty

still could no t b e demons t r a t ed , w e r e e x a m i n e d the e n z y m e in the chola te -ac-

t ivated sys tem. In th i s way , w e w e r e finally able to obse rve measu rab l e

ce reb ros ide sulfatase act ivi ty in the extract from the p r o b a n d (Table III) . Q u a n -

t i ta t ive c o m p a r i s o n s have b e e n difficult b e c a u s e of the poo r reproduc ib i l i ty ,

bu t it d id appear that the ce reb ros ide sulfatase act ivi ty w a s less than wha t

w o u l d b e expec ted from the 4-n i t roca techol sulfate assay.

T h e arylsulfatase A of th i s var iant form of M L D represen t s the first mu tan t

e n z y m e of th i s d i sorder a m e n a b l e for s tud ies of the defect at the molecu la r

level. S u c h s tudies w o u l d b e facil i tated b y an unde r s t and ing of the ac t ion of

cholate on the normal e n z y m e . The re i s e v i d e n c e that t au rodeoxychola te acts

b y d i spers ing ce reb ros ide sulfate mice l les (1) . Chola te m a y also act in th is

capaci ty at i ts lower act iv i ty peak , w h i c h is co inc iden t w i t h that o f taurodeoxy-

chola te . At h i g h e r chola te concen t ra t ions , p rec ip i ta ted chol ic ac id appears to

play a d o m i n a n t role. T h i s unusua l s econd , b roade r ac t iv i ty peak p robab ly

results from surface adsorp t ion of the e n z y m e , subs t ra te , or bo th . Sys tema t i c

s tudies of th i s p h e n o m e n o n have b e e n difficult b e c a u s e w e have no t ye t b e e n

able to ach ieve chola te ac t iva t ion in a re l iably r ep roduc ib le m a n n e r .

506 Hayato Kihara


This investigation was supported in part by Grant Nos. NS-8839, NS-9479, and HD-4612 from the National Institutes of Health.

R E F E R E N C E S

1. Jerfy, A., and Roy, A. B . The sulphatase of ox liver. XVI. A comparison of the arylsulphatase and cerebroside activities of sulphatase A. Biochim. Biophys. Acta, 1973, 293: 178-190.

2. Kihara, H., Porter, M. T. , and Fluharty, A. L. Enzyme replacement in cultured fibroblasts from metachromatic leukodystrophy. Birth Defects, Orig. Art Ser., 1973, 9: 19-26.

3. Percy, A. K., Farrell, D. F . , and Kaback, M. M. Cerebroside sulphate (sulphatide) sulphohydro-lase: An improved assay method. / . Neurochem., 1972, 19: 233-236.

4. Moser, H. W. Sulfatide lipidosis: Metachromatic leukodystrophy. In: The Metabolic Basis of Inherited Diseases. (3rd ed.) (J. B. Stanbury, J . B . Wyngaarden, and D. S. Fredrickson, Eds.). McGraw-Hill, New York, 1972: pp. 688-729.

5. Okada, S., Veath, M. L. , and O'Brien, J . S. Juvenile G M 2 gangliosidoses: Partial deficiency of hexosaminidase. / . Pediat., 1970, 77: 1063-1065.

6. Porter, M. T. , Fluharty, A. L. , de la Flor, S. D. , and Kihara, H. Cerebroside sulfatase determina-tions in cultured human fibroblasts. Biochim. Biophys. Acta, 1972, 258: 769-778.

7. Porter, M. T., Fluharty, A. L. , Harris, S. E . , and Kihara, H. The accumulation of cerebroside sulfates by fibroblasts in culture from patients with late infantile metachromatic leukodys-trophy. Arch. Biochem. Biophys., 1970, 138: 646-652.

8. Porter, M. T. , Fluharty, A. L. , Trammell, J . , and Kihara, H. A correlation of intracellular cerebroside sulfatase activity in fibroblasts with latency in metachromatic leukodystrophy. Biochem. Biophys. Res. Commun., 1971, 44: 660-666.

19 Role of Biochemistry in Research

on Mental Retardation

GEORGE POPJAK Mental Retardation Research Center and Department of Biochemistry,



A few years ago I a t t ended a c l in ical s e s s ion on p sychosoma t i c m e d i c i n e .

T w o cases w e r e p resen ted : o n e w a s that of a w o m a n in he r late th i r t ies w i t h

the s igns and s y m p t o m s of gross thyro tox icos i s , goi ter , exoph tha lmos , cardiac

invo lvemen t , and all the rest; the s econd case w a s that o f a b o y , abou t age 12 ,

w i th the classic s igns and s y m p t o m s of H i r schp rung ' s d i sease , inc lud ing the

X- ray ev idence . It w a s p r o p o s e d dur ing that s e s s ion that t he se two pa t ien ts

should b e g iven psychoana ly t i ca l t rea tment to r id t h e m of the i r thyro tox icos i s

and " e n c a p r o s i s . " O b v i o u s l y not e v e r y b o d y shares the b i o c h e m i s t s ' s Credo ,

that express ions of func t ions are man i fes t a t ions of carefully cont ro l led b o d y

chemis t ry in tegra ted w i th cellular s t ructure , and part icular ly no t w h e n the

p syche or men ta l facult ies m a y b e invo lved—nor , apparent ly , even the m o v e -

m e n t s of the b o w e l .

T h u s men ta l re ta rda t ion m i g h t share in the d i sbe l i e f that i t has any th ing to

do wi th a b i o c h e m i c a l abnormal i ty . W h a t I am go ing to say necessa r i ly m u s t

b e s impl i s t ic b e c a u s e I canno t pos s ib ly roll in to 45 m i n u t e s all o f b i o c h e m i s t r y

and one -ha l f o f S t a n b u r y , W y n g a a r d e n , and F redr i ckson ' s t ex tbook (10) . Cer -

ta inly , up to 1934 n o o n e k n e w , and could not even suspec t , that at least s o m e

of the men ta l re ta rda t ions , or o l igophren ias as I l earned t h e m , we re assoc ia ted

wi th an abno rma l i t y o f m e t a b o l i s m . As eve ryone k n o w s , and as y o u have

hea rd it reaff irmed b y Dr . K a u f m a n th is m o r n i n g , it w a s in 1934 that F0 l l ing

repor ted the p re sence o f pheny lpyruv ic ac id in the u r ine of 10 i m b e c i l e s and

es t ab l i shed the first corre la t ion b e t w e e n a me tabo l i c abno rma l i t y and men ta l

re tardat ion. T h e ti t le o f F0 l l ing ' s paper deserves to b e r e m e m b e r e d : " U b e r

A u s s c h e i d u n g v o n P h e n y l b r e n z t r a u b e n s a u r e in den Harn als S to fwechse lano-

507

508 George Popjdk

mal ie in V e r b i n d u n g mi t Imbez i l l i t a t , " i . e . , " A b o u t the excre t ion of pheny l -

pyruvic ac id in the u r ine as a me tabo l i c anomaly assoc ia ted w i th i m b e c i l i t y "

(3) .

O f course , it w a s Gar rod (4) , before Foi l ing , at the turn of the century , w h o

first r ecogn ized the gene t ic b a s i s of me tabo l i c d i sorders , w h i c h he cal led " i n -

bo rn errors of m e t a b o l i s m . " F r o m h i s s tudies on a lkaptonur ia , cys t inur ia , al-

b i n i s m , and pen tosur ia , Gar rod deve loped the concep t that cer ta in d i seases

can arise b e c a u s e an e n z y m e , cont ro l l ing a s ingle me tabo l i c s tep , is e i ther

m i s s i n g or is severe ly impa i r ed in its funct ion. H e apprec ia ted qu i t e correct ly,

e .g . , that h o m o g e n t i s t i c acid w a s a no rmal in t e rmed ia t e in the m e t a b o l i s m of

tyros ine and that i ts accumula t ion in the b o d y in a lcaptonur ia mus t have b e e n

the resul t o f the a b s e n c e o f the e n z y m e ox id iz ing th i s acid . Gar rod ' s predic-

t ion was p roved correct 50 years later b y La D u and h is associa tes (8) . In the

case of pheny lke tonur i a—aga in , as re la ted b y Dr . Kaufman—it was not unt i l

1953 that Je rv is (6) e s t ab l i shed that a b s e n c e of pheny la l an ine hydroxylase w a s

r e spons ib le for the me tabo l i c abnormal i ty . Bu t w e have also heard from Dr .

Kaufman h o w extraordinar i ly difficult it was even in s u b s e q u e n t years to p in-

po in t p rec ise ly the abnormal i ty . T h e b i o c h e m i s t has m u c h difficulty w h e n

work ing wi th h u m a n b e i n g s or on h u m a n p r o b l e m s .

T h e last 40 years of b i o c h e m i c a l research resul ted in the descr ip t ion of p rob -

ably all the ma jo r me tabo l i c pa thways . In the early 1940 ' s Beadle and Ta tum

es tab l i shed exper imenta l ly the concep t of " o n e gene , one e n z y m e " (1) . T h e n

came the ident i f ica t ion of the structure of D N A b y Cr ick and W a t s o n (13) .

The re fol lowed the ident i f ica t ion of the gene t ic code and of the funct ional

uni t—the c i s t ron—of the D N A control l ing the structure of a s ing le po lypep t ide

cha in . T h u s the concep t of " o n e gene , o n e e n z y m e " w a s t rans la ted in to the

concep t of " o n e c is t ron , o n e p o l y p e p t i d e . " S tudy of bacter ia l gene t i cs pro-

duced ev idence impl i ca t ing the par t ic ipa t ion of two o ther genes in the tran-

scr ip t ion of the structural g e n e in to e n z y m e pro te in . T h e s e are the regulator

and opera tor genes . T h e funct ion of the regula tor gene is to p roduce re-

pressors w h i c h in teract w i t h a locus on D N A adjacent to the structural gene .

Th i s latter locus is k n o w n as the opera tor gene and , w h e n c o m b i n e d wi th an

act ive repressor , does no t pe rmi t the t ranscr ip t ion of the structural gene .

There w a s m u c h specula t ion abou t the o r ig in of h u m a n metabo l i c d isorders

resul t ing from the a b s e n c e or def ic iency of an e n z y m e . In the second ed i t ion

of our " b i b l e , " " T h e M e t a b o l i c Bas i s of Inher i t ed D i s e a s e , " it w a s pos tu la ted

that p robab ly all the abnorma l i t i e s of amino acid m e t a b o l i s m and the s torage

d iseases assoc ia ted w i t h men ta l re tardat ion resul ted from regulator or opera tor

gene muta t ions such as those that p reven ted the t ranscr ip t ion of a structural

gene cod ing for a specific e n z y m e . F r o m wha t y o u have heard from prev ious

speakers it is un l ike ly that regulator or opera tor gene muta t ions are respons i -

b le for defects assoc ia ted w i th the a b s e n c e or i m p a i r m e n t of a s ing le e n z y m e .

T h e defects in all c losely e x a m i n e d ins tances are n o w a t t r ibutable to structural

19. Biochemistry in Mental Retardation 509

gene muta t ions . E v e n the e v i d e n c e prov ided b y the s tudy of bac ter ia l sys t ems

makes it un l ike ly that an abnorma l i ty a r i s ing from the s i lence or l ow act ivi ty

o f an e n z y m e w o u l d resul t from muta t ions in regulator or opera tor genes .

Expe r imen t s w i t h mu tan t s of E . coli con ta in ing the gene for the induc ib l e en-

zyme , /3-galactosidase, have s h o w n that mu ta t i ons in the regula tor or opera tor

gene loci ( excep t ing the de le t ion of the opera tor gene in O - mu tan t s of E. coli)

resul ted no t in the r ep ress ion of the induc ib i l i t y o f the e n z y m e — o n the con-

trary, such mu ta t i ons resul ted in the de repress ion of /3-galactosidase and the

appearance of the e n z y m e as a cons t i tu t ive p ro te in in the bac te r ia . B y ana logy

I wou ld expec t a regula tory or opera tor gene mu ta t i on in m a n , excep t ing aga in

the less l ikely total de le t ion , to mani fes t i tself no t b y r ep res s ion of e n z y m e

syn thes i s bu t b y abnormal ly h igh levels o f cer ta in e n z y m e s . M a m m a l i a n

sys tems and part icular ly h u m a n cellular sys t ems have b e e n m u c h neg lec ted

dur ing the past 1 0 - 1 5 years at the expense of E. coli. H o w e v e r , there is p len ty

of ev idence that a m e n d s wil l b e m a d e dur ing the years to c o m e . T h e recogn i -

t ion that f ibroblas ts in cul ture and l eukocy tes can express the me tabo l i c pat-

te rn—normal or a b n o r m a l — o f a p h e n o t y p e is n o w b e i n g exp lo i ted v igorous ly

in m a n y labora tor ies . S tudy of f ibroblasts and leukocytes taken from ind iv idu-

als afflicted b y a me tabo l i c d i sease should answer m a n y ques t ions of e n z y m e

regula t ion in m a m m a l i a n sys t ems and dec ide conc lus ive ly w h e t h e r regulator

and opera tor genes exis t in m a m m a l i a n cel ls , and , i f they exis t , w h a t m i g h t b e

the c o n s e q u e n c e s o f mu ta t i ons in these . G o r d o n T o m k i n s ' e x p e r i m e n t s w i t h

ty ros ine amino t rans fe rase (12) , and D o n Hagger ty ' s , i n our labora tory , on

pheny la l an ine hydroxylase in cul tures o f h e p a t o m a cells s t rongly sugges t the

ex i s tence of opera tor g e n e s even in m a m m a l i a n cells . Cer ta in ly , that a s s u m p -

t ion is the s imples t o n e that can b e i n v o k e d to expla in the " i n d u c t i o n " of

these e n z y m e s b y cor t icos te ro id ho rmones .* A n even s t ronger ev idence as to

the ex i s t ence of an opera tor gene in m a m m a l i a n cells c a m e to l ight on ly dur ing

the last few m o n t h s from the s tudy of leukocytes and f ibroblas ts t aken from

he te rozygous and h o m o z y g o u s ind iv idua ls afflicted b y famil ia l hype rcho le s -

te ro lemia . Alan F o g e l m a n found in our labora tory that the l eukocy tes of such

he te rozygotes r e s p o n d e d to i ncuba t i on in a l ipid-free se rum w i t h a far greater

induc t ion of hydroxymethy lg lu t a ry l -CoA reduc tase than the leukocytes o f

n o r m a l ind iv idua l s . S imi l a r f indings w i t h f ibroblasts w e r e repor ted b y G o l d -

s te in and B r o w n in the O c t o b e r i s sue of the Proceedings of the National Acad-

emy of Science (5) . Go lds t e in and B r o w n have had an oppor tun i ty to e x a m i n e

also f ibroblasts of h o m o z y g o u s ind iv idua ls and found that in these the H M G -

C o A reductase w a s p e r m a n e n t l y e leva ted and fur thermore that the levels of

* Tomkins and his associates have interpreted their observations as indicating a "posttranscrip-tional" control of enzyme induction by steroid hormones. This interpretation has been chal-lenged repeatedly (2,7). The data of Tomkins and of others are more readily understood on the assumption that the role of steroid hormones in enzyme induction is the derepression of an operator gene thus facilitating the synthesis of specific mRNA.

510 George Popjdk

the e n z y m e could no t b e r ep ressed in the h o m o z y g o u s cel ls , as t h e y can in

normal cel ls , b y l o w dens i ty l ipopro te in . T h e inference from these exper i -

m e n t s is that in famil ia l hype rcho les t e ro lemia a mu ta t ion of the opera to r g e n e

results in the inab i l i ty of the opera tor to b i n d the repressor and h e n c e lead to

elevated levels of the e n z y m e . T h i s is the prec ise ana logy of the de rep ress ion

of /3-galactosidase, or i ts appearance as a cons t i tu t ive e n z y m e in n o n i n d u c e d

E. coli w i t h muta ted regula tor or opera tor genes . O f course , famil ial h y p e r c h o -

les tero lemia has n o t h i n g to do w i t h men ta l re tardat ion, bu t the p r o b l e m s o f

genet ic mu ta t ions are m o s t per t inen t .

A l though it i s dangerous to extrapolate from the s ingular to the genera l , I

suggest that p r o b a b l y all s ing le e n z y m e def ic iencies are the resul t o f structural

gene muta t ions . I a m exc lud ing from th is p rognos t i ca t ion the major c h r o m o -

some abnorma l i t i e s , such as the t r i somies in w h i c h affairs are p robab ly far

more complex , and I doub t if a m e n a b l e to b i o c h e m i c a l analys is at present .

I ven tu red in to these h is tor ica l ref lect ions b e c a u s e they represen t , to m e at

least , the role of b i o c h e m i s t s i n s eek ing an unde r s t and ing of the l iv ing wor ld ,

an explana t ion of life in te rms of c o m p r e h e n s i b l e chemis t ry .

Be l i ev ing as I do that man i fes ta t ions o f a b n o r m a l funct ion result from a b -

normal m e t a b o l i s m , m y concep t o f the role o f b i o c h e m i s t s in the s tudy o f

menta l re tardat ion is the s a m e as they fulfilled h is tor ica l ly in the under -

s tand ing of the l iv ing wor ld . T h i s role w a s great ; never the less b i o c h e m i s t s

engag ing , or p l ann ing to e n g a g e , in the s tudy o f the vast p r o b l e m s of men ta l

re tardat ion in m a n , are severe ly hand icapped : first, b e c a u s e a large n u m b e r of

men ta l re ta rda t ions , those resul t ing , e .g . , from b i r th in jur ies , in t rau ter ine in -

fec t ions , and major c h r o m o s o m a l aber ra t ions , are no t a m e n a b l e to b i o c h e m i c a l

analys is b y our current t e c h n i q u e s . T h e s econd h a n d i c a p m o s t p resen t -day

profess ional b i o c h e m i s t s have is that they canno t plan any rat ional explora t ion

of men ta l re tardat ion unt i l after c l inical col leagues have ident i f ied a syndrome

or d i sease ent i ty , and specif ical ly a d i sease en t i ty w h i c h , b y the s tudy o f the

pa t ien t ' s ped ig ree , is inhe r i t ab le . O n c e a gene t ic backg round is e s t ab l i shed ,

the b i o c h e m i s t is o n fairly firm ground . E v e n so , the s tudy of a me tabo l i c

d isorder in m a n , b e that a syn the t ic or ca tabol ic s tep , is b e s e t w i th m u c h dif-

ficulty, as h u m a n e and e th ica l cons ide ra t ions l imi t the n u m b e r and na ture of

samples a b i o c h e m i s t can take . O n e is largely res t r ic ted to u r ine , b lood , C S F ,

and occas iona l b i o p s i e s from l imi ted s i tes .

Y o u have heard Dr . Kau fman ' s difficult dec i s i on o n h o w to m a k e the bes t

use of 20 m g of a l iver b iopsy . In spi te o f these l imi ta t ions such s p e c i m e n s are

sufficient for the ana lys is o f chemica l compos i t i on and h e n c e recogn i t ion o f a

major me tabo l i c error. In th is respect w e can b e gu ided b y past exper ience

w h i c h s h o w e d that in all me tabo l i c errors o f gene t i c or ig in there were no a b -

normal me tabo l i t e s in the b o d y and b o d y fluids, on ly no rma l me tabo l i t e s in

abnorma l amoun t s . F r o m the k n o w l e d g e of me tabo l i c pa thways o n e can pre-

dict, o f course , that accumula t ion or a b s e n c e of a me tabo l i t e m u s t b e the result


of a b lock in a cha in of reac t ions . S u c h a b lock m a y have a var ie ty of con-

s e q u e n c e s . Le t us a s s u m e three reac t ions in w h i c h subs t ance A is t ransformed

into B , w h i c h goes to C w h i c h is the i m m e d i a t e p recursor o f D . If the e n z y m e

conver t ing C in to D is no t func t ion ing , me tabo l i t e C m a y accumula te if the

reac t ion C—»D is the on ly p a t h w a y of i ts m e t a b o l i s m . H o w e v e r , if the p reced-

ing s teps A—>B—>C are freely revers ib le , the precursors farther away from the

metabo l i t e w h o s e reac t ion is b locked m a y accumula te . A good example of

such a case is the accumula t ion o f m e t h i o n i n e in the b lood of pa t ien t s wi th

cys ta th ion inur ia , a l though the me tabo l i c b lock is several s teps away from

m e t h i o n i n e .

T h e me tabo l i c b lock b e t w e e n C and D m a y resul t i n the exaggera t ion of a

pe rhaps m i n o r a l ternat ive p a t h w a y of c o m p o u n d C and lead to the accumula -

t ion of o ther me tabo l i t e s . T h e classic example i s , o f course , pheny lke tonur i a , in

w h i c h no t on ly pheny la l an ine , bu t also i ts secondary me tabo l i t e s , pheny l -

pyruvic and phenyl lac t ic and phenylace t i c ac ids , accumula te in the b o d y and

lead to the i r excre t ion in the u r ine in a b n o r m a l a m o u n t s .

A failure to form produc t D m a y have several c o n s e q u e n c e s if D i tself i s a

vital me tabo l i t e or a p recursor of a vi tal me tabo l i t e . T h u s , as y o u have hea rd

from Dr . Kaufman th is m o r n i n g , ty ros ine , w h i c h is no t an essen t ia l a m i n o

acid in no rma l ind iv idua l s , b e c o m e s an essen t ia l a m i n o acid in p h e n y l k e -

tonur ics . O r a failure to iod ina te ty ros ine leads to failure of the p roduc t ion of

t r i iodo tyron ine and thyrox ine and resul ts in goi t rous c re t in i sm.

If subs t ance D has the role of a f eedback regula tor in a me tabo l i c cycle or

cha in , i ts a b s e n c e m a y lead to the overproduc t ion of a p recursor w h i c h it con-

trols. T h u s in orot ic ac idur ia the overproduc t ion of orot ic ac id m a y b e a sc r ibed

partly to depr iva t ion of the b o d y of p y r i m i d i n e nuc leo t ides , w h i c h act , at least

cy t id ine t r iphospha te , as f eedback inh ib i to r s of ca rbamyl -phospha te syn the -

tase and aspartate t r ansca rbamylase .

A l though e x a m i n a t i o n of me tabo l i c pa t te rns leads u l t imate ly to the iden-

tif ication of a defec t ive , absen t , or s i lent e n z y m e , th is tells us n o t h i n g abou t

the o r ig ins o f men ta l re tarda t ions assoc ia ted w i t h me tabo l i c errors . If the ab -

norma l me tabo l i c pa t terns have a m e s s a g e , w e are unfor tunate ly unab le to

read that m e s s a g e . W e are totally ignoran t of the fundamenta l causes of the

failure of the ma tu ra t ion of the central ne rvous sys t em or the failure of the

deve lopmen t of the h i g h cogn i t ive powers in any of the i n b o r n errors of m e -

tabo ls im. It was the fond h o p e of m a n y inves t iga tors that a s tudy of pheny lke -

tonur ia m i g h t p rov ide an a n s w e r to th is fundamenta l q u e s t i o n , ye t w e are as

ignoran t on th is po in t after s o m e 30 years of research as w e were of the me ta -

bol ic abnormal i ty i tself before Foi l ing d ropped ferric ch lor ide in to the u r ine of

some i m b e c i l e s .

O u r i gno rance on th i s q u e s t i o n s tems pr imar i ly , I b e l i e v e , f rom our i g n o -

rance of the b i o c h e m i c a l m e c h a n i s m s under ly ing the no rma l deve lopmenta l

and matura t ion p rocesses in the central ne rvous sys t em, i . e . , the p rocesses of

5 1 2 George Popjdk

different ia t ion. M a n y of the p resen ta t ions w e heard at th is conference w e n t a

long w a y in p rov id ing m u c h n e w informat ion on th is ques t ion . W e m a y argue

w h e t h e r it is of any mater ia l in teres t wha t is the m e c h a n i s m of men ta l retarda-

t ion resul t ing from an i n b o r n error of m e t a b o l i s m . M y s tand on th is po in t is

that it is o f pa ramoun t impor t ance , not jus t for an unde r s t and ing of men ta l re-

tardat ion, bu t also for an unde r s t and ing of the normal p rocesses of m e n t a t i o n

that earned us the self-styled rank of Homo sapiens. I nbo rn errors of m e t a b o -

l i sm are na ture ' s expe r imen t s , w h i c h , a l though exac t ing a heavy pr ice from

those afflicted, have taught us vo lum e s abou t h u m a n b iochemis t ry . B e s i d e s ,

r ecogni t ion of the nature of a me tabo l i c error led at least in a few ins tances to

rat ional therapy (PKU, ga lac tosemia , orot ic ac idur ia , me thy lma lon ic acidur ia ,

goi t rous c re t in i sm) .

I feel therefore that o n e of the impor tan t con t r ibu t ions b i o c h e m i s t s can

m a k e in the s tudy of men ta l re tardat ions is the s tudy of me tabo l i c abnorma l i -

t ies and a search for such abnorma l i t i e s a m o n g menta l ly re tarded, and par t icu-

larly a m o n g those w h o s e d isease is o f u n k n o w n e t io logy and is suspec ted of

b e i n g of genet ic or ig in .

The re are at p resen t abou t 150 k n o w n inbo rn errors of m e t a b o l i s m . O n e

canno t he lp suspec t ing that there are m a n y m o r e as ye t und i scovered . I wou ld

like to recall that in 1950 there were k n o w n only seven abnormal i t i e s of amino

acid me t abo l i sm . B y 1970 there were 50 . O u r r ecogn i t ion of me tabo l i c errors

has g rown logar i thmical ly s ince the 1950 ' s . Apar t from the susp ic ion that there

m a y b e m a n y m o r e as yet und i scove red me tabo l i c errors , there is the fact of

spon taneous muta t ion . T h e spon taneous muta t ion rate in ge rm cells is e s -

t imated to b e abou t o n e allele in 1 0 5 loci per genera t ion . T h i s m e a n s in prac-

tical te rms that in each genera t ion o n e n e w muta t ion is added for every 40

preex is t ing o n e s , or a load of 2 . 5 % . O f course , these muta t ions n e e d no t nec -

essar i ly result i n men ta l re tardat ion, bu t some undoub ted ly wil l .

T h e mere e s t ab l i shmen t o f a me tabo l i c error is insuff icient to character ize

the d i sease fully. T h u s it is no t e n o u g h to k n o w that s o m e o n e is excre t ing ex-

cess ive amoun t s of orot ic ac id . It is the b i o c h e m i s t ' s duty to es tab l i sh the

cause of such excess ive excre t ion , i . e . , the ident i f ica t ion of the defect ive en-

z y m e s and the nature of the enzymat ic defect. T h e ident i f icat ion of the defec-

t ive e n z y m e seems to m e mos t impor tan t as th is m a y well sugges t an appro-

priate t rea tment . In the example of orot ic ac idur ia , I quo ted , r ecogn i t ion that

two e n z y m e s are b locked (at least in one form of the d i sease)—orot idy l ic

py rophosphory lase and decarboxylase—led to the rea l iza t ion that pa t i en t s suf-

fer ing from th is d i sease were really suffering from py r imid ine depr iva t ion .

T h e y are wel l t reatable b y the oral admin i s t ra t ion of u r id ine and cy t id ine .

The re are already several examples of successful t rea tments of d i sease b y the

supply of a m i s s i n g pro te in a m o n g w h i c h the latest is the t rea tment of Fabry ' s

d isease w i t h normal h u m a n p lasma, w h i c h con ta ins a -g lucos idase , the en-

z y m e w h i c h is inac t ive in Fab ry ' s d i sease and to w h i c h Dr . Ta l lman had

referred earlier.


T h e final s tep in the descr ip t ion of an e n z y m e defect is the ident i f ica t ion of

the na ture of the defect in the e n z y m e itself. T h i s defect m a y range from i m -

pa i red catalytic ac t iv i ty , impa i r ed affinity for c o e n z y m e or subs t ra te , to total

loss of catalytic act ivi ty . W i t h the great advances that have b e e n m a d e in pro-

te in chemis t ry , the na ture of the abnormal i t i e s can n o w b e p r o b e d . T h i s is no t

an easy task. Firs t , a reference no rma l e n z y m e mus t b e o b t a i n e d from h u m a n

sources , from ind iv idua ls p r e s u m e d or a t tes ted to b e or to have b e e n normal .

W i t h a few excep t ions , a major h u m a n organ m a y b e n e e d e d for such a pur-

pose , i . e . , for the prepara t ion of a reference s p e c i m e n of an e n z y m e in a state

of h o m o g e n e o u s pur i ty . A l though organs are avai lable from p o s t m o r t e m s , no t

m a n y e n z y m e s survive pro teolys is after death . A l s o , phys i c i an co l leagues wil l

at test that death , excep t ing those resul t ing from acute card iovascular acc idents

or from v io lence , inf l icted b y m a n or m a c h i n e s , c o m e s s lowly to m a n . Dur ing

the protracted pe r iod of dy ing m a n y changes occur in o rgans . S o m e e n z y m e s ,

such as pheny la l an ine hydroxylase , m a y decay to unde tec tab le levels dur ing

the agony of dy ing .

T h e n , even if one succeeded in o b t a i n i n g a quan t i ty of the pure no rma l en-

zyme—severa l mi l l igrams—suff ic ient for all the n e e d e d phys ica l measu re -

m e n t s and p roduc t ion of an t i bod i e s , o n e n e e d s an adequa te source of the a b -

normal e n z y m e also. It is no t at all l ike w o r k i n g w i t h E. coli, e i the r the wi ld

type or any of i ts mu tan t s , w h i c h can b e o b t a i n e d , i f necessa ry , in cul tures of

hundreds o f l i ters and reproduced at wil l . Apar t from the difficulties in o b -

ta in ing sources of h u m a n e n z y m e s , ano ther factor mus t govern our cho ice in

s tudying the na ture of an e n z y m e defect . T h i s i s , for t echn ica l r easons , the

molecular w e i g h t of the pro te in , or to b e m o r e p rec i se the molecu la r w e i g h t of

the subun i t s of the pro te in . It w o u l d b e foolhardy at p resen t to under take

research on the molecu la r abnorma l i ty of a p ro te in , w h i c h had a subun i t

molecular w e i g h t of over 6 0 , 0 0 0 and in w h i c h the abnorma l i ty m a y have

ar i sen b y the r ep l acemen t of o n e or two a m i n o acids .

It is n o w o n d e r that the on ly abnormal i t i e s of p ro te in syn thes i s in m a n that

have b e e n dec iphe red so far are those of h e m o g l o b i n . H e m o g l o b i n is readi ly

avai lable w i t h o u t great sacrifice or d iscomfor t on the part of the donor , and its

subun i t s have molecu la r w e i g h t of on ly 1 6 , 0 0 0 . T h e a m i n o ac id c o m p o s i t i o n of

these subun i t s and the i r low molecular w e i g h t l imi t s the n u m b e r of pep t ides

one ob t a in s b y trypt ic or o ther proteolyt ic d iges t ion and al lows the ident i f ica-

t ion of the p resence of a dev ian t pep t ide in the digest . For these r easons , it is

m y pred ic t ion that the nature of an enzyma t i c abnorma l i t y that wi l l b e first

defined wil l b e that o f the t ransferase def ic iency ga lac tosemias . I a m pre-

d ic t ing th is w i th conf idence (a) b e c a u s e of the work of Drs . T e d e s c o and

M e l l m a n on this e n z y m e (11) ; (b) b e c a u s e the ga l ac to se - l -P ur idyl t ransferase

is readi ly ob t a inab l e f rom ery throcytes ; (c) b e c a u s e G e o r g e Dale in our labora-

tory ob ta ins n o w th is e n z y m e in 9 0 - 9 5 % pur i ty in h i g h y ie ld w i t h abou t two

days of work ; and (d) b e c a u s e it con ta ins two ident ica l s u b u n i t s of 3 1 , 0 0 0

molecular we igh t .

5 1 4 George Popjdk

There m a y b e s o m e w h o m i g h t say, "Al r igh t , y o u have e s t ab l i shed that a

g lu tamic acid in the h e m o g l o b i n molecu le w a s replaced b y va l ine ; so w h a t ? "

M y reply i s that I l ea rned that th is r ep lacement caused specific c h a n g e s in the

so lubi l i ty proper t ies of the pro te in caus ing it to quas icrys ta l l ize in the red cell

w i th the c o n s e q u e n t s ick l ing and lysis o f the cell . F r o m the s tudy of the phys i -

cal p roper t ies of th i s H b w e also learned that its so lubi l i ty proper t ies can b e

res tored to near ly normal b y its ca rbamoyla t ion w i t h cyanate . A relat ively

s imple chemica l modi f ica t ion o f an a b n o r m a l pro te in m a y n o w re l ieve the suf-

fering of m a n y Negroes afflicted b y s ickle cell anemia . I have g iven already a

sufficient n u m b e r of examples to s h o w that unrave l ing the na ture of e n z y m e

def ic iencies of i n b o r n errors of m e t a b o l i s m m a y give c lues as to t rea tment .

Before c los ing I should l ike to touch br ief ly on o n e further topic related to

gene t ic abnormal i t i e s and to w h i c h b i o c h e m i s t r y had already m a d e subs tan-

tial con t r ibu t ion . T h i s is the prenatal d i agnos i s of a th rea ten ing genet ic abnor -

mal i ty . Cytologis ts can n o w d i agnose , b y the cul tur ing and mic roscop ic e x a m -

ina t ion of fetal amnio t i c cel ls , c h r o m o s o m a l aber ra t ions assoc ia ted w i t h major

gene t ic defects . B i o c h e m i s t s can also dec ide b y the s tudy of the enzymat i c

c o m p l e m e n t o f such cells w h e t h e r the concep tus is l ikely to b e no rma l o r to

carry the genes of s o m e major i n b o r n error; at least a few of such d i seases can

b e d i agnosed as early as the fifteenth w e e k of ges ta t ion . If one be l i eves in

eugen i c s , then one can assert that no chi ld w i th D o w n ' s syndrome or Tay-

S a c h s ' d i sease n e e d b e bo rn . T h e further s tudy of the pheno typ ic express ion

of genes in amnio t i c fetal cells b y b i o c h e m i c a l t e c h n i q u e s m a y ex tend the

scope of prenata l d i agnos i s o f th rea ten ing d i sease b e y o n d our p resen t capab i l -

ity.

At p resent only a l imi ted n u m b e r of me tabo l i c abnormal i t i e s can b e t reated

even after the full unde r s t and ing of the nature o f the abnormal i ty . All the suc-

cessful t rea tments have b e e n , so far, dietary: res t r ic t ions ( amino acids) and

supp lemen ta t ions ( h o r m o n e s and v i t amins ) . W e have to awai t the evaluat ion

of the long- te rm effects of the t rea tment of Fab ry ' s d i sease b y the t ransfusion

of normal p l a sma referred to b y Dr . Ta l lman .

It is unfor tunate that m o s t o f the me tabo l i c abnormal i t i e s k n o w n to b e as-

socia ted w i th e n z y m e def ic iencies and men ta l re tardat ions are complex reac-

t ions involv ing also cof actors and auxi l iary e n z y m e s . If it were not for such a

complex s i tua t ion , it wou ld b e poss ib l e to " a n c h o r " a m i s s i n g e n z y m e onto an

inso lub le mat r ix , to enc lose such a s tabi l ized e n z y m e in a d ia lys ing b a g , and

inser t it in a b o d y cavi ty . S o m e such t rea tment , h o w e v e r U top i an an idea th i s

m a y s e e m today, m i g h t b e pract icable in years ahead .

I wou ld l ike to m a k e a last specula t ion . Pe rhaps somat ic hybr id i za t ion of

genet ical ly i n c o m p e t e n t cells w i th compe ten t cells could or m i g h t b e , b e y o n d

eugen ic s , an u l t imate so lu t ion to correct ing me tabo l i c abnormal i t i e s that lead

to men ta l re tardat ion. Y o u m i g h t t h ink th is sugges t ion n o w to b e m e r e sc ience

fiction. T h r e e years ago I w a s l aughed out o f court dur ing a si te v is i t w h e n I

p roposed the cul tur ing of l iver cells for the s tudy of factors affecting the

19. Biochemistry in Mental Retardation 5 1 5

pheno typ ic expres s ion of pheny la l an ine hydroxylase . W e were duly d isap-

proved for t ry ing to do s o m e t h i n g that n o one else h a d done before .

In less than 1 year afterward the cul tur ing o f hepa tocy te s b e c a m e a reali ty

and , today, w e have two cell cul ture l ines w h i c h con ta in pheny la l an ine hy-

droxylase as a cons t i tu t ive e n z y m e . W e h a v e also abou t ten cell l ines , reput-

edly of hepa t i c o r ig in , w h i c h con ta in n o de tec tab le levels of th is e n z y m e . D o n

Hagger ty h a s s h o w n in our labora tory that the pheny la l an ine hydroxylase in

the cul tured cells of hepa tocy tes w a s also unde r a h o r m o n a l control ; g lucocor-

t icoids caused at phys io log ica l concen t ra t ion , at abou t 5 x 1 0 - 7 M , a 4-fold

increase in the levels of the e n z y m e in 18 hours b y the induc t ion of the syn-

thes i s of a specific prote in . H o r s e se rum, i . e . , the s e rum of an adult an imal ,

con ta ins an as ye t un ident i f ied non-s te ro ida l factor or factors w h i c h also s t im-

ulate the levels of pheny la l an ine hydroxylase . T h e r e is a ra ther in te res t ing

p h e n o m e n o n in the express ion of pheny la l an ine hydroxylase assoc ia ted wi th

the g rowth cycle of the cel ls . W h e n conf luent cul tures of fully i nduced cells are

d iv ided for subcu l t iva t ion , the levels of the e n z y m e in the sparse ly popula ted

n e w cul tures dec l ine in 18 to 24 hours to very low levels and r e m a i n so unt i l

the cells b e c o m e conf luent . T h e r e u p o n w i t h i n 24 hours there is a sudden r ise

in pheny la l an ine hydroxylase and the cells b e c o m e except iona l ly sens i t ive to

the s t imula t ing ac t ion of cor t icoids and adult se rum. T h e p h e n o m e n o n is not

un l ike the sudden appearance of pheny la l an ine hydroxylase in the l iver of the

n e w b o r n rat; w h e r e a s 2 days before b i r th the e n z y m e is unde tec tab le .

A l though w e have already lea rned a little abou t the regula t ion of pheny la -

lan ine hydroxylase levels in t i s sue cul ture cel ls , w e do no t unde r s t and at all

the m e c h a n i s m of r ep res s ion and de repress ion of th is e n z y m e in m a m m a l i a n

cells. W h a t h a p p e n s in the conf luent H-4 cells that t r iggers off sudden ly the

syn thes i s o f pheny la l an ine hydroxy lase? D i d the R L C and H T C cells o f l iver

o r ig in , w h i c h con ta in no pheny la l an ine hydroxylase , suffer a de le t ion of the

pheny la l an ine g e n e or is the t rans la t ion of that g e n e mere ly repressed? W e are

h o p i n g to ge t s o m e answer s to these ve ry b a s i c q u e s t i o n s b y h y b r i d i z i n g the

act ive H-4 cells w i t h the inac t ive R L C and H T C cells and b y e x a m i n i n g the

proper t ies of the h y b r i d s .

T h i s b r i n g s m e b a c k to the q u e s t i o n o f w h e t h e r i t m i g h t b e feas ib le o n e day

to correct a me tabo l i c error resul t ing from gene t ic defect b y somat ic hybr id iza -

t ion. I mus t leave the ques t i on u n a n s w e r e d and to your imag ina t ion . Correc-

t ion of a gene t i c abno rma l i t y b y cell hyb r id i za t i on in vitro w a s a l ready

ach ieved b y Nadle r ' s g roup 3 years ago (9) . Dr . T e d e s c o has repor ted that

Nadler , C h a c k o , and R a c h m e l e r succeeded in demons t ra t ing ga lac tose -1 -

phospha te ur idyl t ransferase in h y b r i d cells m a d e b y the fusion of h u m a n

diploid f ibroblas ts o b t a i n e d from different ga lac tosemic pa t ien ts . T h e cells

con ta ined no de tec tab le e n z y m e act iv i ty before hybr id i za t ion . A m o n g the

seven l ines of ga lac tosemic cells s tud ied , three c o m b i n a t i o n s , ou t o f the pos -

s ible 2 1 , gave enzymica l ly act ive h y b r i d cel ls . In all three in s t ances the fusion

of the cells of o n e par t icular ind iv idua l [Gal . (—) D ] w i th the cells of 3 o thers (A,

5 1 6 George Popjdk

C, and E) led to the format ion of act ive e n z y m e . T h e s e very remarkab le exper i -

m e n t s sugges t at least two non iden t i ca l po in t muta t ions in t ransferase defi-

c i ency ga lac tosemia and sugges t fur thermore that if somat ic hybr id i za t ion

could b e ach ieved in vivo, me tabo l i c abnormal i t i e s resul t ing from s imple po in t

muta t ions m i g h t b e a m e n a b l e to correc t ion .

In th is ske tchy s u m m a r y o f the very large areas pe r t a in ing to i n b o r n errors

of m e t a b o l i s m and a m e n a b l e to b i o c h e m i c a l analys is I w i s h e d to convey the

ph i losophy that gu ided m e and m y col leagues in p repar ing a research pro-

gram for the b i o c h e m i s t r y sec t ion of the Men ta l Re ta rda t ion Resea rch Cen te r

at U C L A for the c o m i n g years . T h e y are also the though t s that parts o f th is

conference have evoked in m e . I should also l ike to leave the m e s s a g e that we

are conce rned no t w i th m i c e bu t wi th m e n .

REFERENCES

1. Beadle, G. W. , and Tatum, E. L. Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 1941, 27: 499-506.

2. Butcher, F. R., Bushnell, P. E. , Becker, J. E. , and Potter, V. R. Effect of cordycepin on induc-tion of tyrosine aminotransferase employing hepatoma cells in tissue culture. Exp. Cell Res., 1972, 74: 115-123.

3. Foiling, A. Uber Ausscheidung von Phenylbrenztraubensaure in den Harn als Stoffwechselano-malie in Verbindung mit Imbezillitat. Hoppe-Seyler's Z. Physiol. Chem., 1934, 227: 169.

4. Garrod, A. E. "Inborn Errors of Metabolism." (Croonian Lectures). Lancet, 1908, 2: 1, 73, 142 and 214.

5. Goldstein, J. L . , and Brown, M. S. Familial hypercholesterolemia: Identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol. Proc. Natl. Acad. Sci. USA, 1973, 70: 2804-2808.

6. Jervis, G. A. Phenylpyruvic oligophrenia: Deficiency of phenylalanine-oxidizing system. Proc. Soc. Exp. Biol. Med., 1953, 82: 514-515.

7. Kenney, F. T., Lee, K. L. , Stiles, C. D., and Fritz, J. E. Further evidence against post-transcriptional control of inducible tyrosine aminotronsferase synthesis in cultured hepatoma cells. Nature (London), 1973, 246: 208-210.

8. La Du, B. N., Zannoni, V. G., Laster, L., and Seegmiller, J. E. The nature of the defect in tyrosine metabolism in alcaptonuria. / . Biol. Chem., 1958, 230: 251-260.

9. Nadler, H. L. , Chacko, C. M., and Rachmeler, M. Interallelic complementation in hybrid cells derived from human diploid strains deficient in galactose-l-phosphate uridyl transferase activity. Proc. Natl. Acad. Sci. USA, 1970, 67: 976-982.

10. Stanbury, J. B . , Wyngaarden, J. B . , and Fredrickson, D. S., Eds. The Metabolic Basis of Inherited Disease. (3rd ed.) McGraw-Hill, New York, 1972.

11. Tedesco, T. A. , and Mellman, W. J . Galactosemia: Evidence for a structural gene mutation. Science, 1971, 172: 727-728.

12. Tomkins, G. M., Gelehrter, T. D. , Granner, D. , Martin, D. M., Samuels, H. H., and Thompson, E. B . Control of specific gene expression in higher organisms. Science, 1969, 166: 1474-1480.

13. Watson, J . D. , and Crick, F. H. C. Genetical implications of the structure of deoxyribonucleic acid. Nature (Lond.), 1953, 171: 964-967.

Epilogue

ALBERT DORFMAN Joseph P. Kennedy, Jr., Mental Retardation Research Center,

Department of Pediatrics, Pritzker School of Medicine,

The University of Chicago, Chicago, Illinois

In the fo l lowing remarks I will try to g ive m y impre s s ions of the w i n d o w

into neu rob io logy as seen first from the po in t of v i e w of a b i o c h e m i s t and cell

b io log i s t and finally from the po in t o f v i e w of a ped ia t r ic ian conce rned wi th

the care of ch i ldren w i t h men ta l re tardat ion.

In the recen t past , b i o c h e m i s t s have b e c o m e m o r e and m o r e c o n c e r n e d w i t h

the in tegra t ion of me tabo l i c sys t ems . T h e earl iest b i o c h e m i c a l s tud ies w e r e

conce rned wi th the end resul ts of me tabo l i c sys tems as e v i d e n c e d b y the anal-

yses o f w h o l e o r g a n i s m s , b l o o d , u r ine , etc. W i t h the deve lopmen t s s tar t ing

jus t before W o r l d W a r II and expand ing rapidly thereafter , such sys t ems were

d i ssec ted b y the i so la t ion of e n z y m e s and de te rmina t ion of me tabo l i c path-

ways . W h i l e such isola ted sys t ems s e e m e d or ig inal ly dis tant from phys io l -

ogy , it has n o w b e c o m e qu i te clear that in format ion der ived from such s tudies

was neces sa ry for the unde r s t and ing of phys io log ica l sys t ems . Pe rhaps o n e of

the great m i l e s t o n e s of scient i f ic a c h i e v e m e n t has b e e n the d e v e l o p m e n t of the

under s t and ing of the m e c h a n i s m b y w h i c h gene t ic in format ion is t r ansmi t ted

from genera t ion to genera t ion as wel l as the m e c h a n i s m b y w h i c h th is infor-

ma t ion controls the expres s ion of cell act ivi ty . For the m o s t part , th i s p rogress

has b e e n m a d e u t i l i z ing prokaryot ic sys t ems . W e are n o w en te r ing an era of

more in t ens ive s tudy of the eukaryot ic cell . Resea rch on prokaryot ic cells has

p rov ided a wea l th of in fo rmat ion of the ba s i c b i o c h e m i c a l and gene t ic m e c h a -

n i s m s character is t ic o f l iv ing mat ter . W e n o w m u s t ut i l ize th is in format ion to

de t e rmine w h i c h m e c h a n i s m s ob t a in in eukaryot ic cells and w h i c h n e w m e c h -

an i sms have b e e n evolved to carry out the funct ions u n i q u e to eukaryot ic

cells.

5 1 7

5 1 8 Albert Dorfman

T w o s t r iking differences b e t w e e n the two types of sys t ems are immed ia t e ly

apparent . Prokaryot ic cells are se l f -conta ined en t i t i es w h i c h carry out all the i r

funct ions as ind iv idua l cells w h i l e eukaryot ic cells exis t in o r g a n i s m s wi th

spec ia l iza t ion of funct ion for ind iv idua l types of cells . Fu r the rmore , the spe-

c ia l ized cells of the o rgan ized sys t ems are mutua l ly i n t e rdependen t and ap-

pear to send m e s s a g e s to each o ther , that is they engage in cell talk. At

present , a large part of b io log ica l research is conce rned wi th th is p rob lem. T h e

impl ica t ions of such c o m m u n i c a t i o n are profound. Involved is a de te rmina-

t ion of: (a) the s t imulus w h i c h results in s end ing of m e s s a g e s , (b) the na ture of

the m e s s a g e , (c) the receptor for the m e s s a g e , and (d) the m a n n e r in w h i c h the

receptor conver ts the m e s s a g e to change the b e h a v i o r of the rece iv ing cell. T h e

deve lopmen t of all these funct ions represents the p r o b l e m of different ia t ion of

eukaryot ic cells .

It has b e c o m e increas ing ly apparent that the cell m e m b r a n e is of cri t ical i m -

por tance in all of these p h e n o m e n a . Al ready great p rogress has b e e n m a d e in

d i ssec t ing the s t ructure o f m e m b r a n e s . M o s t impor tan t has b e e n the r ecogn i -

t ion of the complex and dynamic structure of such m e m b r a n e s , par t icular ly

the real izat ion of the p resence of macromolecu les w h i c h serve to act as re-

ceptors for par t icular m e s s a g e s .

In the case of cer ta in types of cells such as the e ry throcyte , a clearer pic ture

of th i s in tegra ted sys t em is b e g i n n i n g to emerge . It s e e m s l ikely that an un-

ders tanding of the s tructure of m e m b r a n e s of a w i d e var ie ty of o ther cells will

eventual ly lead to greater c o m p r e h e n s i o n of the ne rvous sys tem. If one adopts

a deve lopmenta l and evo lu t ionary po in t of v i e w , one m a y regard the ne rvous

sys tem as the u l t imate in a sy s t em of cells spec ia l ized for the purpose of com-

munica t ion . T h e ne rvous sys t em p r o b a b l y ut i l izes the genera l m e c h a n i s m s

bu t has addi t ional ly evo lved n e w and spec ia l ized m e c h a n i s m s .

T h e great progress in unde r s t and ing c o m m u n i c a t i o n in cel ls , o ther than in

the ne rvous sys t em, has occur red largely as a result o f i m p r o v e d m e t h o d s of

t i ssue culture. In the case o f the cells o f the ne rvous sys t em th is is as yet

l imi ted , a l though progress is b e i n g m a d e . T h e reports o f Dr . de Vel l is and Dr .

Dich te r g ive us an ins igh t in to the status o f such s tudies . W i t h the develop-

m e n t of t i ssue culture l ines o f m o u s e neu rob l a s toma and rat glial cells there

has b e e n in t ense act ivi ty in m a n y labora tor ies . M u c h valuable in format ion

regard ing the me tabo l i c proper t ies of such cells has b e e n ob ta ined . W i t h use

of explants s o m e in format ion regard ing in tegra t ion is b e g i n n i n g to e m e r g e .

H o w e v e r , the use of cell or o rgan cul ture of the ne rvous sys t em con t inues to

have severe l imi ta t ions compared wi th w o r k on o ther t i s sues . T h e failure of

normal different iated neu rons to d iv ide in cul ture represen ts a se r ious s tum-

b l ing b lock . T h e avai lable sys tems of culture are no t ye t sat isfactory for the

s tudy of different ia t ion of the ne rvous sys tem.

I wou ld l ike to d igress to po in t ou t the k i n d of s tudies w h i c h are b e i n g

carr ied out in cul ture in o the r t i s sues . In ou r o w n labora tory w e have b e e n

Epilogue 519

pr imar i ly c o n c e r n e d w i t h the different ia t ion of car t i lage. W e have u sed th i s

m o d e l b e c a u s e of the ex i s t ence of a cons ide rab le b a c k g r o u n d of in format ion

regard ing the b i o c h e m i c a l pa ramete rs w h i c h cons t i tu te the pheno typ i c expres -

s ion of th is t i s sue and b e c a u s e different ia t ion of cart i lage m a y b e obse rved in

cell culture. T h e s e s tudies have b e e n recent ly r e v i e w e d (4) . W h e n ch ick l i m b -

b u d cells at s tage 24 are d i ssoc ia ted and cul tured on plast ic d i shes at usual cell

dens i t i es , cart i lage different ia t ion does no t occur w h e n m e a s u r e d b y morpho l -

ogy, format ion of me tach romat i c mat r ix , or syn thes i s of large a m o u n t s of

chondro i t in sulfate pro teoglycan . If, h o w e v e r , such cells are cul tured at ve ry

h i g h dens i ty or over agar for 48 hours and then subcu l tu red on plast ic d i shes ,

d i f ferent ia t ion to cart i lage is rapid . Apparen t ly dur ing th is shor t pe r iod of

t ime s o m e even t occurs w h i c h act ivates the m a c h i n e r y for express ion of the

cart i lage p h e n o t y p e . It has b e e n pos s ib l e to s h o w that cells cul tured over agar

or at h i g h dens i ty go o n to p roduce large a m o u n t s o f chondro i t i n sulfate pro-

teoglycan and cart i lage specific col lagen.

In recent years a large l i terature has appeared demons t r a t i ng that the t hymi -

d ine ana log , 5 ' - b r o m o d e o x y u r i d i n e (BrdU) , i n h i b i t s the p h e n o t y p i c expres -

s ion of a n u m b e r of spec ia l i zed cells . T h i s effect appears to b e due to the

r ep lacement of t h y m i d i n e in D N A b y BrdU. W h e n l i m b b u d s are sub jec t ed to

B r d U dur ing the early pe r iod of cul ture and s u b s e q u e n t l y subcu l tu red in the

a b s e n c e of B r d U , dif ferent ia t ion does no t s u b s e q u e n t l y occur e v e n t hough no

de tec tab le B r d U r e m a i n s in the D N A . In contras t , w h e n different ia ted cart i-

lage cells are sub jec ted to B r d U , the suppress ion of different ia ted funct ion is

revers ib le . S imi la r expe r imen t s have n o w b e e n per formed in a n u m b e r of

o ther sys t ems . T h e s e resul ts sugges t that in the course of di f ferent ia t ion of

eukaryot ic cells there is a specific s e q u e n c e of even t s w h i c h is t i m e ordered .

O n c e th is s e q u e n c e is d i s tu rbed , the oppor tun i ty m a y no t aga in occur .

In the case of the ne rvous sys t em the p r o b l e m of di f ferent ia t ion appears to

b e more complex . T h e s tudies p resen ted b y D r s . Rak ic and A l tman b e g i n to

g ive s o m e in s igh t in to s o m e o f the phase s o f d i f ferent ia t ion. It s e e m s l ikely

that the mul t ip l ica t ion and in i t ia l di f ferent ia t ion of neuroep i the l i a l cells m a y

b e m o r e ak in to the dif ferent ia t ion of cart i lage or musc l e . S u b s e q u e n t l y , o ther

impor tan t even t s m u s t occur before an in tegra ted ne rvous sys t em is fo rmed. It

is impor tan t to no te that at least two deve lopmen ta l p rocesses , mye l ina t i on

and mig ra t ion , r equ i re the coopera t ion of at least two different cell types . T h e

expe r imen t s of Dr . Rak ic ind ica te that mig ra t ion m a y b e gu ided b y glial e le -

m e n t s . A mos t in te res t ing ques t i on that has not to m y knowledge b e e n

addressed is the nature of the mi l i eu th rough w h i c h cells migra te . It m i g h t b e

expec ted that the mat r ix b e t w e e n cells plays an impor t an t role in migra t ion .

T h e ex i s tence of gene t ic mu tan t s w h i c h apparen t ly s h o w d i s tu rbances of

mig ra t ion m a y offer a useful tool for the s tudy of the chemica l s t ructure of the

glial p rocesses w h i c h appear to gu ide mig ra t ion as well as the m i l i e u th rough

w h i c h such mig ra t ion occurs .

520 Albert Dorfman

W h e n one th inks of the m a n y specific cell surface c o m p o n e n t s that have

recent ly b e e n ident i f ied , it is un l ike ly that s tudies b y classic m e t h o d s of

s ta in ing wil l g ive us answers w i t h respec t to deve lopmenta l dev ia t ions in

b ra in deve lopment . It also s e e m s un l ike ly that ana tomica l s tudies o n pa t ien ts

l abe led as menta l ly re tarded wil l p roduce mean ingfu l answers unt i l the pa-

t ien ts are adequa te ly sor ted out from a cl inical po in t of v i e w .

Dr . Pop jak has a l ready s u m m a r i z e d the current state of the art as wel l as fu-

ture d i rec t ions of research c o n c e r n i n g me tabo l i c d i seases w h i c h result in

menta l re tardat ion. I wou ld l ike to e m p h a s i z e the impor t ance of a genet ic

approach. W h e n e v e r w e can ident i fy a c l inical s y n d r o m e for w h i c h clearcut

Mende l i an t r ansmis s ion ex is t s , w e have a po ten t tool for the inves t iga t ion of

e t io logy. In the case of recess ive d i seases , it appears safe to a s s u m e that the

d isease s t ems from an abnorma l i t y of a s ing le gene product . N o mat te r h o w

bizar re the c o m b i n a t i o n of cl inical man i fes t a t ions , exp lana t ion wil l b e found

eventual ly to have a s ingle b i o c h e m i c a l b a s i s w h i c h m a y in turn lead to m a n y

complex abnormal i t i e s . T h e self -destruct ive b e h a v i o r of pa t ien ts w i t h the

L e s c h - N y h a n s y n d r o m e is an excel lent example of th is pr inc ip le .

In the case of d o m i n a n t d i seases the b i o c h e m i c a l ba s i s is less clear. O u r o w n

work on M a r i a n ' s d i sease (3 ,5) sugges ts an increase of hya luron ic ac id syn the -

sis w h i c h m i g h t involve a regula tory m e c h a n i s m . T h e recent s tud ies b y Go ld -

s te in and B r o w n (1,2) on T y p e II hype r l i p idemia s t rongly ind ica te a regulatory

defect in th i s d o m i n a n t d i sease w h i c h appears to b e due to an a b s e n c e of a cell

receptor . I a m e m p h a s i z i n g th is po in t because I be l i eve the road to e luc ida t ion

of specific s y n d r o m e s o f m e n t a l re tardat ion m a y b e the careful c l inical descr ip-

t ion w h i c h m a y in turn lead to apprec ia t ion of the gene t i cs and s u b s e q u e n t

e luc ida t ion of the b i o c h e m i c a l defect.

E luc ida t ion of b i o c h e m i c a l m e c h a n i s m s is no t conf ined to gene t ic d i seases .

T h e p resen ta t ions o f Drs . Hel ler and T e n n y s o n po in t the w a y to a n e w level of

under s t and ing of t r ansmis s ion of s ignals w i t h i n the ne rvous sys t em. W h e t h e r

or not the exact m e c h a n i s m s pos tu la ted turn out to b e correct , t hey represent a

s tep forward in the a t tempt to fa thom the m e c h a n i s m s b y w h i c h m e s s a g e s are

t ransmi t ted .

I wou ld l ike n o w to j u m p ahead to the con t r ibu t ions that we have heard

regard ing b e h a v i o r s tud ies . A n y o n e w h o h a s dealt w i t h re tarded ch i ld ren

real izes the difficulties of classif icat ion. Desp i t e great faith in b i o c h e m i s t r y

and cell b io logy , w e all k n o w that a large part of the p rob l em l ies in social ,

e c o n o m i c , and educa t iona l p rob l ems . T h e extent of th is is no t clear. A s is

apparent from the p resen ta t ions , w e canno t ye t define adequa te ly the m e c h a -

n i s m s b y w h i c h these factors p roduce re tardat ion. I refer, of course , to impor -

tance of in utero nu t r i t ion or early infant nu t r i t ion as c o m p a r e d to the effects of

env i ronmen t on early deve lopment . He re , I b e l i e v e , great p rogress can b e

m a d e b y psycholog is t s in def in ing specific character is t ics that m a y b e related

to e t io logy. W e all n o w accept that defects in per formance m a y b e very spe-

Epilogue 521

cific. T h e unrave l ing of these defects m a y he lp us define e t io logy. W h i l e w e as

phys i c i ans and sc ien t i s t s canno t control the e c o n o m i c and social e n v i r o n m e n t ,

w h e n e v e r w e can d i scover specific e t io logical factors , the c ha nc e s of soc ie ty

l i s ten ing to us are great ly e n h a n c e d . W i t n e s s the great p rogress that has b e e n

m a d e in the last few years in the control o f lead p o i s o n i n g , an impor tan t cause

of men ta l re tardat ion. A l though t rea tment has b e e n of s o m e impor t ance in

decreas ing the effects o f th is tox in , m o s t o f the progress has b e e n m a d e b y

control of the env i ronmen t .

Resea rch in men ta l re tardat ion is in m a n y w a y s frustrat ing b e c a u s e of the

very na ture of the p rob lem. S i n c e men ta l re tardat ion invo lves so m a n y et iol-

o g i e s , each of us has had to conf ine ourse lves to a very l imi ted sphere . In

turn, in each sphere progress is dependen t on the evolu t ion of fundamenta l

knowledge . In the pas t few years w e have h a d inc reas ing pressure on research

b o t h from l imi ta t ion of funding and threats of inc reas ing regula t ion of c l inical

research. P r o p o s e d legis la t ion and admin i s t ra t ive regula t ions w o u l d severe ly

h a m p e r work on fe tuses w h i c h m a y b e of cri t ical impor t ance in s tudies of

deve lopmen t of the bra in . Regu la t ion o f t es t ing m i g h t m a k e further vacc ine

deve lopmen t vir tual ly i m p o s s i b l e . I am not c o n v i n c e d that e i the r the de-

creased funding or the regula t ion s t ems from pub l ic outcry, bu t I t h ink ra ther

from a smal l g roup of peop le w h o do not under s t and the i s sues . I be l i eve it is

e n c u m b e n t o n us to a t tempt to coun te r t he se t h ings bu t no t b y unrea l i s t ic

p romise s or exaggera ted c la ims . M o s t o f all w e m u s t b e t rue to ourse lves as

sc ien t i s t s and phys i c i ans and con t inue to seek the t ruth for i ts o w n sake as

wel l as to al leviate h u m a n suffering.

R E F E R E N C E S

1. Brown, M. S., Dana, S. E. , and Goldstein, J . L. Regulation of 3-hydroxy-3-methylglutaryl coen-zyme A reductase activity in cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. / . Biol. Chem., 1974, 249: 789-796.

2. Goldstein, J . L . , and Brown, M. S. Familial hypercholesterolemia: Identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol. Proc. Natl. Acad. Sci. U. S., 1974, 70: 2804-2808.

3. Lamberg, S. I., and Dorfman, A. Synthesis and degradation of hyaluronic acid in the cultured fibroblasts of Marfan's disease. / . Clin. Invest., 1973, 52: 2428-2433.

4. Levitt, D. , Ho, P.-L. , and Dorfman, A. Differentiation of cartilage. In: Cell Surface in Develop-ment. (A. A. Moscona, Ed.). Wiley, New York, 1974: Chapter 6, pp. 101-125.

5. Matalon, R., and Dorfman, A. The accumulation of hyaluronic acid in cultured fibroblasts of the Marfan syndrome. Biochem. Biophys. Res. Commun., 1968, 32: 150-154.

Author Index

Numbers in parentheses are reference numbers and indicate that an author's work is referred to although his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed.

Actic, L . , 172(3), 178

Adachi, M. , 485(30), 498

Adam, A. , 487(18), 497

Adametz, J. H., 273(1), 307

Adey, W. R., 375, 377

Adinolfi, A. M. , 191(1), 200, 233(1, 2) , 253(1, 2) , 259, 266(1, 2) , 267, 270

Adlard, B . P. F . , 402(1), 419

Adornato, B . , 485(19), 497

Aghajanian, G. K. , 183, 207(64), 217, 252(9), 253(3), 259, 260

Akert, K., 233(53), 257(53), 262, 273(2), 298(2),

299(2), 300, 302(82), 305, 307, 310, 393(12), 398

Albe-Fessard, D. , 304(38), 309

Alberici, M. , 269(10), 270

Al-Janabi, J . M. , 462(4), 465

Allen, L. , 432(13), 441

Allerand, C. D. , 121

Altman, J . , 42, 43, 44, 45 , 46, 47, 49, 50(5, 6), 51 , 52, 53(10, 11 , 14, 15), 55, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 68(19), 69(19), 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81(24), 83 , 84, 85, 86, 87, 88, 89, 90, 91, 123(25), 139, 187, 200,

381(1, 2) , 390

Amassian, V. E. , 393(1), 397(1), 398

Anastasi, A., 426(1), 428(2), 441

Anden, N. E. , 191(3, 4) , 202, 206(1-4) , 214,

227(4, 5) , 258(5), 259, 265(4, 5) , 270

Andersen, P., 142(1, 2, 3) , 150, 151(1), 164(1, 3) , 166, 167

Anderson, B . , 273(2), 298(2), 299(2), 300, 305,

307

Anderson, J . W. , 47(40), 90

Anderson, M., 223, 224

Anderson, W. J . , 52, 53(10, 11, 14, 15), 55,

57, 58, 59, 60, 61 , 62, 65, 66, 73(12), 77, 78,

79, 89

Andrade, A. N. , 191(10), 199(10), 201, 232(30, 31), 233(30, 31) , 253(30), 257(31), 261

Andrea, U., 483(28), 486(28), 498

Angevine, J . B . , Jr . , 6(2), 7(2), 9(1, 2 ) , 15(2), 37, 381(3), 390

Anton, A. H., 249, 260 Applebaum, M. I. , 432(19), 441 Appleman, M. M. , 269(6), 270

Appleman, W. J . , 269(25), 271 Arbib, M. A., 338(1), 340

Arnaiz, G. R., 269(10), 270

Artom, G., 9(3), 37

Astrom, K. E . , 32(4), 37 Atlas, M. , 7(5), 37 Auld, R. M. , 450(23), 458 Ayling, J . E . , 459(3), 462(1, 2 , 4 ) , 464, 465

Bailey, P. , 283(3), 299, 307

Bak, I. J . , 189(26, 32, 44) , 202, 229(7), 231(7),

233(7), 251 , 252(7, 72), 253(7), 260, 262 Bakerman, S., 485(6), 497 Balazs, R. , 87(21), 89 Bard, P. , 276, 302(4), 307, 335(1), 340 Barkley, D. S., 102(1), 114, 116(2), 121 Barlow, P. , 415(2), 420

523

524 Author Index

Barnes, D. , 86, 87, 89, 90 Barrett, R. E . , 189(50), 192(50), 203, 227(95),

228(83, 84, 88, 89), 233(83, 84), 243(88, 89), 253(17, 83, 84), 256(84), 260, 263, 264, 265(23), 271

Barrnett, R. J . , 251, 263, 267(11), 270 Bartha, A., 17(77), 40 Battig, K., 296(5), 298(5), 301(5), 307, 394(2),

398 Beach, F. A. , 393(3), 398 Beadle, G. W., 508, 516 Beaver, D. L . , 229(67), 262 Beck, E. C., 336(3), 340 Becker, J. E . , 508(2), 516 Bedard, P., 206(5), 214, 256(8), 260 Beilin, H., 380(4-6), 390 Benhamida, C., 141(26), 168 Benjamin, R. M., 393(4), 398 Bensch, K. G., 124, 125, 139 Benton, C. E . , 123(1), 138 Benton, J . W., 134(2), 138 Bergren, W. R., 469(2), 474(13), 475(13), 477 Berman, A. J . , 317(1), 332 Berman, D. , 317(1, 17), 332 Berman, J . , 317(17), 332 Bernardi, G., 192(5, 20, 21) , 196(5, 20, 21),

197(21), 198(21), 201, 265(16), 271 Berne, R. M. , 210,215 Berry, H. K., 459(5), 465 Berry, M., 6(6), 15(6), 37 Bertler, A . , 206(6), 214 Beutler, E. , 495(32), 496(32), 498 Bhatnagar, R. K., 189(40), 191(40), 192(40), 202,

206(26, 48, 49), 207(49), 215, 216, 227(65), 262

Bidder, T. G., 273(5), 303(5), 307 Bignall, K. E . , 397(5), 398 Birch, H. G., 419(14), 420 Bird, M. M., 117(3), 121 Bjdrklund, A., 228(68), 262 Black-Cleworth, P., 336(34), 337(34, 36, 40),

338(4, 34, 36), 339(34), 340, 341 Blackstad, T. W. , 142(2, 4, 5, 6, 7) , 164(4), 167 Blomquist, A. J . , 188(13), 201 Bloom, F. E. , 183, 224, 251, 252(9), 253(3), 259,

260 Blot, W. J . , 415(15), 420 Boches, F . , 477 Boehm, G. R., 462(1), 464 Bolles, R., 439(3), 441 Bolzhina, N. S., 273(37), 300, 308 Bond, D. D. , 273(5), 303, 307

Bond, Y. R. , 7(5), 37 Bonin, G., 24(7), 30(7), 37 Boreus, L. O., 228(70), 262

Bornstein, M. B . , 102(6), 224, 117(4, 12), 120(5), 121

Boucher, R. , 206(56), 227, 227(73), 263 Bouvier, G., 206(56), 227, 227(73), 263 Bowden, D. M., 381(7), 390 Bower, B. D. , 137(22), 139 Brady, J. V . , 283(7), 307

Brady, R. O., 479(1-3, 35, 36, 40), 481(38), 483, 484, 485(35), 486(39), 487(22, 37), 488(37, 39), 489, 490(37), 492(37), 493(37), 494(37), 495(37), 497, 498

Bray, D. , 116(6), 222 Breckinridge, B . , 267(7), 268, 269(7), 270 Breen, G. A. M. , 115(7, 9) , 116(9), 117(7, 8, 9),

118, 222 Brenner, P., 485(19), 497 Bridgman, P. W., 422, 442 Brink, K. , 142(7), 267 Broadhurst, P. L. , 380(24), 384(24), 392 Brodmann, K., 4, 5, 11, 14, 37 Brooker, G., 117(16), 119(16), 222 Brown, J. H., 267(8), 270

Brown, K. A. , 274(57), 296(57), 309, 319(4), 320(21), 322(21), 323(21), 325(21), 326(8), 327(2, 3) , 328, 329, 332, 333

Brown, M. S., 509. 526, 520, 522 Brown, R., 495(4), 497 Brown, W. J . , 164(8), 267

Browning, R. A. , 194(23), 195(23), 202, 209(38), 226

Brozek, G., 336(35), 337(35), 338(35), 342 Bruner, J. S., 380(8), 390 Brunner, R. L . , 80, 81(24), 83, 84, 85, 90 Bruppacher, H., 233(53), 257(53), 262 Brutkowski, S., 296(8), 300(9), 302, 307 Buchwald, J . S., 274(57), 296(57), 309, 319(4, 5) ,

320(21), 322(21), 323(21), 325(21), 326, 327(2, 3) , 328, 329, 330, 332, 333, 338(5, 6) , 340

Buchwald, N. A., 192(5, 6, 20-22 , 34, 35), 193(35), 194(23), 195(23, 35), 196(5, 6, 20-22) , 197(21), 198(11, 21 , 34), 200(34, 36, 49), 202, 202, 203, 209(38), 226, 223, 224, 225(1), 226, 265(16), 272, 274(44), 302(44), 305(10, 44), 307, 309, 394(19), 397(19), 398

Buist, N., 487(44), 497(44), 499 Bunge, M. B . , 117(10), 222, 243(10), 260 Bunge, R. P., 117, 222, 222 Burgen, A. S. W. , 189(7), 202 Burt, C., 434, 442

Author Index 525

Bushnell, P. E . , 508(2), 516

Butcher, F. R., 508(2), 526 Butcher, R. W., 209(60), 227 Butters, N. , 309

Cajal, S. Ramon Y . , 142(9), 150(9), 151, 161, 164(9), 267

Callens, M., 298(25), 303(25), 308

Campbell, B . A. , 172(4), 178

Campbell, L. B . , 189(8), 202 Carlsson, A. , 191(3), 202, 206(1, 7) , 224, 225,

227(4, 11, 12), 259, 260 265(4), 270

Carman, J . B . , 227(29), 262, 301(11), 307

Carpenter, D., 337(36), 338(36), 342 Carpenter, M. B . , 191(9), 192(9), 202, 227(13,

14), 256, 260

Carr, S., 137(27), 239 Carroll, M. , 495(5), 496(5), 497

Cattell, J . M . , 4 2 3 , 442 Cauthen, J. C , 300(84), 320 Cavanaugh, M. C , 123(18), 239 Cavanaugh, M. W., 102(2), 224 Caviness, V. S., 15(10), 20(10), 36(59), 37,

39

Chacko, C. M., 471, 477, 515, 526 Chaffee, J. K., 102(1), 224, 116(2), 222 Chambers, W. W., 94, 98, 317(27, 28), 318(27,

28), 333

Chase, H. P., 86(26, 49) , 90, 91

Cheek, D. B . , 86(27), 90

Cheramy, A. , 189(25), 202, 226

Chio, K. S., 231(15), 260

Chipman, L. M. , 189(7), 202 Christensen, E. , 135, 138

Clark, W. E. Le Gros, 12(11), 37 Cocks, W. A. , 87(21), 89

Cohen, G. , 189(50), 192(50), 203, 228(83, 84),

233(83, 84), 253(16, 17, 83-85) , 255(86), 256(84), 260, 263, 265(23), 272

Cole, R. , 115(9), 116(9), 117(9, 19), 119(19), 222 Coleman, P. D. , 124(4), 235 Colle, J . , 298(25), 303(25), 308

Collins, J. P., 174(7), 175(10), 178

Colonnier, M. L. , 151(11), 156(13), 161(11), 163(10, 11, 13), 164(12, 13), 165(11), 267

Combs, C. M., 316(32), 317(32), 333

Comme-de Broth, R., 228(39), 262 Conel, J . L. , 123(5), 137(5), 235, 156(14), 160,

267, 381(9), 390

Connor, J. D. , 224, 265(9), 270

Cools, A. R. , 305(12), 307

Cooper, E. R., 235(18), 260

Cooper, T., 209(40, 65), 226, 227

Costa, E . , 2 2 4

Cote, L. J . , 189(50), 192(50), 203, 228(19, 83, 84),

233 (83 ,84 ) , 253 (83 ,84 ) , 256(84), 260, 263,

265(23), 272 Cotman, C. W. , 96(1, 10, 11, 12, 13, 14, 17, 18),

97(12), 98(14, 17, 18, 19), 98, 99

Cotzias, G. C , 227(20, 21) , 260

Cowan, W. M., 142(53), 269, 301(11), 307,

381(10), 390

Cox, V. C , 384(50, 51), 392

Coy, M. A. , 20(19), 38

Coyle, J. T. , 255(51), 258(51), 262

Cragg, B . G. , 180, 184

Craigie, E. H., 316(9), 332

Crain, S. M., 101(5), 102(6), 111(3, 4) , 112(4), 224,

117(11-13) , 222 Crandall, P. H., 137(31), 140

Crawford, H. T. , 386, 392 Crick, F. H. C , 508, 526 Cripps, R. , 459(5), 465

Criswell, H. E . , 273(27), 308

Crome, L . , 20(12), 36(12), 37 Crow, T. , 336(7), 337(36, 40) , 338(36, 40) , 340,

341

Culley, W. J . , 86(28), 90

D'Agostino, A. J . , 229(22), 260

Dahlstrom, A. , 191(3, 4) , 201, 205(8), 206(1 -3 , 8), 224, 225, 227, 258(5), 259, 260, 265(4, 5) ,

270

Dalby, D. A., 303(55), 309

Dalhouse, A. , 174(8), 178

D'Amato, C. J . , 6(18), 9(18), 15(18), 20(19), 22(18), 38, 52(36), 60(36), 90, 123(16, 17), 239

Dana, S. E . , 520(1), 522

Dance, N. , 496(21), 498

Das, G. D . , 87(20), 89

Davis, E. W. , 283(3), 299, 307

Davis, G. D. , 296(13, 14), 298(14), 299(13), 300, 308, 309, 394(6), 398

Davison, A. N. , 403(3), 412(4), 420

Daw, J. C , 210, 225 Deadwyler, S. A. , 96(10, 19), 99, 189(37), 202

Dean, W. H., 296(14), 298(14), 308

de Arellano, R., 316(32), 317(32), 333

Dekaban, A. S. , 20(13), 37, 381(11), 390

de la Flor, S. D. , 504(6), 505(6), 506

Delgado, J . M. R. , 273(63), 296(63), 298(63), 301(63), 309, 394(21), 398

De Long, G. R., 102(7, 8) , 224, 116(14, 15), 122, 381(12), 390

Dembitzer, H., 55(37, 38) , 90

Denenberg, V. FL, 387(13), 390

526 Author Index

Denny-Brown, D. , 273(15), 298, 299(15), 308

Deol, G., 384(38), 391

De Robertis, E. , 269(10), 270

Descarries, L. , 252, 260

de Vellis, J . , 115(7, 9) , 116(9), 117(7, 8, 9, 16, 17,

19), 118, 119, 120, 121, 122

Dichter, M. , 102(9, 12), 103(9, 12), 106, 108, 109,

110, 111(9), 112(9), 114

Dickerson, J . W. T., 404(5), 420

Disterhoft, J . F . , 3 4 8 ( 1 - 3 , 1 1 , 1 5 ) , 360(2), 370, 371

Divac, I., 273(16), 296(16, 17), 298(17), 301, 308

Dixon, M., 462(6), 465

Dobbing, J . , 86(29), 90, 137, 139, 402(1, 6, 8, 11, 13), 403(3, 7, 9, 10, 12), 404(5, 9, 11, 12), 407-411(12), 413(12), 414(12), 419, 420

Dobrzecka, C , 439(7), 441

Dodge, P. R. , 134(2), 138, 139

Donat, J . , 393(1), 397(1), 398

Donnell, G. N. , 467(6), 468(6), 469(2), 474(13), 475(13), 477

Dorfman, A. , 519(4), 520(3, 5) , 521

Doty, R. W. , 336(3), 337(8), 340, 393(7), 398

Dragoin, W., 439(30), 442

Dresse, A. , 208(45), 216, 228(64), 262 Droz, B . , 252, 260

Duby, S., 227(20), 260

Duffy, P. E . , 189(50), 192(50), 203, 229(25),

231(25), 260

Dupree, A. L . , 283(54), 295(54), 300(54), 309

Duvoisin, R. C., 227(95), 264

Dvornik, D. , 468(3), 477

Eayrs, J . T., 87(21), 89, 123(8), 139

Ebels, E. J . , 60(30), 90

Eccles, J. C., 94(2), 98, 142(3), 164(3), 167,

256(26), 260

Edds, M. V . , 94, 98

Edwards, D. A., 384(14), 390

Ehringer, H., 206(10), 225, 227(11), 260

Ellingson, R. ] . , 156(15), 159(15), 163(15), 267, 180(3), 184

Engel, J . , Jr . , 337(9, 37, 38), 339(9, 37, 38), 340,

341

Engel, R. , 159(16), 267 Engelbert, V . , 102(18), 111(18), 224 Erinoff, L . , 208(11), 225 Ermolenko, S. F . , 301(18), 308

Ernhart, C., 317(10), 332 Ervin, F . , 337(12), 340

Evangelista, I., 125(13), 239 Fagen, L. , 174(2), 178

Fahn, S., 228(19), 260

Fairchild, M. D. , 178

Falck, B . , 205(12, 13), 206(7, 12, 13), 225, 227(12,

28), 228(28), 260, 261

Farber, J. L . , 117(41), 222 Farel, P. , 338(10), 340

Faro, M. D. , 316(11), 332

Farrell, D. F . , 505(3), 506

Faull, R. L. M. , 206(14), 225, 227(29), 262 Federov, V. K. , 373 ,377 Feldman, M., 29(42), 30(42), 32(42), 39

Fibiger, H. C., 208(15), 225, 252(46), 253(46),

257(46), 258(46), 261

Field, P. M. , 384(42), 392

Fillerup, D. L . , 485(19), 497 Finger, S., 273(19), 308

Fink, R. P., 206, 215

Fisch, L. , 317(12), 332

Fischbach, G. D. , 102, 103(9-12) , 106, 108, 109,

110, 111(9), 112(9), 224, 117(20, 21), 222 Fish, I. , 86(31), 90

Fisher, D. B . , 449(1, 2) , 450(20), 451(1, 3) ,

453(3), 457 Fisher, K., 102(18), 111(18), 224 Fishman, M. A. , 139

Flechsig, P. E . , 9(24), 38, 381, 390

Fleischhauer, K. , 30(14), 37 Flood, P., 142(4), 164(4), 267 Florendo, N. T. , 267(11), 270

Fluharty, A. L . , 501(7), 502(2, 8) , 504(6), 505(6),

506

Foiling, A. , 507, 526 Fonberg, E . , 300(9), 307

Forstr0nen, P. F . , 47(48), 92 Fountain, G., Jr . , 308

Fox, C. A. , 191, 199(10), 202, 229(79), 231(79), 232(30-32), 233(30-32, 75), 253(30), 257(31), 262, 263

Fox, J. H., 139

Fox, M. W. , 286(20), 308, 397(8), 398

Fox, S. S., 273(21), 295, 296(21), 298(21), 303, 304(22), 308

Frank, K., 343(4), 370

Freeman, J . A., 336(11), 340

Freeman, T. , 472(11), 477 Frenette, G., 219(9), 220(7-9), 224 Freytag, E . , 123(10), 239 Friar, L . , 178

Friedman, P. A. , 450(5), 451(3-5) , 453(3), 455(4), 457

Fritz, J . E . , 508(7), 516 Fujita, S., 42(32, 33) , 90

Fukuda, A. , 209(62), 227 Fuller, D. R. G. , 198(11), 201, 223(2), 224

Author Index 527

Fulton, J . F . , 273(33), 296(33), 297(33), 308 Funtze, W. , 209(35), 216 Fuortes, M. G. F . , 343(4), 370 Fuxe, K., 191(3, 4) , 201, 205(8, 17), 206(1-4, 8,

18), 208(55), 224, 225, 227, 227, 228(71), 233(35), 251, 252(33), 253(35), 258(5, 33), 259, 260, 261, 262, 265(4, 5) , 270

Gaballah, S., 269(12), 270 Gabbay, K. H., 468(3), 477 Gal, A. E . , 487(37), 488(37), 489(37), 490(37),

492(37), 493(37), 494(37), 495(37), 498 Galambos, R. , 373, 377 Galkin, T. W., 386, 392 Gall, C., 96(13), 99 Galton, F . , 423, 442 Ganote, C. E . , 229(67), 262 Garber, B. B . , 116(22, 23), 222 Garber, H., 434(11), 442 Garcia, J . , 337, 340, 438(10), 440(9, 20), 442 Garelis, E. , 213(19), 225 Garey, L. J . , 4(15), 38, 163(17), 164(17), 267 Garrod, A. E. , 508, 526 Gartner, L. M., 155(18), 267 Gauchy, C., 189(25), 202, 226 Gelehrter, T. D. 509(12), 526 Gerald, P. S. , 455(10), 457 Geschwind, N. , 381, 390 Giarman, N. J . , 251, 263 Gilman, A. G., 267(22), 272 Ginos, J . , 227(20), 260 Gitzelman, R., 468, 477 Glees, P., 47(45), 92 Glendenning, K. K. , 303(55), 309 Globus, A. , 124(11), 239, 343(5), 370 Glowinski, J . , 189(25), 201,226 Goddard, J . , 459(17), 465 Godina, G., 32(16), 38 Golden, G. S., 228(37, 38, 39), 261 Goldensohn, E. S., 125(14), 134, 138, 239 Goldfischer, S., 155(18), 267 Goldman, P. S., 188, 202, 273(23), 296(23),

298(23), 300(23), 308, 381(7, 19, 20, 36), 382, 383(19, 21 , 22, 36), 384(17, 21), 385(21), 386(19-21 , 36), 387(18, 36), 388, 390, 391, 393(10, 11), 394(9), 398

Goldstein, J . L . , 509, 526, 520, 522 Goldstone, M. W., 102(16), 224, 117(30), 222 Golgi, C., 32(17), 38 Gomez, J . A. , 273(24), 283(54), 295(24, 54),

300(24, 54), 305(24), 308, 309 Gonatas, J . , 481(25), 498 Gonatas, N. K., 125, 134, 138, 239

Goodman, D. C., 97, 98 Gormezano, I., 335(13), 337(14, 23), 340, 341 Gottfried, A. W. , 315(13), 332 Goy, R. W. , 384(23), 392 Graham, F . , 317(10), 332 Granner, D. , 509(12), 526 Granoff, D. M., 86(39), 90 Graves, R. , 485(6), 497 Gray, E. G. , 231,262 Gray, J . A. , 380, 384(24), 392 Graystone, J. E . , 86(27), 90 Green, K., 438(10), 442 Greenfield, J . G., 206(20), 225 Greengard, O., 459(14), 465 Greengard, P., 213(41), 226, 267(11, 13, 17, 18),

270, 271 Greenstreet, R. L. , 381(7), 390 Grillo, M. A. , 251 ,262 Grimm, U. , 459(8), 461(7), 465 Grofova, I. , 229(42, 78), 231(78), 232, 263, 251,

252(42), 262, 263 Gruenau, S. P., 173(9), 174(9), 178 Guillery, R. W., 205(21), 225 Gulley, R. L. , 229(43, 44) , 231(43), 232, 251, 262 Gumulka, W. , 206(22), 225 Guth, L. , 211(23), 225 Guyenet, P., 189(25), 202, 226 Gybels, J . , 298, 303(25), 308 Hager, J. L . , 438, 442 Halas, E. S., 319(5), 332 Hall, J. G . , 3 1 5 , 332 Hamberger, B . , 206(4), 224 Hamlyn, L. H., 142(19, 20) , 267 Hanaway, J . , 235, 262 Handa, S . , 497 Hankins, W. G., 440(20), 442 Hanlon, C. R., 209(65), 227 Hara, K., 159(63), 163(63), 269 Harik, S. I. , 295, 308

Harlow, H. F . , 188(13), 202, 380, 392, 393(12, 20), 398

Harlow, M. K., 188(13), 202 Harper, C., 459(5), 465 Harrington, S., 434(11), 442 Harris, S. E . , 501(7), 506 Harvey, J. A. , 205(24, 27), 207(24), 225 Hasselberger, F. X . , 210(44), 211(44), 212(44), 226 Hassler, R. , 189(26, 32, 44), 202, 252(72), 262 Hattori, T., 252, 253(46), 257(46), 258, 262 Hebb, C., 189(14), 202 Hebb, D. O., 339(15), 340 Heber, R. , 434, 442

528 Author Index

Heikkila, R., 189(50), 192(50), 203, 228(83, 84), 233(83, 84) , 253(83-85), 255(86), 256(84), 263, 265(23), 272

Heilman, K. M. , 300(84), 320

Heimer, L . , 190, 202, 206, 225 Helfand, G. D. , 459(3), 462(2, 4) , 465 Heller, A. , 189, 191(16, 40) , 192, 194(23),

195(23), 202, 202, 205(24, 25 , 27, 28, 30), 206, 207(24, 25, 29, 30, 49) , 208(11, 58), 209(38), 210(34), 213(31, 50, 52), 214(25, 29, 30), 225, 226, 227, 227(65), 262

Hem, J . , 142(7), 267

Herman, M. K. , 124, 125, 239 Hernandez-Peon, R., 373, 377

Herndon, R. M. , 55(34), 90

Herrnstein, R., 431, 442 Hersh, L. B . , 477 Hertzig, M. E. , 419(14), 420 Herz, A., 224

Heuser, G., 305(10), 307 Hewer, E. E. , 9(30), 38

Hickman, S., 496(8), 497

Hicks, L . , 373(6), 377 Hicks, S. P., 6(18), 9(18), 15(18), 20(19), 22(18),

38, 52, 60(36), 90, 123(16, 17, 18), 239 Hillarp, N.-A., 191(3, 4 ) , 202, 205(13), 206(1, 7,

13), 224, 225, 227(4, 12, 28), 228(28), 259, 260,

262, 265(4), 270 Hillman, D. E . , 55(43), 92, 232(30), 233(30),

253(30), 262 Himwich, H. E . , 305(86), 320 Hinds, J . W., 9(20), 17(21, 22), 20(21), 24(21,

22), 38

Hinds, P. L. , 17(21), 20(21), 24(21), 38

Hines, H. M., 94(5), 98

Hines, M., 296(60), 309

Hirano, A., 55(37, 38) , 90

Hirosawa, K., 229(47), 262 Hirsh, R., 348(11), 372 His, W. , 3(23), 30, 38

Hjort-Simonsen, A. , 142(22), 267 Ho, P.-L. , 519(4), 522 Hoehn, M. M., 227(95), 264 Hokfelt, T. G. M., 206(4, 18), 224, 225, 227(34),

233(35), 251(36), 253(35, 4 8 - 5 0 ) , 256, 262, 262, 267(14), 272

Hoffman, C., 434(11), 442 Hoffman, H., 94(6), 98

Hoffmann, P. C., 189, 192, 202, 210, 226 Hogarty, P. S., 432(19), 442 Holtzapple, P. G., 473, 477 Holzer, H., 209(35), 226

Honzik, M. P. , 432(13), 442 Hooper, F . H., 384(27), 392

Hopewell, J . W. , 86(29), 90, 402(8), 420 Horel, J . A. , 97, 98

Horn, A. S., 255(51), 258(51), 262 Horn, G., 123(19), 134(19), 239

Hornykiewicz, O., 206(10, 36, 37) , 225, 226, 227(11), 260, 265(15), 272

Hoshino, K. , 20(39), 38

Housepian, E. M., 141(49), 142(49), 143(49),

153(49), 160(49), 269 Hovde, C. A. , 283(54), 295(54), 300(54), 309 Howard, E . , 86(39), 90 Howe, R. C., 178

Hrbek, A. , 159(23, 24) , 163(23), 267 Hsia, D. Y . , 459(9), 461(9), 465 Huang, C.-M., 329(16), 332 Huang, C. Y . , 449(6, 7) , 450(7), 457 Hubel, D. H., 4(25, 26, 27), 30(26, 27), 38,

343(17), 372

Hull, C. D. , 192(5, 6, 20-22 , 34, 35), 193(35), 194, 195(23, 35), 196(5, 6, 20-22) , 198, 200(34, 36, 49) , 202, 202, 203, 209(38), 226, 223(2), 224, 225(1), 226, 265(16), 272, 274(44), 302(44), 305(44), 309, 394(19), 397(19), 398

Humphrey, G. L. , 326(6, 7) , 328(7), 329(7), 332 Hunt, J. M. , 189(38), 202 Hunt, J . McV. , 380(28), 382(28), 392 Huttenlocher, P. R. , 123(26), 124, 125(15, 20,

21), 239, 188, 202

Huxley, A. , 437, 442 Hyman, A., 317(17), 332

Inglish, D . , 117(17, 19), 119, 222,

Ingram, W. R. , 205(39), 226 Irwin, D. A. , 273(27), 308

Isaacson, R. L. , 296(66), 298(66), 320, 393(13), 398

Ito, M. , 94(2), 98, 113(13), 224 Iwase, K., 159(63), 163(63), 269 Jackson, R. , 493(31), 498 Jacobsen, H. N. , 316(32), 317(32), 333

Jacobson, S., 9(28), 38 James, D. W. , 117(3), 222 Jankowska, E . , 373, 374, 377 Jarvik, L. F . , 382(29), 384(29), 392 Jatzkewitz, H., 483(28), 486(28), 498 Jeannerod, M., 274(28), 301(28), 302, 308

Jeavons, P. M., 137(22), 239 Jellinek, M. , 209(40, 65), 226, 227 Jencks, W. P., 477 Jenden, D. J . , 189(8), 202 Jenne, B . , 142(7), 267

Author Index 529

Jensen, A. R., 422, 442 Jerfy, A., 505(1), 506 Jerison, H. J . , 93(7), 98

Jervis, G. A. , 445, 457, 508, 526 Joftes, D. L . , 20(19), 38

Johnson, A. B . , 155(18), 267 Johnson, A. L. , 87(21), 89

Johnson, R. E . , 267(7), 268, 269(7), 270

Johnson, R. T. , 20(40), 39

Johnson, T. N. , 301(29), 305, 382(30), 387(30), 392

Johnson, W. G., 483(39), 484(39), 485(9), 486(9, 39), 488(39), 497, 498

Jones, B . E . , 189(25), 202, 226 Jones, E. G., 164(25), 267 Jones, M., 55(38), 90

Jonsson, G., 249, 253(49, 50), 262 Jouvet, M. , 373(3), 377 Jouvet-Mounier, D. , 172(3), 275 Justice, P. , 459(9), 461(9), 465 Kaback, M. M. , 485(10), 497, 505(3), 506 Kado, R. T. , 375, 377 Kahle, K., 381(31), 382(31), 392 Kaiser, G. C., 209(65), 227

Kakolewski, J . W. , 273(27), 305, 384(50, 51), 392 Kalckar, H. M. , 467(6), 468(6), 477 Kallen, R. G., 472(20), 477 Kameyama, Y . , 20(39), 35 Kamin, L. J . , 434, 442

Kandel, E. R. , 336(16), 340

Kanfer, J. N. , 483(13, 15), 497 Kang, E. S., 451(3, 4) , 453(3), 455(4, 10), 457 Karalitzky, A. R., 317(1), 332 Karlberg, P. , 159(23), 163(23), 267 Kataoka, K., 189(26), 202

Kauffman, S. L . , 7(29), 24(29), 35 Kaufman, S., 447(11, 12, 16), 448(14, 17), 449(1,

2 , 6, 7, 14, 15, 17, 18, 19), 450(5, 7, 13, 20) , 451(1, 3 -5 , 19), 453(3), 455(4, 10, 21), 457, 459(10, 12), 461(11, 12), 465

Kawana, E. K. , 233(53), 257(53), 262 Kaye, M. P. , 209(40), 226 Kebabian, J. W. , 213(41), 226, 265(13, 17, 18),

270, 272 Kemp, J . M. , 189(27), 190(27), 191, 192(28-31),

198(28-30), 202, 223, 224, 231(57), 232(54, 55, 57-59) , 233, 234(57), 252, 253(55), 256, 257, 258(57), 262, 266(19-21) , 272, 301(30), 308

Kennard, M. A. , 273(32, 33), 296(33), 297, 305, 305, 394(14), 395

Kenne, M. F. L . , 9(30), 35 Kenney, F. T., 508(7), 526

Kerr, F. W. L . , 97, 95 Kihara, H., 501(7), 502, 504(6), 505(6),

506

Kilham, L . , 55(34), 90

Kim, J. S., 189(26, 32, 44) , 202

Kim, S. U. , 117(37), 222 Kimble, D. P., 273(21), 295(21), 296(21), 298(21),

305(36), 305 Kimura, D. , 382(32, 33), 384, 392 King, F. A., 300(84), 320 Kinoshita, J. H., 467(6), 468(3, 6) , 477 Kirkby, R. J . , 296(35), 297, 305(36), 305 Kirkwood, R., 449(2), 457 Kitsikis, A. , 219(9), 220(7-9) , 224 Kiyono, S., 274(28), 301(28), 302(28), 305 Kjaerheim, A., 142(5), 267 Kleinman, J . , 210(34), 226 Klenk, E . , 481 , 497

Kling, A. , 188(33), 202,393, 394(16, 18), 397(17), 395, 399

Klosovskii, B. N. , 273(37), 300, 305 Knapp, A. , 459(8), 461(7), 465 Knipsel, J. D. , 337(36), 338(36), 342 Knox, W. E . , 459(14), 465 Koch, R., 469(2), 477 Kodman, F . , 315(18), 333 Koelliker, A. , 3(31), 9(31), 35 Koelling, R. , 440(9), 442 Koenig, H., 231 ,262 Kolb, L. C., 302(39), 309

Kolodny, E. H., 483(13-15), 484(14), 486(12), 497

Konorski, J . , 296(43), 309, 439(7), 442 Korey, S., 481(25), 495

Kornblith, C., 348(6, 11), 350(6), 370, 372

Kornguth, S. E . , 47(40), 90

Kostovic, I., 9(37), 35 , 142(30), 149(30), 165

Kovacs, S., 87(21), 59 Kozak, L. P., 469(7, 8) , 477 Krai, P. , 439(30), 442 Krami, M., 468(3), 477 Krauthamer, G. M., 304(38), 309 Kupalov, P. S., 373, 374, 377 Lacote, D. , 172(3), 275 LaDu, B . N. , 461(13), 465, 508, 526 Laemie, L . , 141(26), 265 Lamberg, S. I. , 520(3), 522 Langman, J . , 6(65), 9(65), 15(65), 20(32, 78), 35,

40 Langworthy, O. R., 274(40), 296, 298(40),

302(39), 309 Lannholm, G. V. , 429(17), 442

530 Author Index

Larochelle, L . , 206(5, 56), 224, 227, 227(73),

256(8), 260, 263 Larramendi, L. M. H., 47(41), 90

Larsell, O., 90

Larsson, K. , 191(3, 4) , 202, 206(1-3) , 224, 227(4, 5), 258(5), 259, 265(4, 5) , 270

Lashley, K. S., 339(17), 340, 437, 442 Laster, L . , 508(8), 526 Lauber, S. M., 303(55), 309

Laurell, C. B . , 471(9), 477 Laurendeau, M., 380(40), 392 Laursen, A. M., 273(42), 299(42), 309

Laverty, R. , 206(14), 225 Lawicka, W., 296(43), 309

Leeours, A. R. , 9(80), 40, 381, 392 Ledeen, R. , 482(16), 497 Lee, K. L. , 508(7), 526 Legrand, J . , 87(42), 90

Lenn, N. J . , 262

Lenneberg, E. H., 380, 382(34), 384, 392 Leroy, J . , 485(19), 497

Levine, M. S., 192(34, 35) , 193, 194(23), 195(23, 35), 198(34), 200(34), 202, 202, 209(38), 226, 223(2), 224, 274(44), 302(44), 305(44), 309, 394(19), 397(19), 398

Levine, S., 380(24), 384(24), 387(35), 392 Levitt, D. , 519(4), 522

Levitt, M., 317(28), 318(28), 333

Leyhausen, P., 304(45), 309

Li, S. C , 491(17), 497 Li, Y. T., 491, 497

Lickey, M. E. , 273(21), 295(21), 296(21), 298(21), 308

Lidbrink, P. , 253(50), 262 Lidsky, T. I. , 200(36), 202 Liles, S. L. , 300, 309

Lindenberg, R. , 123(10), 239 Lindsley, D. B . , 180(6), 184, 301(65), 302(68),

320

Lindsley, W. F. B . , 86(26), 90 Lineberger, R. O., 86(28), 90

Linesman, M. A. , 348(7), 350(7), 370 Ling, G., 225(1), 226 Liu, C. N. , 94, 98, 317(28), 318(28), 333 Llinas, R., 55(43), 92, 343(8), 372 Lloyd, L. L. , 315(19), 333 Lloyd, T. , 450(5), 451(5), 457 L0mo, T., 142(2), 267 L0yning, Y . , 142(3), 164(3), 267 Loizou, L. A., 208(42, 43), 226, 228(62, 63),

262 LoPresti, R. W., 178

Lorente, de No, R. , 142(27), 150(27), 164(27), 168

Lowry, O. H., 210(44), 211(44, 54), 212(44), 226, 227

Lucao, P. A. , 189(37), 202

Luciani, L . , 81(44), 92 Lund, J . , 4(33), 38

Luqui, J. J . , 191(10), 199(10), 202

Luse, S. A. , 229(22), 260 Lynch, A. , 86(29), 90, 402(8), 420 Lynch, G. S., 96(1, 10 -14 , 17, 18), 97(12), 98, 99,

189(37), 202

Lytle, L . , 172(4), 178

McAfee, D. A. , 267(13), 270 McCall, R. B . , 432(19), 442 McCaman, R. E . , 189(38), 202 McCandless, D. , 459(5), 465 McConnell, J . A. , 235(45), 262 McCormick, N. , 87(20), 89

McCrady, B . , 87(18), 89

MacDonald, L. R., 275 McDonald, T. , 231(60), 262 Macfarlane, J . W., 432(13), 442 McGee, M. M., 459(14), 465 McGeer, E. G., 208(15), 225 McGeer, P. L . , 208(15), 225, 252(46), 253(46),

257(46), 258(46), 262 McGinnis, J . , 120, 222 McGinty, D. J . , 299(50), 309

McGowan, B . , 438(10), 442 McGowan, B . K., 440(20), 442 Macht, M. B . , 335, 340

McMasters, R. E . , 227(13), 260 McMorris, F. A. , 116, 117(26), 222 Maeda, T., 208(45), 226, 228(64), 262 Magnini, G., 32(34), 38

Magoun, H. W. , 274(47), 296(47), 299(47), 301, 309

Mains, R. E . , 116(24), 222 Makman, M. H., 267(8), 270 Malliani, A. , 192(47), 202 , 224

Manunes, P. , 485(6), 497 Marco, L. A. , 189(50), 192(50), 203, 233(87),

253(87), 257(87), 263, 266(24), 271 Marcus, R. J . , 189, 195, 203, 274(48, 49 , 79 -81) ,

277(49, 80), 282(80), 294(49, 80), 296(81), 297(49, 80), 305(81), 309, 310, 394(27), 395(27, 28), 397(27), 398(28), 399

MareS, P., 159(24), 167 Margolis, G., 55(34), 90 Marin-Padilla, M., 9(35), 32(35), 38, 125(23, 24),

139, 155, 156, 158, 159, 164, 168

Author Index 531

Marsh, J . T . , 373(6), 377 Martin, D. M. , 509(12), 526 Matalon, R. , 520(5), 522

Matthews, D. A. , 96(1, 11), 95, 99

Max, E. E. , 449(7), 450(7), 455(21), 457, 459(12), 461(12), 465

Mayes, J. S., 469, 477 Mazzotta, M., 491(17), 497

Mehler, W. R. , 24(7), 30(7), 37, 191(39, 42) , 195(42), 202

Meister, A. , 450(24), 458, 459(18), 465 Melchior, J. C , 135, 138 Meller, K., 47(45), 92

Mellman, W. J . , 469(17), 470(18), 473(18), 477, 513, 526

Melzack, R., 318 ,333 Mempel, E . , 300(9), 307 Mering, T. A., 335, 342 Merola, L. O., 468(3), 477 Merrill, M. A. , 424, 442 Mettler, C. C , 273(53), 283(53), 295(53), 296(53),

297(53), 299(53), 300(53), 309 Mettler, F. A. , 273(24, 51 , 53, 74), 283(53, 54),

295, 296, 297, 299(51, 53), 300, 305, 308, 309, 310

Meulders, M., 298(25), 303(25), 308 Meyer, D. R., 303(55), 309 Meyer, P. M., 303(55), 309

Meynert, T. Von Gehirne der Saugethiere, 11, 12, 38

Miale, I. L . , 17(67), 40, 42(46), 92 Miller, E. A., 381(36), 383(36), 386(36), 387(36),

392

Miller, H. R. , 274(70), 283(70), 303(70), 320 Miller, L. R. , 469, 477 Miller, R. W. , 415(15), 420 Mills, J . A. , 296(88), 305(88), 312 Miltoma, C., 447(22), 450, 458 Minchin Clark, H. G. , 472(11), 477 Minsky, M., 338(19), 342

Mishkin, M. , 296(5), 298(5), 301(5, 29), 307, 308, 382(30), 387(30), 391, 393(11), 394(2), 398

Mitcham, J . C , 273(56), 305(56), 309 Model, P. G., 117(4), 222 Moldave, K. , 450(24), 458, 459(18), 465 Molinoff, P. B . , 206(59), 227 Molliver, M. E . , 9(37), 38, 142(30), 149(30), 168 Molson, J . , 117(19), 119(19), 222 Montplaisir, J . , 348(9), 361(9), 372 Moorcroft, W. A. , 172(4), 275 Moore, C. L. , 155(18), 267

Moore, R. Y . , 189(18, 19, 40), 191(40), 192(40),

202, 202, 205(24, 27, 28, 30, 47) , 206(26, 28, 30-32 , 46, 48-52) , 207(24, 29, 30, 47, 49), 213(31, 47, 50, 52), 214(29, 30), 225, 226, 227, 227(65), 262

Morest, D. K. , 24, 30(38), 38, 93, 99, 142(32), 143, 147(31, 32, 33), 168

Mori, S., 231(66), 233(66), 251 , 252(66), 262 Morrell, F . , 159(61), 163(61), 269 Morris, J. E . , 117(27,28), 119(27, 28) , 222 Morris, P. L. , 295, 308

Moscona, A. A. , 102(14), 224, 116(22,23) , 117(27, 28), 119(27, 28), 222

Moser, H. W. , 134(2), 137(27), 235, 239, 501(4),

506 Moses, H. L. , 229(67), 262 Mosko, S., 96(11, 12), 97(12), 99 Mouret, J . , 274(28), 301(28), 302(28), 305 Mugnaini, E . , 47(47, 48), 92 Murakami, V . , 20(39), 35 Mytilineou, C , 189(50), 192(50), 203, 228(83, 84,

89), 233(83, 84), 243(88, 89), 253(17, 83 -85) , 255(86), 256(84), 260, 263, 265(23), 272

Nadler, H. L. , 471, 477, 515, 526 Nafstad, P. H. J . , 143(34), 265 Naifeh, K., 175(10), 275 Nakamura, T., 42(33), 90 Narayan, O., 20(40), 39 Nauta, W. J . H., 191(42), 195(42), 202, 205(53),

227, 283(7), 307, 387(37), 392 Navon, R., 487(18), 497 Neff, N. H., 213(19), 225 Neilson, D. R., J r . , 338(26-28), 342 Nellhaus, G., 231(60), 262

Nelson, P. G., 102(15, 16), 224, 116, 117(26, 30), 222

Nelson, S. R. , 211(54), 227 Netsky, M. G., 235(45), 262 Neufeld, E . , 496(8), 497 Neville, H. W. , 86(49), 92 Ng, W. G., 474(13), 475(13), 477 Nichols, K., 459(5), 465 Nicholson, C , 343(8), 372 Nicholson, J . L . , 87, 88, 92, 123(25), 239 Niemer, W. T. , 276, 278, 320 Nigh, C. A. , 209(40), 226 Nilsson, O., 206(18), 225, 227(34), 233(35),

251(36), 253(35), 262 Nitsch-Hassler, C , 189(44), 202 Noback, C. R. , 141(49), 142(49), 143(49),

153(49), 160(49), 269 Nobin, A. , 228(68), 262 Noble, A. , 416, 420

532 Author Index

Nonneman, A. J . , 393(13), 398

Norman, J . L . , 173(5), 178

Norman, R. J . , 175(10), 178, 274(57), 296(57), 309, 320, 322, 323, 325, 326(8), 332, 333

Norton, W. T. , 155(18), 167

Nowakowski, R. S., 6, 7(41), 39

Obata, K. , 189(43), 202 O'Brien, D. , 86(26), 90

O'Brien, E. D. , 20(19), 38, 123(18), 139

O'Brien, J . H., 303(22), 304(22), 308

O'Brien, J . S., 484, 485(19), 487(44), 491(45), 497,

498, 499, 501(5), 506 Oda, M. A. S., 123(26), 139

Oden, M. H., 430(26), 442

O'Flynn, M. E . , 459(9), 461(9), 465

Okada, S., 484, 485(19), 491(45), 497, 498, 499,

501(5), 506

Okada, Y . , 189(32, 44), 202 Okuma, T., 305(10), 307

Olds, J . , 348(2, 3, 6, 7, 9, 10, 11, 15, 16), 350(6, 7), 360(2), 361(9), 370, 371

Olds, M. E . , 348(12), 371

Olivier, A. , 206(56), 217, 227(73), 232(69), 262, 263

Olmstead, C. E . , 274(81), 296(81), 305(81), 310,

395(28), 398(28), 399

Olson, L . , 191(4), 201, 206(3), 208(55), 224, 227,

227(5), 258(5), 259, 265(5), 270 Olson, M. I. , 117, 222 Olsson, T. , 159(23), 163(23), 267 Oppenheimer, M. J . , 274(70), 283(70), 303(70),

320 Orr, A., 296(87), 322 Orth, R. , 491(17), 497 Owens, J . , 337(40), 338(40), 342 Padeh, B . , 487(18), 497 Page, E. B . , 434, 442 Palay, S. L . , 251 ,262 Pallie, W. , 385, 392

Papavasiliou, P. S., 227(20), 260 Papert, S., 338(19), 342

Pappas, G. D. , 141(35, 46, 55, 56, 62), 142(55, 62), 151(46, 55), 153(46, 55), 155(62), 168, 169,

233(2), 253(2), 259, 266(2), 270

Parent, A. , 206(5), 224, 232(69), 256(8), 260,

262

Parizek, J . , 252(72), 262 Parker, B . , 317(17), 332 Parks, T., 96(11, 12), 97(12), 99 Parmeggiani, P. L. , 296(58), 303(58), 309

Passonneau, J. V . , 210(44), 211(44, 54), 212(44), 226, 227

Patterson, P. H., 116(24), 222 Pavlov, I. P., 336, 339(20), 342, 438, 442 Peacock, J . H., 102(15, 16), 224, 117(30), 222 Pentchev, P. G., 479(40), 498

Percy, A. K. , 505(3), 506 Persson, H. E. , 143(36), 160(36), 168

Peter, P., 227(14), 2 5 6 , 2 6 0 Peters, A., 29(42), 30(42, 43), 32(42), 39, 412(4),

420

Peterson, E. R. , 117(10, 13), 222

Peterson, G. R. , 117(40), 222 Petsche, H., 30(14), 37

Petzold, G. L. , 213(41), 226, 267(18), 272 Phillips, A. G., 384(38), 392 Piaget, J . , 380,392 Pinard, A. , 380(40), 392 Pirson, R. A. , 462(1), 464

Pirson, W. D. , 459(3), 462(4), 465

Poirier, L. J . , 206(5, 56, 57), 224, 227, 227(73, 74), 232(69), 256(8), 260, 262, 263

Poliakov, G. I . , 9(44, 45 , 46), 39, 142(37), 168

Popoff, C., 269(12), 270 Porcher, W. , 206(32), 208(58), 226, 227 Porter, M. T. , 501(7), 502(2, 8) , 504(6), 505(6),

506

Potter, V. R., 508(2), 526

Powell, T. P. S. , 142(53), 163(17), 164(17, 25), 267, 269, 189(27), 190(27), 191, 192(28-31), 198(28-30), 202, 223, 224, 231(57), 232(55, 57 -59 ) , 233, 234(57), 252, 253(55), 256, 257, 258(57), 262, 266(19-21) , 272, 301(11, 30), 307,

308

Precht, W. , 55(43), 92, 189(45), 202 Prevelic, S. , 141(47), 268 Prensky, A. L . , 137(27), 239

Price, D. D. , 192(6, 21), 196(6, 21), 197(21), 198(21), 202, 224

Price, R. , 496(21), 498

Proctor, F . , 273(72), 297(72), 320 Puck, T. T. , 459(17), 465 Pudritz, R. E. , 208(15), 225 Purpura, D. P. , 141(26, 38-50 , 55-57, 60, 62),

142(49, 55, 62), 143(38, 40, 44, 49) , 149(62), 151(46), 153(46, 49, 55), 155(62), 160(49), 165(43), 168, 169, 179, 181(5), 183, 184, 188, 192(47), 202, 224

Quirk, J . M. , 483(13, 15), 484(22), 487(22, 37), 488(37), 489(37), 490(37), 492(37), 493(37), 494(37), 495(37), 497, 498

Rabin, A., 223, 224 Rabinowicz, T., 160, 269, 381(41), 392 Rachmeler, M. , 471, 477, 515, 526

Author Index 533

Rafols, J . A., 191(10), 199(10), 201, 232(31, 32),

233(31, 32, 75), 257(31), 261, 263 Raiborn, C. W. , Jr. , 102(21), 111(21), 124, 116(38,

39), 222 Raiha, N. C. R. , 461(15), 465 Raines, C. S. , 120(5), 222 Raisler, R. L. , 393(20), 395 Raisman, G., 142(53), 269, 394(42), 392

Rakic, L . , 102(1), 224

Rakic, P., 4(49, 50, 51 , 52), 6, 7(41, 52), 9, 10(52), 13(52), 14, 15(49, 52), 18, 20(47, 48, 49, 53, 68), 23, 24, 25, 27, 28, 29, 30(47), 31(52), 32(47, 49, 52, 63, 64), 34(48, 49, 64, 69), 39, 40, 47(53, 54), 55(55), 92, 381(43), 392

Rakin, L. L. , 116(2), 222 Rail, T. W. , 267(22), 272 Ramirez del Angel, A. , 206(22), 225 Ramon y Cajal, S., 9(56), 20(55), 32, 34(56), 39,

46, 92

Randt, C. T. , 273(5), 303(5), 307

Ranson, S. W. , 274(47), 296(47), 299(47), 301, 309

Rapin, I. , 155(18), 267

Regan, D. , 326(22), 329(22), 333 Reid, M. J . , 315(19), 333

Reinius, S., 251(36), 262

Reinoso-Suarez, F . , 278, 309 Reis, D. J . , 206(59), 227 Renshaw, B . , 338(21), 341 Rescorla, A. , 346 ,372 Retzius, G., 32(57, 58), 39

Richards, J . G. , 253(76), 263

Richardson, K. C , 251, 263

Richardson, S. A. , 419(14, 16), 420 Richman, D. P., 36(59), 39

Richter, C. P., 274(40), 296, 298(40), 309 Riesen, A. H., 124(4), 138, 318(23), 333

Rinvik, E . , 229(42, 78), 231(78), 232, 251, 252(42), 262, 263

Rio Hortega, P. , 32, 39

Ritch, R. H., 155(18), 267 Roberge, A. G. , 219(9), 220(7-9) , 224 Robinson, D. , 484(23), 495(5), 496(5), 497, 498

Robison, G. A., 209(60), 227 Robson, K., 317(28), 318(18), 333 Rocha-Miranda, C. E . , 192(48), 203

Rogers, A. W. , 6(6), 15(6), 37 Rogers, S., 473, 477 Romanovskaya, E. A. , 273(61), 309

Rose, G., 96(13), 99

Rose, G. H., 173(6, 9), 174(7-9), 175(10), 178, 180(6), 184

Rose, J . E . , 381(44), 392

Rosen, J . , 309

Rosenblatt, F . , 338(22), 342

Rosengren, E . , 206(6), 224 Rosenthal, D. , 435(23), 442

Rosenzweig, M. R. , 387(45), 392 Rosman, N. P., 137(28), 239 Ross, R. , 393(1), 397(1), 398

Rossignol, S. , 156(13), 163(13), 164(13), 267

Rosvold, H. E . , 273(23, 63), 296(5, 23, 63), 298(5, 23, 63), 300(23), 301(5, 29, 63), 307, 308,

309, 381(7, 36), 382(30), 383(22, 36), 386, 387(30, 36), 390, 391, 393(10, 11), 394(2, 21), 398

Roth, R. H., 207(64), 227 Rowe, R. D. , 86(27), 90 Rowland, V. , 273(5), 303(5), 307

Roy, A. B . , 505(1), 506 Ruch, T. L . , 296(64), 320 Ruddle, F. H., 116, 117(26), 222 Ruffett, T. L . , 17(22), 24(22), 38

Russell, T. R. , 269(6), 270 Sachs, B . , 480, 498

Saiga, M., 113(13), 124 Salmoiraghi, G. C , 224

Salsman, K., 482(16), 497 Samanin, R. , 206(22), 225 Samuels, H. H., 509(12), 526 Samuels, S., 481(25), 498

Sandhoff, K., 482(29), 483(28), 485(26, 29), 486(28, 29), 487(29), 489(27), 491(29), 498

Sands, J . , 402(11), 403(9, 10, 12), 404(9, 11, 12), 407-411(12), 413-414(12), 420

Santini, M. , 141(47), 265 Satz, P . , 3 8 0 , 385(46), 392 Sauer, F. C , 17(61), 24(61), 39 Sauer, M. E . , 17(62), 39

Saxon, S. V . , 317(24, 25), 333 Sayre, D. F . , 249, 260

Sayre, E. P., 18(54), 27(54), 28(54), 29(54), 39 Scarff, T. , 141(50), 169, 181(5), 254 Schade, J . P., 123(29), 126(29), 137(29), 239 Schapiro, S., 123(30), 239 Scharlock, D. P. , 393(22), 399 Schear, M. J . , 227(95), 264 Scheibel, A. B . , 124(11), 137(11), 239, 240

147(54), 164(54), 169, 343(5), 370 Scheibel, M. E . , 147(54), 164(54), 269 Scherrer, H., 373(3), 377 Schlitz, K. A. , 188(13), 202, 393(12), 395 Schlag-Rey, M. , 301(65), 320 Schlenzka, K. , 459(8), 465

534 Author Index

Schmaltz, L. W. , 296(66), 298(66), 320, 393(13), 398

Schmechel, D. E . , 24, 32(63, 64), 33(64), 34(64), 40

Schmidt, S. Y . , 267, 270

Schneck, L . , 485(30), 498

Schneider, G. E . , 393(23), 399

Schneiderman, N . , 337(23), 341

Schneirla, T. C , 304(67), 310

Schrader, W. B . , 429(17), 442 Schramm, L. , 397(5), 398

Schramm, S., 319(5), 332 Schrier, B . K. , 117(31, 35 , 41) , 222 Schuffman, S. S. , 229(67), 262 Schulz, D. W. , 210(44), 211(44, 54), 212(44), 226,

227

Schwafel, J . A. , 274(57), 296(57), 309, 320(21), 322(21), 323(21), 325(21), 326(8), 332. , 333

Schwartz, I. R. , 141(55), 142(55), 151(55), 153(55), 269

Schwyn, R. C , 229(79), 231(79), 232(30, 31) ,

233(30, 31), 253(30), 257(31), 262, 263 Scott, B . , 102(18), 111(18), 224 Scott, G., 47(40), 90 Sechzer, J . A. , 317(26), 333 Seeds, N. W. , 116(32), 117(32, 33, 34), 222 Seegmiller, J . E . , 508(8), 526 Seelbach, S. , 439(3), 442

Segal, M. , 348(11, 14 -16) , 365(14), 366(14), 372

Segal, S., 473, 477

Segrest, J . , 493(31), 498

Seiden, L. S., 206(31, 32) , 213(31), 226

Seiger, A., 208(55), 227, 228(70), 262

Selfridge, O. G., 338(24), 342 Seligman, M. E. P. , 438, 442 Sestauj, K., 468(3), 477

Shapiro, D. L . , 117(35), 222 Shaskan, E. G., 249, 255(80), 258(80), 263 Shenkin, H. A. , 296(64), 320 Shiffer, L . , 227(21), 260 Shimada, M., 6(65), 9(65), 15(65), 20(32, 78), 38,

40, 42(33), 90

Shimazu, T. , 209(61, 62), 227 Shofer, R. J . , 141(48-50, 56, 57), 142(49),

143(49), 153(49), 160(49), 269, 181(5), 184

Sholl, D. A. , 126, 240, 164(58), 269 Shuster, L . , 117(40), 222 Sidbury, J . B . , Jr. , 469(15), 477 Sidman, R. L. , 6(2, 66), 7(2, 66), 9, 15(2, 10),

17(67), 18(54), 20(10, 53, 68), 27(54), 28(54), 29(54), 31(66), 34(69), 37, 39, 40, 42(46),

55(55), 92, 102(8), 224, 116(15, 36), 222, 222, 381(12, 47) , 390, 392

Siebert, W. M. , 339(25), 342 Silver, A. , 189(14), 202 Simard, H., 232(69), 262 Simard-Duquesne, N. , 468(3), 477 Singh, P. , 206(56), 227, 227(73), 263 Skinner, J . E . , 302(68), 320 Sluckin, W. , 387(48), 392 Smart, J . L . , 402(13), 420 Snider, R. S., 276, 278, 320 Snyder, S. H., 249, 255(51, 80) , 258(51, 80), 262,

263

Soltysik, S., 200(49), 203 , 373 , 374, 377 Sotelo, C., 252, 263

Sourkes, T. L. , 206(57), 227, 227(74), 263 Spence, S., 308

Spencer, J. W. , 173(9), 174(9), 178

Spencer, W. A. , 336(16), 338(26-28), 340, 342

Spiegel, E. A. , 274(70), 283(70), 303(70), 320 Spiro, A. J . , 155(18), 267 Spooner, B . S. , 243(96), 264 Sprague, J . M. , 317, 318, 333 Srivastava, S., 495(32), 496(32), 498

Stanfield, B . , 96(13, 14), 98(14), 99 Steck, A., 227(20), 260

Stein, D. G., 273(19), 308, 309

Stellar, E . , 317(27, 28) , 318(27, 28), 333

Stensaas, L. J . , 9(70), 18(54), 24(72), 27(54), 28(54), 29(54), 32, 39, 40

Stensaas, S. S., 24(72), 32(72), 40 Stepien, I., 283(71), 305(71), 320 Stepien, L . , 283(71), 305(71), 320 Sterman, M. B . , 178, 299(50), 309

Stern, J . , 20(12), 36(12), 37 Stevens, C. F . , 336(29), 342 Steward, O., 96(17, 18), 98(17, 18), 99 Stewart, R. M., 36(59), 39 Stiles, C. D. , 508(7), 526 Stirling, J. L . , 484(23), 498

Stokes, L. P., 386, 392 Stone, J . , 336(11), 340

Strominger, N. L. , 191(9), 192(9), 202, 393(22), 399

Strop, M., 73(12), 77, 78, 79, 89

Stuart, D. K. , 348(3), 370 Sudarshan, K. , 68(19), 69(19), 70, 71 , 72, 73, 74,

75, 87(20), 89

Sutherland, E. W., 209(60), 227 Suzuki, K., 481(38), 498

Svennerholm, L . , 482(33), 498

Sychowa, B . , 283(71), 305(71), 320

Author Index 535

Szabo, J . , 227(82), 263 Szentagothai, J . , 94(2), 95, 161, 163(59, 164(59),

165(59), 169 Szwejkowska, G. , 439(7), 442 Tagini, G., 113(19, 20) , 224 Tallman, J . F . , 479(35, 36, 40) , 481(38), 483, 484,

485(35), 486(39), 487(22, 37) , 488, 489, 490, 492, 493, 494, 495, 498

Tappel, A. L . , 231(15), 260 Tatum, E. L. , 508, 526 Tauc, L . , 338(30), 342 Tay, W . , 4 8 0 , 498 Taylor, D. , 96(1), 98

Tedesco, T. A. , 469(17), 470(16, 18), 471(16), 472(20), 473(18), 477, 513, 526

Teichmann, W. , 461(7), 465 Tennyson, V. M. , 189, 192, 203, 228(83, 84, 89),

229(25), 231(25), 233(83, 84, 87) , 243(88, 89), 253(83-35, 87) , 255(86), 256(84), 257(87), 260, 263, 265, 266, 272

Terman, L. M. , 424, 430(26), 442, 442 Terry, R. D. , 481(25, 42) , 498 Teuber, H. L . , 179, 254, 273(72), 297, 320 Textor, S. C., 462(1), 464 Thatcher, R. W. , 141(60), 269 Thieme, G., 205(13), 206(13), 225, 227(28),

228(28), 262 Thoenen, H., 253(90, 91), 263 Thomas, R. K. , Jr . , 273(56), 305(56), 309 Thompson, C. I . , 188(13), 202 Thompson, E. B . , 509(12), 526 Thompson, R. F . , 338(26-28), 342, 393(4), 395 Thompson, R. L. , 273(24, 73, 74), 283(54),

295(24, 54, 73, 74), 296, 300(24, 54), 305(24, 74),308, 309, 310

Thompson, W. J . , 269(6, 25), 270, 272 Thorndike, E. L . , 374, 377 Thurston, D. , 317(10), 332 Thurston, J . S., 20(19), 35 Tilney, F . , 40 Tischer, W. , 459(8), 465 Tizard, J . , 419(14), 420 Tobin, A., 188(51), 203 Tomkins, G. M. , 509, 526 Toon, R., 210(34), 226 Torp, A., 205(13), 206(13), 225, 227(28), 228(28),

262 Tourian, A. , 459(17), 462(16), 465 Towbin, A. , 315(29), 333, 385(49), 392 Trammell, J . , 502(8), 506 Tranzer, J . P., 253(90, 91), 263 Tryon, R. C., 422, 436, 442

Tsang, Y . -C . , 393(24), 399 Tsunekawa, T. , 209(65), 227 Tucker, T. J . , 188(33), 202 , 393(25), 394(18),

397(17), 395, 399 Tunnicliff, G. , 117(37), 222 Udenfriend, S. , 450(23), 455 Umezaki, H., 159(61), 163(61), 269 Ungerstedt, U. , 191(4), 202, 205(63), 206(3), 224,

227, 227(5), 228(92), 258(5), 259, 263, 265(5), 270

Valenstein, E. S. , 384(50, 51), 392 Valsamis, M. P. , 155(18), 267 Valverde, F . , 4(75), 40, 124(33), 132(33), 140 Valzelli, L . , 206(22), 225 Van der Loos, H., 9(37), 35 , 142(30), 149(30),

265 van Groenigen, W. B . , 123(29), 126(29), 137(29),

239 Van Norstrand, G. K. , 380, 385(46), 392 Van Orden, L. S., Il l , 251 , 263 Van Woert, M. H., 227(21), 260 Varma, D. S . , 468(3), 477 Varon, S., 102(21), 111(21), 224, 116(38, 39), 222 Vassilevsky, N. N. , 337(38), 339(38), 342 Vatter, A. E . , 117(34), 222 Veath, M. L. , 485(19), 497, 501(5), 506 Veneziano, P. , 17(77), 40 Vernon, L. , 192(5, 6, 22) , 196(5, 6, 22) , 202, 224 Verpoorte, J . , 491(43), 493(43), 499 Vidgoff, J . , 487(44), 497, 499 Vignal, W. , 3(76), 9(76), 32(76), 40 Villablanca, J . R. , 189, 192(35), 193(35), 195(35),

202, 203, 273(75), 274(44, 48, 49 , 57, 77, 79 -81) , 276(75), 277(49, 80), 282(80), 294(49, 80), 295(75), 296(57,81) , 297(49, 7 8 , 8 0 ) , 299(76, 78), 302(44), 304(78), 305(44, 81), 309, 320, 320(21), 322(21), 323(21), 325(21), 326(8), 332, 333, 394(27), 395(27, 28) , 397(27), 398(28), 399

Villalba, M. , 487(37), 488(37), 489(37), 490(37), 492(37), 493(37), 494(37), 495(37),495

Voeller, K., 141(62), 142(62), 149(62), 155(62), 269

Volk, B . W. , 483(14), 484(14), 485(30), 497, 495 von Reuss, A. , 467, 477 Wagner, A. , 189(26), 202 Walbran, B . , 273(19), 305 Walker, B . E . , 17(62), 39 Wallace, H. W. , 450(24), 455, 459(18), 465 Wallace, R. B . , 72(57, 58), 76, 92 Wallach, L . , 380(52), 392 Walsh, G. O., 125(13), 239 Walsh, T. M., 30(43), 39

536 Author Index

Walters, J . R., 207(64), 217 Wan, C , 491(17), 497 Ward, A. A., Jr . , 137, 140, 164(65), 169

Warren, H. B . , 302(82), 310 Warren, J. M. , 302, 310

Wassle, W. , 482(29), 485(29), 486(29), 487(29), 491(29), 498

Watanabe, K. , 159(63), 163(63), 169 Watkins, E. S., 298, 310 Watson, J. D. , 508, 516 Watson, P. K. , 211(23), 215 Watson, R. T., 300(84), 310 Watterson, R. L . , 17(77), 40 Webb, E. C , 462(6), 465 Webster, K. E . , 190(53), 203 Webster, W., 20(78), 40 Wechsler, D. , 430(29), 442 Weinstein, S., 179, 184 Weinzimer, S. H., 143(64), 269 Weiss, M., 481(25), 498 Weiss, P., 20(79), 40 Weiss, R., 481(42), 498 Wells, W. W., 469(7, 8) , 477 Wender, P. , 385(53), 392 Wenger, D. , 491(45), 499 Werner, I., 117(40), 222 Wessells, N. K., 243(96), 264 West, J . , 98(19), 99

Westrum, L. E. , 137, 240, 164(65), 269 Wheatley, M. D. , 274(85), 296(85), 301, 320 White, L. E . , 137, 240, 164(65), 269 White, R. P., 305(86), 320 Whittaker, V. P. D. , 253(94), 264 Whittier, J . R., 296(87), 322

Wiesel, T. N. , 4(25, 26, 27), 30(26, 27), 38, 343(17), 372

Wilcott, R. C , 180(3), 184 Wilcoxon, H., 439(30), 442 Willman, V. L. , 209(65), 227

Wilson, P. P. , 173(5), 178 Wilson, R. D. , 190(15), 202 Wilson, S. H., 117(41), 222

Windle, W. F . , 316, 317(30-32), 332, 333, Winick, M. , 55(59, 60), 86(31), 90, 91, 137(28),

240, 410(17), 416, 420 Winocur, C , 296(88), 305(88), 322 Winters, W. D. , 375, 377 Wisniewski, H. K., 155(18), 267 Witelson, S. F . , 385, 392 Wittowski, W., 30(14), 37 Wong, D. L . , 102(1), 214, 116(2), 221 Wong, S. L. R. , 206(52), 213(52), 217 Wood, R. L. , 229(44), 251, 261 Woody, C. D. , 336(7, 34, 35), 337(9, 3 2 - 3 9 , 40),

338(32, 3 4 - 3 6 , 39, 40), 339(9, 3 1 - 3 4 , 37, 38), 340, 341

Worden, F. G., 373(6), 377 Wright, K. A. , 52, 53(14, 15), 55, 57, 89 Wu, J . , 472(20), 477 Wyers, E. J . , 305(10), 307 Yahr, M. D. , 227(95), 264 Yakovlev, P. I. , 9(80), 40, 381, 392 Yakovleva, V. V . , 373, 377 Yamada, K. M. , 243(96), 264 Yamadori, T., 381(56), 392 Yamakama, T., 497

Yarowsky, P. J . , 337(39, 40), 338(39, 40), 341 York, D . , 2 2 4 Yoshida, M., 189(43, 45), 202, 223, 224 Zannoni, V. G., 461(13), 465, 508(8), 516 Zanocco, G., 296(58), 303(58), 309 Zeiger, R. , 485(10), 497 Zeki, S. M., 4(81), 40 Zeman, W., 231(97), 264 Zernicki, B . , 304 ,311 Zieglgansberger, W. , 224 Zimmer, J . , 96(20), 99, 142(66), 169 Zucker, I., 303(90), 311

SUBJECT INDEX

A Acetylcholine, 189-190, 205, 225-226, 253, 257 Acetylcholinesterase, 95-98 , 228, 232 Action potentials, 107, 111-113 Adenylate cyclase, 209-213, 267, 270 Alcaptonuria, 508 Alternation, spatial, 220-221 y-Aminobutyric acid (GABA), 189, 205, 228, 232,

253

Amygdala, 327

Anoxia, neonatal, 123, 137, 315-317, 319, 331 Arylsulfatase, 501-503, 505 Astrocytes, 30, 33, 37

Auditory system, 316-317, 326-331 , 353-354, 358-359, 363

cochlear microphonic, 329

cochlear nucleus, 316, 326, 329, 347, 354

inferior colliculus, 316, 326, 329, 355-359, 361 medial geniculate, 353, 360 trapezoid body, 359, 364

Autopsy, 123, 126, 143 Autoradiography, 6 - 7 , 9 - 2 2 , 24, 3 0 - 3 1 , 36, 4 2 -

45, 5 1 - 5 3 , 55, 57, 60, 6 5 - 6 8 , 7 3 - 8 1 , 8 5 - 8 6 Axonal growth, 123-125 , 134

B

Basal ganglia, 187-190, 192, 200, 205-206, 316 caudate nucleus, 187-194, 196, 205, 207-213,

219, 225-226 , 2 3 2 - 2 3 3 , 240, 247 -249 ,

252 -253 , 2 6 5 - 2 6 9 , 2 7 3 - 2 7 5 , 279, 3 2 1 -322, 351, 375, 393 -395

globus pallidus, 187, 190-192 , 196, 198-200 , 233, 375

neostriatum, 209, 228, 2 3 2 - 2 3 3 , 255-256 nigroneostriatal pathway, 189, 192, 194-195 ,

207 -210 , 213 -214 , 228, 232, 239, 2 5 6 - 2 5 8 , 265 -266

putamen, 187, 190-192 , 207, 232, 240, 245, 247, 249, 265

striatum, 1 8 8 - 1 9 1 , 195, 197-200 substantia nigra, 187, 189-192 , 198, 200, 2 0 6 -

207, 2 2 8 - 2 3 5 , 240, 243, 2 5 1 - 2 5 2 , 258, 265, 270

Basket plexus, 151, 153, 164 Behavior, 171-173 , 294, 300, 302 -304 , 306,

3 1 7 - 3 1 9 , 327, 3 4 4 - 3 4 5 , 3 4 9 - 3 5 3 , 365 -368 , 383, 389, 393-396

arousal, 277, 296-297 compulsory approach, 283-287, 298-306 contact placing reaction, 287, 293, 302, 305 exaggerated treading, 286, 300, 304 hyperactivity, 286, 292, 294-297 motor activity, 276-277, 282, 287, 292, 296,

301

neural substrates, 379-382, 384 orienting reaction, 276, 307

postural abnormalities 276, 287, 305 vocalization, 278, 283, 286, 299

Biopsies, 126-128, 133 Blink reflex, 320-325 Body weight, 405, 407-408

Brain growth, 403-404 , 406-407 , 410, 412-413 , 4 1 7 ^ 1 9

Brain edema, 413-414

Brainstem, 190-192 , 235, 239-240 , 316-329 , 338, 349-350, 358-359, 361

Brain weight, 406-407

c

Carbohydrate metabolism, 214, 226 Cataract, 468

537

538 Subject Index

Catecholamine metabolism, 226 Cell multiplication, 4 0 3 ^ 0 5 , 408-410, 412-417,

419

Cerebellum, 42, 52-55 , 81-34 , 94, 40^-410

basket cells, 42-47, 55, 68 cortex, 42, 47, 52, 63, 68

development, 4 1 , 53 , 68, 73 -81 , 85 , 95 germinal layer, 42, 46-47, 52-53 , 60, 86-88

granule cells, 42-46 , 50-68 , 81 , 88 migration of cells, 42 -45 , 47 -50 , 60 molecular layer, 42-47 , 50, 60-65

mossy fibers, 50, 55, 60, 65 neurogenesis, 42-47, 51 , 55-60

parallel fibers, 46-50 , 55, 60, 65

Purkinje cell, 42, 47-50 , 53, 55, 63-68 regenerating cells, 53, 60, 63 , 68

stellate cells, 42, 55 vermis, 45 , 63 , 68

Cerebral edema, 413 Cerebrum, 3-4 , 7-15, 17, 19, 22-24, 30-32 , 36,

94, 103, 109, 111, 125, 129-133 , 137, 147, 155, 163, 165, 190, 192, 195, 197, 363-364 , 368

Cholesterol, 412

Choline acetyltransferase, 225-226

Cholinesterase, 189 Chromosomal anomalies, 415 Cognitive development, 379-381 , 383-385 Conditioning, 174-175, 335-340, 345-347, 354,

360-361, 365-367 Cortical development, 381, 387 Cortical layers, 9-10, 15 Corticogenesis, 3 -6 , 22-30 Cyclic AMP, 209, 213, 267-269 Cystathionuria, 511

D

Deafness, 315, 317-319, 331 Decerebration, 321-328 Dendrites, 47, 50 -51 , 55, 63, 123-137, 141-150,

153, 155-159 , 161-164 , 179, 191, 196, 229, 2 3 2 - 2 3 5 , 266, 4 0 2 - 4 0 3 , 405, 413, 416

Demethylimipramine, 255 Deoxyribonucleic acid (DNA), 9, 404, 410, 412,

4 1 5 - 4 1 6 , 508 Developmental retardation, 155-156 , 159, 163,

165 Dopamine, 1 8 9 - 1 9 1 , 194, 2 0 5 - 2 1 4 , 2 1 9 - 2 2 8 ,

239-240 , 2 4 5 - 2 5 3 , 2 5 5 - 2 5 8 , 265-267 , 270 Dorsal root ganglion, 103, 110-113 Down's syndrome, 142, 153-159, 435, 514

E Electroencephalogram (EEG), 134, 295, 303,

305-306

Electron microscopy, 42, 46, 53

Electromyogram (EMG), 321, 324

Encephalopathies, 137

Entorhinal lesions, 95-98

Environmental factors, 402, 424, 433-437 , 520

Enzyme deficiencies, 501, 508-514

Evoked potentials, 142, 159-163, 165, 173-181,

183

Excitation, 182, 331, 346, 364, 367

F Fabry's disease, 512, 514 False transmitters, 253, 257-258 Fetal growth, 403, 405-406, 408, 413, 415 Filopodia, 145, 156, 158, 161, 164

G

Galactosemia, 467 -476 , 512 -513 , 515

GABA, see -y-Aminobutyrie acid

Gangliosides, 479, 4 8 2 - 4 8 3 , 487

G A 2 , 482, 485-486

G M 2 , 479-493 , 496

Gaucher's disease, 482

Genetics, 113, 423, 433-437 , 439, 453, 456, 461,

467, 4 7 0 - 4 7 1 , 508 -510 , 514 -515 , 517, 520,

Glia, 1 8 - 2 0 , 2 4 - 3 6 , 50, 55, 93, 103, 404, 412,

414, 416

Glucocorticoids, 117

Glycerolphosphate dehydrogenase, 117-120

Glycogen, 209-213

Glycosphingolipids, 479, 486-487

Growth rate, 402, 405 -407 , 410, 412, 417 -419

H

Habituation, 327-328, 331, 361, 364 Hexosaminidases, 4 8 4 - 4 8 5 , 4 8 7 - 4 8 8 , 491,

493-497 Hippocampus, 9 4 - 9 6 , 142-143 , 1 4 5 - 1 5 8 , 1 6 3 -

164, 327, 351, 353, 3 6 5 - 3 6 6 , 3 6 8 - 3 6 9 Hodgkin's disease, 461 Hormones, 51 , 87, 117-120, 123, 134 Human brain, 142-143

fetal 147-148 , 151, 163-164

immature, 142-143, 153, 163-164 Hydroxylation, 206, 228, 253, 447-456 Hypercholesterolemia, 509 Hyperlipidemia, 520

Subject Index 539

Hyperphenylalaninemia, 455-456, 459

Hypothalamus, 240, 255, 349, 358-359

Hypothyroidism, 123, 134, 137

I

Inhibition, 182-183, 189, 194, 196, 198-199, 222-224, 304, 346, 362-363

Intelligence, 422, 427-431 , 436-438, 440 Intelligence tests, 421-435 Interneurons, 191, 196 Iron-enzyme, 449-450 Irradiation, 123, 415

K

Kinase activity, 468-470

Klinefelter's syndrome, 435

L

Lactate, 211 Learning, 173-177, 220, 336, 345, 347-353,

363-365, 369, 384-387, 389 Lesions, 327, 440

of caudate nucleus, 189, 195, 209, 211-213,

278, 2 8 3 - 2 8 7 , 2 9 3 - 3 0 6 , 383, 393 cortical, 188, 2 7 3 - 2 7 5 , 279, 296, 3 0 1 - 3 0 3 , 306,

3 8 2 - 3 8 3 , 385 -387 , 393-397 of internal capsule, 210 of medial forebrain bundle, 210, 213 of medial hypothalamus, 207-208 of nigrostriatal pathway, 207, 210 recovery from, 289-390 of substantia nigra, 207, 227 thalamic, 195, 208, 233, 283

Limbic system, 327 Lipofuscin, 229, 231 Locomotor skills, 4 1 , 68-87 Lysosomes, 231, 245, 252, 481 , 488, 491, 496,

501

M

Malnutrition, 51 , 86-87, 123, 137, 401-406, 408,

412, 414-419 Maturation, 94, 171-173, 177, 183, 200, 511 Medial forebrain bundle, 205-206 , 208, 228 Melanin, 229, 231 Memory deficits, 317 Mental age, 421 , 432 Mental retardation, 121, 12S-134, 137, 179,

187-189 , 315, 3 1 8 - 3 1 9 , 331, 336, 4 1 5 - 4 1 6 ,

421 , 435, 445, 4 5 5 - 4 5 6 , 459, 468, 480, 503, 508, 510 -512 , 514, 517, 5 2 0 - 5 2 1

Metabolic diseases, 415, 520 Metabolism, inborn errors of, 137 Metachromatic leukodystrophy, 187, 501-505

Microcephaly, 128, 415 Mitosis, 18, 24

Mongolism, 128

Morphology, 103, 141-142, 159 Motor activity, 394-397 Motor responses, 173, 175-177, 188, 337 Myelination, 137, 180, 411-112, 416

Myoclonic seizures, 132, 134-137

N

Neuraminidase, 483-485, 487, 491

Neuron

circuitry, 94, 98

firing rates, 192-195, 198

genesis, 6-20, 22-24, 30, 34-36

migration, 4 , 17, 20 -30 , 32, 36-37

Neuropathology, 125, 129, 137

Niemann-Pick's disease, 482

Norephinephine, 2 0 5 - 2 0 9 , 2 1 2 - 2 1 4 , 232, 2 5 1 -

253, 255, 2 5 7 - 2 5 8 , 266

Nucleus

entopenduncular, 190, 192, 198-199

habenular, 192, 200

parafascicular, 191

subthalamic, 191

o

Oligodendroglia, 403 Operant sensitization, 373-374 Orienting reflex, 324, 326

P

Performance, 220-222, 382 Phenylalanine, 445-447, 449, 451, 453, 455, 462,

511

Phenylalanine hydroxylase, 4 4 6 - 4 5 6 , 4 5 9 - 4 6 3 , 5 0 8 - 5 0 9 , 511 , 513, 515

Phenylketonuria (PKU), 435, 4 4 5 - 4 4 6 , 4 5 0 - 4 5 6 4 5 9 - 4 6 1 , 463, 508, 5 1 1 - 5 1 2 , 514

Phosphodiesterase (PDE), 267-270 Phospholipids, 449-451 , 453 Postsynaptic potentials, 107-109 , 113, 179,

181-183, 336, 338 Postural control, 69, 82 Premature births, 317, 319

540 Subject Index

Presynaptic inhibition, 338, 362 Pseudoconditioning, 345-347, 3 5 5 - 3 6 1 , 364-365 Pterin compounds, 447-453 , 455-456 , 462 Pyridine nucleotide, 449-450

R

Recovery, 393-394, 397 Reserpine, 251-252

Response latencies, 338, 344-345, 351, 354-355, 360

Resting potentials, 106-109 Rubella, 415

s

Sandhoff's disease, 485-487, 496 Sensory motor deficit, 287, 304 Serotonin, 191, 206-207, 210, 232, 251-253, 258 Sleep-wakefulness, 277, 295, 297 Sodium mechanism, 111 Species difference, 403, 406, 462 Spinal cord, 103-111 Stanford-Binet test, 424-425, 429, 431 Steroid therapy, 415 Sulfohydrolase system, 502 Switching operation (neuronal), 344-346, 362,

364, 369

Synapses, 9, 4 7 - 5 2 , 55, 60, 1 0 6 - 1 1 1 , 113, 115, 117

asymmetrical, 231, 233, 243, 252-253, 257-258 axodendritic, 142, 149, 161, 191 connectivity, 266, 413, 416 development, 123-125, 137, 179-183

"en passage," 233, 247, 252, 258 EPSP-IPSP sequence, 196, 198-199 growth, 402, 405 symmetrical 231, 233, 243, 256-257

Synaptic delay, 338 Synaptic pathways, 153, 155, 164

axodendritic, 142, 147, 149 axosomatic, 142, 150-151, 153, 165 inhibitory, 164-165

Synaptogenesis, 42, 47-52

T

Tay-Sachs disease, 187, 479-485, 487, 494, 496, 514

Tetralogy of Falot, 153, 158 Thalamus, 190-193, 195-196, 200, 266, 351, 353,

360-361, 363, 368-369 Tissue culture, 101-113, 115-117, 120, 518-519 Transferase deficiencies, 468-470 Transneuronal degeneration, 316 Trisomy 21 , 155-158

Tyrosine, 445-448, 451-153, 455, 508-509, 511 Tyrosine hydroxylase, 194, 206-208

u

Unit recording, 326-329, 344, 361-363, 374-375

V Vesicles, 47-50 Visual cortex, 179-180 Visual discrimination, 173-174, 220-221

A 5 B 6 C 7 D 8 E 9 F 0 G 1 H 2 I 3 J 4

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Brain Mechanisms in Mental Retardation