THE ANATOMICAL RECORD 199:287-308 (1981)

SOUTHERN SOCIETY OF ANATOMISTS

lbentieth Annual Meeting October 16-18, 1980

Augusta, Georgia

Host MEDICAL COLLEGE OF GEORGIA

287

SOUTHERN SOCIETY OF ANATOMISTS OFFICERS 1979-80

President DALE E. BOCKMAN

(Medical College of Georgia)

President-Elect RICHARD J. WEYMOUTH (South Carolina-Columbia)

Secretary-Peasurer HERBERT SCHAPIRO

(Eastern Virginia)

Councilors W.J. DOUGHERTY

(South Carolina) M.L. ZIMNY

(LSU) R.P. STURTEVANT

(Lo yola) M. SHARAWY

(Medical Collge of Georgia)

This meeting was dedicated to the memory of

DR. B . RAJ BHUSSRY President of the Southern Society of Anatomists, 1978-79

288

CORPORATE SPONSORS The following firms have generously supported this, the 20th Annual

Meeting of the Southern Society of Anatomists, by their participation in the commercial exhibits and/or sponsoring of social events:

AMERICAN OPTICAL CORPORATION AMMHAY, INC.

BECKMAN INSTRUMENTS, INC. CAROLINA BIOLOGICAL SUPPLY, CO.

E.I. Du PONT DE NEMOURS & COMPANY, INC. ELECTRON MICROSCOPY SCIENCES

HAIXO INSTRUMENTS, INC.

ISI, INC. JEOL USA, INC.

GEORGIA INSTIlUMENTS, INC.

H l T A C H l SCIENTIFIC INSTRUMENTS

L.K.B. INSTRUMENTS, INC. MEDICAL INTERNATIONAL SOUTHEASTERN, INC.

NEWCOMB ASSOCIATES PGT

PH I L I tJs ELECTRON IC I N STRU M E NTS, IN c . PH Y SI M E T HI c s , IN c .

SOUTIIEI~N MICRO INSTRUMENTS, INC. VASHAW SCIENTIFIC, INC.

POK1’ER INSTRUMENTS S E R V I C E , INC.

289

OPENING SESSION

Dr. Dale E. Bockman, Professor and Chairman Department of Anatomy, Medical College of Georgia

President, Southern Society of Anatomists Dr. William H. Moretz

President, Medical College of Georgia

A total of 54 free papers were presented in 7 sessions as follows

SESSION I

“Neurobiology”

(Chairpersons; E.G. Hamel, Jr. and M.L. Kirby)

SESSION I1

“Neuromuscular”

(Chairpersons: D.W. Sickles and G.S. Sohal)

SESSION I11

“Special Senses, Salivary Glands”

(Chairpersons: W.K. O’Steen and M. Sharawy)

SESSION IV

“Methods in Anatomy”

(Chairpersons; R.R. Cowden and J.B. Longley)

SESSION V

“Cell Biology, Immunology, Tissue Culture”

(Chairpersons: M.L. Zimny and D.B. Lause)

SESSION VI

“Reproduction, Endocrinology”

(Chairpersons: W.P. Jollie and W.J. Dougherty)

SESSION VII

“Pathology, Environmental Pathology”

(Chairpersons: D.E. Hinton and J.M. Barrett)

290

AUGUSTINE, James R . , Depa r tmen t o f Anatomy, U n i v e r s i t y o f S o u t h C a r o l i n a , Schoo l of Medi- c i n e , Co lumbia , Sou th C a r o l i n a . A L u c i t e p l a t e method f o r t h r e e - d i m e n s i o n a l r e c o n s t r u c - t i o n o f n e u r o n a l p o p u l a t i o n s .

The combined u t i l i z a t i o n of HRP h i s tochem- i s t r y c o u p l e d w i t h a ] . “ c i t e p l a t e r e c o n s t r u c - t i o n method p r o v i d e s a n e x c e l l e n t means of s t u d y i n g t h e f u n c t i o n a l o r g a n i z a t i o n of n e u r o - n a l p o p u l a t i o n s . T h i s i s a n a d a p t a t i o n of a method p r e v i o u s l y employed by B a e t e n s c t a l . (1979) t o d e m o n s t r a t e t h e two t y p e s of i s l e t c e l l s i n t h e e n d o c r i n e p a n c r e a s of t h e r a t . C o l o r p h o t o m i c r o g r a p h s w e r e made o f e v e r y s i x t h s e c t i o n ( 4 0 micron s e c t i o n s w i t h 200 m i c r o n s be tween s e c t i o n s ) t h r o u g h t h e baboon o c u l o - moto r n u c l e u s . Each o f t h e r e s u l t i n g 18 s l i d e s was t h e n p r o j e c t e d w i t h t h e a i d of a phot i i - g r a p h i c e n l a r g e r o n t o a 9” X 12” L u c i t e p l a t e and t h e c o n f i g u r a t i o n of t h e o c u l o m o t o r n u c l e u s a t e a c h r e p r e s e n t a t i v e l e v e l drawn t o s c a l e on t h e p l a t e s . The L u c i t e p l a t e s were t h e n s t a c k e d one i n f r o n t of t h e o c h e r y i e l d i n g a s e e - t h r o u g h , 3-D r e c o n s t r u c t i o n (it t h e e n t i r e n u c l e u s . C o l o r t r a n s p a r e n c i e s of e v e r y s i x t h HRP-processed s r c t i o n were made and t h e image o f e a c h s e c t i o n t h e n p r o j e c t e d o n t o t h e i . u c i t e p l a t e s on which t h e c o n f i g u r a t i o n of t h e o c u l o - moto r n u c l e u s w a s p r e v i o u s l y o u t l i n e d . Us ing d i f f e r e n t c o l o r e d s t a r s t u r e p r e s e n t t h e d i f f - e r e n t o c u l o m o t o r m u s c l e s , t h e number and l o c a - t i o n o f HRP-pos i t i ve n e u r o n s was t h e n p l o t t e d . A c o m p o s i t e 3-D r e c o n s t r u c t i o n n f t h e e n t i r e l a b e l e d p o p u l a t i o n of t h e o c u l o m o t o r n u c l e u s on L u c i t e p l a t e s was t h e end r e s u l t . T h i s method p r o v i d e s a n u n p a r a i l e d a p p r e c i a t i o n o f t h e e n t i r e n u c l e a r complex t h a t is ana tomi - c a l l y a c c u r a t e and t h a t c a n he viewed from any s i d e o r a t any a n g l e . T h i s method c a n be a p p l i e d t o any n e u r o n a l p o p u l a t i o n i n a n y a n i m a l fo rm. R e c o n s t r u c t i o n s from s e r i a l e l e c t r o n mic r i jg raphs migh t be p o s s i b l e w i t h t h i s method. F i n a l l y , w i t h a d e q u a t e r e f e r e n c e p o i n t s a v a i l - a b l e on t h e L u c i t e p l a t e s t o a l i g n s e c t i o n s and m a i n t a i n s p a t i a l r e l a t i o n s , t h i s d a t a c a n be e n t e r e d v i a A two-d imens iona l d i g i t i z e r , s t o r e d i n a compute r d a t a b a s e and l a t f , r viewed and m a n i p u l a t e d i n 3-7) f a s h i o n u s i n g a n a p p r o p r i a t r compute r g r a p h i c s d e v i c e . ( S u p p o r t e d by n U n i v e r s i t y o f Sou th C , i r o l i n a , Schoo l of Medi- c i n e I n t r a m u r a l C r a n t ) .

_ _

BELTZ*, A l v i n J . and James BUGGY”, D e p a r t m e n t s o f Anatomy and P h y s i o l o g y , U n i v e r s i t y r i f Sou th C a r o l i n a , S c h o o l of M e d i c i n e , Co lumbia , Sou th C a r o l i n a . ( I n t r o d u c e d by James A . H igh tower ) Mapping of c h a n g e s i n b r a i n f u n c t i o n a l a c t i v i t y f o l l o w i n g m i d b r a i n p e r i a q u e d u c t a l g r a y s t i m u - l a t i o n : A 3H-2-deoxyglucose s t u d y .

The m i d b r a i n p e r i a q u e d u c t a l g r a y (PAG) h a s b e e n i m p l i c a t e d as a c e n t r a l s i t e o f morph ines a n t i n o c i c e p t i v e a c t i o n and a c e n t r a l l o c u s f o r s t i m u l a t i o n p roduced a n a l g e s i a . The p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n t o d e t e r m i n e wh ich CNS s i tes show change i n t h e i r f u n c t i o n a l a c t i - v i t y f o l l o w i n g PAG s t i m u l a t i o n . T h i s w a s accom- p l i s h e d by p r o d u c i n g f u n c t i o n a l maps o f CNS a c t i v i t y u t i l i z i n g a m o d i f i c a t i o n of t h e 2- deoxyg l u c o s e t e c h n i q u e d e v e l o p e d by S o k o l o f f and c o l l a b o r a t o r s ( 1 9 7 7 ) . C h r o n i c e l e c t r o d e s

w e r e i m p l a n t e d s t e r e o t a x i c a l l y i n t o t h e I’AG o f 20 ma le S p r a g u e Dawley r a t s (200-250 gm). Each a n i m a l was t e s t e d 4 8 h o u r s a f t e r s u r g e r y t o d e t e r m i n e if PAC, s t i r n u l a t i o n r e s u l t e d i n a n a l g e s i a t o i n c r e a s i n g i n t e n s i t i e s of e l e c t r i - c a l s h o c k . An ima l s which d i s p l a y e d s t i m u l a t i o n p roduced a n a l g e s i a were s u b s e q u e n t l y i m p l a n t e d w i t h a l e m o r a l venous c a t h e t e r . Twen ty - fou r h o u r s a f t e r c a t h e t e r i z a t i o n t h e a n i m a l s were i n j e c t e d i n t r a v e n o u s l y w i t h 3H-Z-deoxygl u c o s e (167 pci / IOOgm body w e i g h t ) and t h e PAG was s u b s e q u e n t l y s t i m u l a t e d f o r a p e r i o d o f 4 5 min. The a n i m a l s were i m m e d i a t e l y s a c r i c i c e d by i n t r a c a r d i a c p e r f u s i o n w i t h c o l d f i x a t i v e ; t h e i r b r a i n s were removed and f r o z e n i n l i q u i d n i t r o g e n ; and 1 5 p t h i c k s e c t i o n s o f b r a i n were c u t on a c r y o s t a t . The s e c t i o n s were exposed t o LKB u l t r o f i l m f o r a p e r i o d of 3 weeks and t h e r e s u l t i n g a u t o r a d i o g r a p h i c images were a n a l y z e d u s i n g a c o m p u t e r i z e d image p ro - c e s s i n g s y s t e m . ‘The most s t r i k i n g c h a n g e s i n i u n c t i o n a l a c t i v i t y f o l l o w i n g PAC s t i m u l a t i o n o c c u r r e d i n t h e v e n t r a l hippocampus. n u c l e u s p a r a g i g a n t o c e l l u l a r i s , n u c l e u s c u n e i f o r m i s , zona i n c e r t a , n u c l e u s c e n t r a l i s s u p e r i o r , n u c l e u s ambiginis , n u c l e u s s o l i t a r i u s and t l lc d o r s a l hu rn of tiif s p i n a l c o r d . The d a t a ob- t a i n e d from t h i s s t u d y s u g g e s t t h a t many o f t h e above n u c l e a r g r o u p s a r e i n v o l v e d i n t h e p r o d u c t i o n o f a n a l g c s i n b r o u g h t a b o u t by PAC s t i m u l a t i o n . (Suppor t ed by NSF (:rant BNS 7906486 and USC C r a n t 11040 E-133.)

BICKNELL, H. R.* and J. A . Beal, Depar tmen t o f Anatomy, L o u i s i a n a S t a t e U n i v e r s i t y M e d i c a l C e n t e r , S h r e v e p o r t , L o u i s i a n a . A t y p i c a l n e u r o n a l c e l l t y p e s i n t h e s u b s t a n t i a g e l a t i n o s a ( l a m i n a 11) o f t h e a d u l t c a t l u m b o s a c r a l s p i n a l c o r d .

The n e u r o n s o f t h e s u b s t a n t i a g e l a t i n o s a (SG) w e r e examined i n G o l g i p r e p a r a t i o n s t a k e n from t h e l u m b o s a c r a l s p i n a l c o r d o f t h e a d u l t cat . I n a d d i t i o n t o s e v e r a l t y p e s o f c h a r a c - t e r i s t i c SG n e u r o n s , a few n e u r o n s were found w i t h a t y p i c a l c h a r a c t e r i s t i c s wh ich h a v e n o t b e e n p r e v i o u s l y r e p o r t e d i n t h e a d u l t s p i n a l c o r d . The a t y p i c a l c e l l s f a l l i n t o two c a t e g o r i e s : 1) n e u r o n s w i t h a s t a r s h a p e d , r a d i a t i n g d e n d r i t i c p a t t e r n and 2 ) n e u r o n s w i t h d u a l a x o n s . The s ta r s h a p e d n e u r o n s w e r e o b s e r v e d i n Rexed‘ s ( ‘ 5 2 ) i n n e r zone o f l a m i n a 11. These n e u r o n s h a v e a s m a l l , p o l y g o n a l s h a p e d c e l l body (14 x 18 p) p o s s e s s i n g numerous , s h o r t p r i m a r y and s e c o n d a r y d e n d r i t e s wh ich e x t e n d r a d i a l l y f rom t h e soma i n a s t a r - l i k e f a s h i o n . Both t h e c e l l body a n d d e n d r i t e s o f t h e s t a r s h a p e d n e u r o n h a v e an i r r e g u l a r c r a g g y c o n t o u r . A s t r o n g s t r u c t u r a l s i m i l a r i t y e x i s t s be tween t h e s t a r shaped n e u r o n and immatu re , u n d i f f e r e n t i a t e d SG n e u r o n s p r e v i o u s l y d e s c r i b e d i n t h e k i t t e n ( F a l l s and G o b e l , ’ 7 9 ) . The s i m i l a r i t y s u g g e s t s t h a t t h e s ta r s h a p e d c e l l h a s n o t c o m p l e t e d i t s d i f f e r e n t i a t i o n i n t o a m a t u r e n e u r o n a l form. Neurons p o s s e s s i n g d u a l a x o n s were found i n b o t h t h e i n n e r and o u t e r zones of Rexed’s ( ‘ 5 2 ) lamina 11. The two axons o r i g i n a t e f rom d e n d r i t e s a t v a r i o u s d i s t a n c e s f rom t h e c e l l body and g e n e r a t e a n e x t e n s i v e p l e x u s t h a t e x t e n d s f o r s e v e r a l hundred m i c r a . The two a x o n a l a r b o r s d i s p l a y

c o n s i d e r a b l e o v e r l a p . S i n c e d u a l axon c e l l s a r e few i n number and s t r u c t u r a l l y s i m i l a r t o o t h e r SG neurons w i t h s i n g l e axons i t is most p r o b a b l e t h a t t h e y r e p r e s e n t a deve lopmenta l a b e r r a n c y r a t h e r t h a n a f u n c t i o n a l l y d i s t i n c t c l a s s of neurons . (Supported i n p a r t by N I N C D S , NS 16642-01).

BOSHELL, J . L . , Department of O r a l Bio logy/ Anatomy, Medica l C o l l e g e of G e o r g i a School of D e n t i s t r y , Augusta, Georg ia . A Comparison of t h e E f f e c t s of I s o p r o t e r e n o l and T e s t o s t e r o n e P r o p i o n a t e on Acinar C e l l R e g e n e r a t i o n i n Suomandibular Glands of R a t .

chymal r e g e n e r a t i o n of submandibular g l a n d s of r a t s a r e c u r r e n t l y ongoing i n o u r l a b o r a t o r i e s . The purpose of t h i s r e p o r t is t o compare t h e e f f e c t s of i s o p r o t e r e n o l ( ISO) and t e s t o s t e r o n e p r o p i o n a t e (TP) on a c i n a r c e l l m i t o s e s fo l low- i n g s u r g i c a l removal of a p o r t i o n of t h e l e f t submandibular g l a n d . Male Sprague-Dawley r a t s , 325-350 grams i n w e i g h t , were a n e s t h e t i z e d and a wedge was removed from t h e m e d i a l s u r f a c e of t h e l e f t submandibular g l a n d . Beginning on t h e t h i r d day a f t e r t h e o p e r a t i o n , t h e a n i m a l s were i n j e c t e d I P w i t h IS0 (16 mglkg) i n s a l i n e o r TP (100 u g l a n i m a l ) i n sesame o i l . C o n t r o l groups were g i v e n i n j e c t i o n s of e i t h e r s a l i n e o r sesame o i l . A p p r o p r i a t e e x p e r i m e n t a l and c o n t r o l a n i m a l s were k i l l e d on 5 , 1 0 , 1 5 , 20, 2 5 and 30 days p o s t o p e r a t i o n . Submandibular g l a n d s were removed, f i x e d i n f o n a l i n and pro- c e s s e d f o r l i g h t m i c r o s c o p i c e x a m i n a t i o n . One thousand a c i n a r c e l l s were counted i n two a r e a s of t h e o p e r a t e d g l a n d , and t h e number of m i t o t i c f i g u r e s was r e c o r d e d . A t t h e wound s u r f a c e , m i t o s e s of a c i n a r c e l l s i n s a l i n e i n - j e c t e d c o n t r o l s were h i g h through 1 0 d a y s . A f t e r IS0 o r sesame o i l , m i t o t i c a c t i v i t y on t h e wound s u r f a c e d e c l i n e d s h a r p l y and remain- ed n e a r z e r o from day 10 t h r o u g h day 30. I n c o n t r a s t a f t e r T P , t h e r e i s d drop i n m i t o t i c a c t i v i t y a t day 5 i n comparison t o s a l i n e con- t r o l s b u t then a s h a r p r i s e a t day 10 f o l l o w e d by a d e c l i n e t o n e a r z e r o t h r o u g h day 30. Only a f t e r I S 0 was a b u r s t of m i t o s e s of a c i n a r c e l l s observed i n t h e remainder of t h e g l a n d . I t a p p e a r s t h a t sesame o i l and IS0 s u p p r e s s t h e induced m i t o s e s on t h e c u t s u r f a c e , where- a s , a f t e r TP a marked d e p r e s s i o n on day 5 i s fo l lowed by an i n c r e a s e nn day 10. T h i s sug- g e s t s t h a t t h e s e exogenous a g e n t s s u p p r e s s t h e normal r e g e n e r a t i v e p r o c e s s i n r a t suhmandibu- l a r g l a n d s . Suppor ted by NIDR Grant ii DE 05072 -02.

S t u d i e s on f a c t o r s c o n t r o l l i n g paren-

BRINN*, Jack E.; Carol SMITH* and Carl R.MORGAN, Department o f Anatomy, East Caro l ina U n i v e r s i t y School o f Medicine, Greenv i l l e , Nor th Caro l ina . F i x a t i o n o f p a n c r e a t i c i s l e t s f o r morphometric ana lys is .

Sampling o f t h e pancreat ic i s l e t s f o r morphometric ana lys i s i s o f t e n a problem because o f t h e i r uneven d i s t r i b u t i o n i n the pancreas and because o f t he d i f f e r e n t c e l l types. For those i n t e r e s t e d i n i s l e t morphometry a t bo th the EM and LM l e v e l s , t he

problem i s f u r t h e r compl icated by the necess i t y o f us ing d i f f e r e n t f i x a t i v e s f o r t he two types o f microscopy. Being convinced o f t h e e f f i c a c y o f pe r fus ion f i x a t i o n we have devised a procedure t h a t p rov ides an i n i t i a l f i x a t i o n , sampl ing each reg ion o f t he pancreas, and process ing o f those samples f o r bo th LM and EM. The pe r fus ion cons is t s o f a very b r i e f washout w i t h a balanced s a l t s o l u t i o n fo l l owed by a m ix tu re o f 4-5% paraformaldehyde, 0.05% p i c r i c ac id and 0.01% t o 0.05% g lu ta ra ldehyde i n 0.05 M phosphate b u f f e r f o r 3-4 minutes. pancreas by the p i c r i c a c i d i s i n d i c a t i v e o f adequate pe r fus ion . p laced i n Bou in ' s f l u i d f o r 2-3 hours f o r LM o r i n 2.5% g lu ta ra ldehyde f o r EM. p rocess ing i s rou t i ne . I n the LM prepara t ions we have s u b s t i t u t e d a l c i a n b lue f o r aldehyde f u c h s i n t o ob ta in a more d e l i c a t e s t a i n i n g o f t he beta c e l l s . Add i t i ona l exper iments a re be ing done t o determine t h e a p p l i c a b i l i t y o f t he techn ique t o o the r t i ssues , p a r t i c u l a r l y t h e hypophysis. procedure i s t he reduc t i on o f animal cos ts by e l i m i n a t i n g the necess i ty o f us ing separate animals f o r LM and EM morphometry w h i l e p r o v i d i n g good f i x a t i o n f o r bo th l eve l s .

Ye l low ing o f t he

Samples o f t i s s u e are

Subsequent

A ma jor advantage o f t h i s

BURDEN, H. W., M. L. CAPPS*, and I . E. LAWRENCE, JR., Department o f Anatomy, School o f Medicine, East Caro l ina U n i v e r s i t y .

p e r i f o l l i c u l a r t i s s u e has been demonstrated i n numerous p h y s i o l o g i c a l and pharmacological exper iments. It has been suggested t h a t c o n t r a c t i o n o f p e r i f o l l i c u l a r t i s s u e may a i d i n the ovu la to ry process. s p e c i a l i z e d j u n c t i o n s between c o n t r a c t i l e c e l l s may p l a y a r o l e i n t h e spread o f e l e c t r i c a l i n fo rma t ion between c e l l s and f a c i 1 i t a t e synchron iza t ion o f c o n t r a c t i 1 i ty o f p e r i f o l l i c u l a r t i ssue . I n t h e present study, ova r ian p e r i f o l l i c u l a r t i s s u e was examined a t s p e c i f i e d i n t e r v a l s p r i o r t o the expected t ime o f ovu la t i on . Emphasis was p laced on the type and r e l a t i v e number o f c e l l - t o - c e l l con tac ts . Rat ova r ian t i s s u e was taken f o r e l e c t r o n mic roscop ic s tud ies a t d i e s t r u s , a t 1500 hours on the a f te rnoon o f p roes t rus ( 0 hour group), a t 2000 hours ( 5 hour group) and 0100 hours (10 hours group). Three types o f j u n c t i o n s were observed between c e l l u l a r components i n t h e theca ex terna : c lose contac ts , i n te rmed ia te j unc t i ons , and gap junc t i ons . A l l groups s tud ied e x h i b i t e d c lose contac ts and in te rmed ia te j unc t i ons . The number and leng th o f i n te rmed ia te j u n c t i o n s was increased i n t h e p reovu la to ry pe r iod as compared t o the d i e s t r u s per iod . The average leng th o f c lose con tac ts was g rea te r a t 5 and 10 hours than a t d i e s t r u s o r 0 hours. There was an abrupt i nc rease i n t h e number o f gap j u n c t i o n s a t 10 hours. These observa t ions p rov ide morpho log ica l evidence f o r a dynamic r o l e o f p e r i f o l l i c u l a r t i s s u e i n t h e o v u l a t o r y

The fo rma t ion o f

process. No. HO 06899.

"Supported i n p a r t by USPHS Grant

CHIBUZO, G . A. AND J . F. CUMMINGS

Department of Anatomy, School o f V e t e r i n a r y Medic ine , Tuskegee I n s t i t u t e , Alabama 36088

L i n g u a l P r o p r i o c e p t i o n : a n enzyme t r a c e r s t u d y .

It h a s been assumed t h a t t h e tongue , a n o r g a n of d i v e r s e and r e f i n e d movements would be en- dowed w i t h a n e x t e n s i v e p r o p r i o c e p t i v e i n n e r - v a t i o n . However, t h e r e e x i s t s no c o n c l u s i v e e x p e r i m e n t a l e v i d e n c e f o r t h e s o u r c e of s e n s o r y ( p r o p r i o c e p t i v e ) i n n e r v a t i o n of t h e tongue mus- c u l a t u r e . Although some a u t h o r s r e p o r t e d t h e p r e s e n c e of l i n g u a l musc le s p i n d l e s ( p r o p r i o - r e c e p t o r s ) i n some a n i m a l s , o t h e r i n v e s t i g a t o r s a rgued t h a t a t l e a s t i n some a n i m a l s l i n g u a l p r o p r i o c e p t i o n was subserved by r e c e p t o r s o t h e r t h a n musc le s p i n d l e s . Us ing t h e method of induced c h r o m a t o l y s i s accompl ished by selec- t i v e c r a n i a l n e r v e n e u r e c t o m i e s , d i f f e r e n t in - v e s t i g a t o r s s u g g e s t e d t h e mesencephal ic n u c l e u s of t r i g e m i n a l n e r v e , i n t r a h y p o g l o s s a l n e r v e g a n g l i a , d i s t a l v a g a l ( n o d o s a l ) g a n g l i a and c e r v i c a l s p i n a l g a n g l i a as p o s s i b l e s o u r c e s f o r l i n g u a l p r o p r i o c e p t i v e i n n e r v a t i o n .

The r e s u l t b e i n g p r e s e n t e d h e r e r e l i e d on horse- r a d i s h p e r o x i d a s e r e t r o g r a d e l a b e l i n g of s e n s o r y n e u r o n s t o t h e e x t r i n s i c l i n g u a l m u s c l e s . The enzyme (30% s o l u t i o n ) was i n j e c t e d i n micro- l i t e r amounts i n t o t h e g e n i o g l o s s u s , s t y l o g l o s - s u s and h y o g l o s s u s i n v e r y young dogs . Twenty- f o u r t o f o r t y - e i g h t h o u r s p o s t i n j e c t i o n , t h e dogs were e u t h a n a t i z e d and p e r f u s e d t r a n s car - d i a l l y w i t h a Karnovsky-type f i x a t i v e . S e v e r a l c r a n i a l n e r v e g a n g l i a , c e r v i c a l s p i n a l g a n g l i a , t h e b r a i n stems and h y p o g l o s s a l n e r v e s were c o l l e c t e d , s e r i a l l y s e c t i o n e d a t 6 0 N m w i t h f r e e z i n g s l i d i n g microtome and p r o c e s s e d accord- i n g t o t h e method of Nauta e t a l . ( B r a i n Res. 67 ( 1 9 7 4 ) : 219). H o r s e r a d i s h p e r o x i d a s e re- a c t i o n p r o d u c t s were demonst ra ted h is tochemi- c a l l y i n t h e i p s i l a t e r a l upper c e r v i c a l s p i n a l , p roximal v a g a l ( j u g u l a r ) and t r i g e m i n a l g a n g l i a . The r e s u l t t h e r e f o r e c o n t r a d i c t e d t h e sugges- t i o n t h a t t h e mesencephal ic n u c l e u s o f t r i g e m i - n a l n e r v e , t h e i n t r a h y p o g l o s s a l n e r v e g a n g l i a and t h e d i s t a - l v a g a l ( n o d o s a l ) g a n g l i a c o n t a i n - ed t h e l i n g u a l p r o p r i o c e p t i v e n e u r o n a l somata.

T h i s r e s e a r c h was s u p p o r t e d by N I H Grant 15F34- GMO6013-02.

CLARK, George, V e t e r a n s A d m i n i s t r a t i o n Medica l C e n t e r ; and Department o f Anatomy, Medica l U n i v e r s i t y of South C a r o l i n a , C h a r l e s t o n , South C a r o l i n a . The B i o l o g i c a l Stain Commis s i on.

P r i o r t o World War I t h e Gi-iibler l a b e l was a c c e p t e d as q u a l i t y a s s u r a n c e f o r b i o l o g i c a l d y e s . The w a r and i t s a f t e r m a t h e l i m i n a t e d t h i s s o u r c e . I n t h i s c o u n t r y a d o m e s t i c dye i n d u s t r y deve loped . T h e i r p r o d u c t s were o f t e n q u i t e i n f e r i o r and sometimes m i s l a b e l e d . A committee headed b y H . J . Conn found t h a t some samples w e r e f u l l y e q u a l t o t h e pre-war dyes s o t h e problem c o u l d h e s o l v e d b y s e l e c t i o n . Out o f t h i s t h e B i o l o g i c a l S t a i n Commission f i n a l l y developed. P r o c e d u r e s were d e v i s e d f o r c h a r a c t e r i z a t i o n o f many of t h e commonly

used dyes . A s y s t e m o f l a b e l s , i d e n t i f y i n g good h a t c h e s w a s deve loped . I would l i k e t o summarize t h e c u r r e n t work o f t h e B i o l o g i c a l S t a i n Commission. F o r some dyes chemica l tes ts a r e a d e q u a t e h u t many a r e a l s o t e s t e d as s t a i n s . No chemica l tests have been d e v i s e d f o r hematoxyl in , t h e most c o m n l y used d y e , so it i s t e s t e d s o l e l y as a s t a i n . F i f t y - s i x O t h e r s t a i n s are now a v a i l a b l e as c e r t i f i e d s t a i n s . The Comnission c h a r g e s a s i n g l e f e e f o r e a c h b a t c h o f dye t e s t e d and a n o t h e r f o r t h e c e r t i f i c a t i o n l a b e l s which are a f f i x e d t o e a c h b o t t l e o f c e r t i f i e d dyes s o l d b y t h e v a r i o u s s u p p l i e r s . I n t h e p a s t few y e a r s l a b e l sales have d e c r e a s e d . T h i s , i f c o n t i n u e d , w i l l s e r i o u s l y l i m i t t h e work of t h e Commission. T h i s loss i n l a b e l sales is p r o b a b l y due t o t h e i n c r e a s e d sale of com- m e r c i a l l y p r e p a r e d s t a i n i n g s o l u t i o n s . I n a d d i t i o n t o t h e r e g u l a r membership t h e r e are r e p r e s e n t a t i v e s o f v a r i o u s s o c i e t i e s ; A r t h u r LaVel le i s t h e r e p r e s e n t a t i v e from t h e American A s s o c i a t i o n of Anatomis ts . T r u s t e e s a r e e l e c t e d f o r terms o f t h r e e y e a r s . “ P a r t i a l l y s u p p o r t e d b y t h e V e t e r a n s A d m i n i s t r a t i o n and by t h e S t a t e A p p r o p r i a t i o n f o r Biomedica l Research t o t h e Medica l Un i v e rs i t y of South C a ro 1 i n a . C O L E , T . B . , J R . and SCHWEISTHAL, M . H . , Department of Andtomy, O r a l R o b e r t s Uni- v e r s i t y , T u l s a , Oklahoma. A c h a l a s i a : ~ H i s t o l o g i c a l f i n d i n g s of a c a s e s t u d y .

Gross a n a t o m i c a l f i n d i n g s on necropsy o f an 8 5 - y e a r - o l d male w i t h s e v e r e d r h a l a - s i a have heen r e p o r t e d i n a n acrompariying a b s t r a c t . H i s t o l o g i c a l e x a m i n a t i o n of t h e d i l a t e d esophagus was conducted i n o r d r r t o i n v e s t i g a t e t h i s c a s e more t h o r o u g h l y . Approximate ly twenty t i s s u e samples t a k e n from t h e w a l l o f t h e e s o p h a g e a l d i l a t i o i i and a d j a c e n t r e g i o n s were c o l l e c t e d f o r e x a m i n a t i o n . The t i s s u e spec imens were p r o c e s s e d and o b s e r v a t i o n s made employing r o u t i n e l a b o r a t o r y p r o c e d u r e s . Examinat ion o f t h e t i s s u e s e c t i o n s r l e a r l y d e m o n s t r a t e d t h e f o u r g e n e r a l l a y e r s of t h e a l i m e n t a r y t r a c t . The innermost mucosa a p p e a r e d normal and d i s p l a y e d n o n - k e r a t i n i z i n g s t r a t i f i e d squamous e p i t h e l i u m which occa- s i o n a l l y appeared t h i c k e n e d . Lymphocyte i n f i l t r a t i o n of t h e mucosal and submucosal l a y e r s was ronunoii. S o l i t a r y and a g g r e g a t e d l y m p h a t i c nodules were observed i n t h e submucosa. Specimens t a k e n from t h e open- i n g t o t h e d i l a t e d esophagus and t h e t r a n s - v e r s e r i d g e p r o j e c t i n g i n t o t h e esophagea l lumen demonst ra ted a t h i c k m u s c u l a r i s e x t e r n a composed of numerous s k e l e t a l musc le f a s c i c u l i . T h i s t h i c k e n e d muscu- l a r i s e x t e r n a r e p r e s e n t s t h e c r i c o - pharyngeus , t h u s c o n f i r m i n g g r o s s ana- t o m i c a l o b s e r v a t i o n s . R e l a t i v e t o t h e c r a n i a l end o f t h e d i l a t e d esophagus , t h e m u s c u l a r i s e x t e r n a appeared t h i n i n t h e remain ing a r e a s examined. T h i s t h i n n i n g o f t h e muscular l a y e r mdy b r i n r e s p o n s e t o d i l a t i o n of t h e esophagus . The o u t e r a d v a r i t i t i a l l a y e r o f t h e esophagus appeared normal . The h i s t o l o g i c d l f i n d i n g s s u p p o r t the c o n r l u s i o n t h a t t h e tipper e s o p h a g e a l s p h i n c t e r p l a y e d a n i m p o r t a n l r o l e 111 t h i s r a s e of a t h a l d s i a .

293

COWDEN, RONALD R. AND S.K. CURTIS, DEPARTMENT OF BIOPHYSICS, COLLEGE OF MEDICINE, EAST TENNESSEE STATE UNIVERSITY, JOHNSON CITY, TENNESSEE 37614

Microfluorometric Comparison of DNA Specific Fluorochromes

A series of six dyes, putatively selective f o r DNA under certain conditions, were compared using isolated mouse thymic lymphocyte and hepatocyte nuclei. The list included: mithramycin (M) (G-C specific), Hoechst 33258 (H), D.A.P.I., proflavine (PR), quinacrine mustard (QM) (all A-T specific), and pro- pidium iodide (PI) (double-stranded nucleic acid specific). Since PI fluorochromes double-stranded RNA and DNA, these prepara- tions were pretreated with RNase.

All the methods used, except QM, produced values for thymus lymphocyte nuclei and 2c hepatocytes that were within the C.V. f o r the measurement. In general, QM values for thymocyte nuclei were greater than those for 2c hepatocytes, and values for 4c and 8c hepatocyte nuclei were lower than the antici- pated values. While pretreatment with RNase reduced PI values by ca. lo%, the reduction with M and particularly with PR and QM were substantially greater than would be expected from known proportions of DNA:RNA. RNase appears to interfere with either binding or fluorescence emissions when used with these dyes.

CURRY*, T. E., JR., C. A. HODSON*, D. E. D. JONES*, P. D. MOZLEY*, M. L. LAPPS*, and I . E. LAWRENCE, JR. ( In t roduced by H. W. Burden) Departments o f Anatomy and Obs te t r i cs and Gynecology, School o f Medicine, East Caro l i na U n i v e r s i t y , Greenv i l l e , North Caro l ina . A morpho log ica l s tudy o f t h e human ova r ian l igament.

I n t h e human female, t h e ovary i s at tached t o the l a t e r a l angle o f t he u te rus , p o s t e r o - i n f e r i o r t o t h e u t e r i n e tube, by a round cord termed the ovar ian l igament . The ova r ian l igament has been s tud ied p rev ious l y w i t h h i s t o l o g i c a l techniques and has been repor ted t o be a h i g h l y vascu la r f i b r o - muscular s t ruc tu re , r i c h l y supp l ied w i t h nerve elements (Mahran e t a l . , 1971, Obstet. Gynec., 3 7 : 7 1 1 ) 7 5 t h e present study, t h e f i n e s t r u c t u r e o f t he human ova r ian l igament was i n v e s t i g a t e d w i t h t ransmiss ion e l e c t r o n microscopy. Emphasis was placed on c h a r a c t e r i z a t i o n o f t h e muscular and neura l components i n t h e l igament. Specimens were ob ta ined by hysterosalpingectomy from women o f c h i l d b e a r i n g age. T issue was f i x e d i n phosphate bu f fe red g lu ta ra ldehyde- paraformaldehyde, s ta ined en b loc w i t h u rany l ace ta te , sect ioned, and s ta ined w i t h lead c i t r a t e be fo re examinat ion. The ova r ian l igament cons is t s o f smooth muscle c e l l s and f i b r o b l a s t s d ispersed i n a col lagenous mat r ix . Close contac ts and in te rmed ia te j u n c t i o n s were observed between ad jacent smooth muscle c e l l s . Large nerves embedded i n the col lagenous

m a t r i x con ta ined bo th mye l ina ted and nonmyel inated axons. r o l e i n f u n c t i o n a l r e l a t i o n s h i p s between t h e ovary and u terus . Furthermore, j u n c t i o n a l complexes between smooth muscle c e l l s i n t h e ova r ian l igament may coo rd ina te muscular con t rac t i ons i n c e r t a i n rep roduc t i ve processes.

These nerves may p l a y a

DA\’ENPORT, W i l l i a m D. , J r . and C a r r o l l R. B a l l , Department o f Anatomy, U n i v e r s i t y o f M iss i ss ipp i Medical Center, Jackson, M i s s i s s i p p i . Endo the l i a l damage by h i g h f a t - l o w p r o t e i n d i e t seen by scannin e l e c t r o n microscopy.

Mice fed a’semipurif ied, h i g h f a t ( l a r d ) - low p r o t e i n ( case in ) d i e t show an ex t remely h i g h inc idence o f a t r i a l thrombosis. A f t e r s i x weeks o f d i e t feed ing , exper imenta l animals were p a i r k i l l e d w i t h con t ro l s , a t weekly i n t e r - va l s . Mice which d ied a t i r r e g u l a r i n t e r v a l s were a l s o s tud ied .

The e n d o t h e l i a l su r face o f a t r i a was examined by scanning e l e c t r o n microscopy (SEM). The f i r s t s igns o f damage were prominent nuc lear b u l g i n g and a l t e r a t i o n s i n the rhomboidal shape o f t he endo the l i a l c e l l s . More severe damage inc luded c r a t e r - l i k e ho les i n t h e cytoplasm o f e n d o t h e l i a l c e l l s w i t h exposure o f sub-endo- t h e l i a l t i s s u e t o which p l a t e l e t s w i t h pseudo- pod ia were adher ing. t h e l i a l c e l l s has been repo r ted when l e t h a l doses o f f a t t y ac ids were i n j e c t e d i n t o r a b b i t ear veins. Th is a l t e r e d e n d o t h e l i a l i n t e g r i t y p robab ly under l i es the s u r c e p t i b i l i t y o f these s i t e s t o fo rmat ion o f d ie t - i nduced th rombot ic l es ions .

S i m i l a r i n j u r y t o endo-

Supported by NIH Biomedial Research Support Grant #5-S07 RR05386)

D ian i , A r t h u r R. and D. Davis*, Department o f Anatomy, Eas t Caro l i na U n i v e r s i t y School o f Med ic ine and Department o f B io logy , East Caro l i na U n i v e r s i t y . A l t e r a t i o n o f C a l i b e r Spectra and Numerical Dens i t y A s s E i a t e d t h Nerve F i b e r s m y m p a t h e t i c Trunk o f The Ketonur i c D i a b e t i c Chi nese Hamster .

t h e sympathet ic t runk (ST) o f 7 k e t o n u r i c d iab- e t i c Chinese hamsters and 7 age - matched non- d i a b e t i c s were q u a n t i t a t i v e l y analyzed t o de- te rmine frequency d i s t r i b u t i o n and numerical dens i t y . Animals were per fused w i t h Karnov- s k y ’ s f i x a t i v e and each ST was embedded i n Durcupan. For mye l ina ted f i b e r s , s e r i a l c ross sec t i ons were c u t a t 1 pm and randomly s e l e c t - ed t o e l i m i n a t e observer b ias . Fasc i c les o f ST were photographed and combined i n t o photo- montages (875X). A p l a s t i c g r i d w i t h 16 u n i - form sec to rs was p laced ove r the geometr ic cen- t e r o f each f a s c i c l e . Ex te rna l d iameter and d e n s i t y o f mye l i na ted axons w i t h i n 2 randomly se lec ted sec to rs were measured. For unmyel in- a ted f i b e r s , s e r i a l c ross sec t i ons were c u t a t 60-90 um and randomly se lec ted f o r ana lys i s . Every t h i r d g r i d space ( w i t h nervous t i s s u e ) was photographed (1400X) i n an e l e c t r o n mic ro- scope u n t i l each f a s c i c l e was sampled. Dia- meter and numerical d e n s i t y o f a l l unmyel in- a t e d f i b e r s i n each p r i n t (5DOOX) was recorded.

-___- ~ ~ ~ _ _ _

Myel ina ted and unmyel inated f i b e r s f rom

294

Myel ina ted and unmyel inated f i b e r s i n d i a - b e t i c hamsters d i sp layed a s i g n i f i c a n t reduc- t i o n i n d iameter which was r e l a t e d t o p ro - g r e d i e n t ke tonur ia . Mean numerical d e n s i t y o f mye l ina ted f i b e r s was s i g n i f i c a n t l y re - duced whereas t h a t o f unmyel inated f i b e r s was increased. The a l t e r a t i o n s i n sympathet ic nerve popu la t ions a re be l i eved t o be a mani- f e s t a t i o n o f p r e v i o u s l y observed demye l ina t ion and "dy ing back" neuropathy. On the bas is o f axon diameter, i t appeared t h a t bo th v i s c e r a l a f fe ren t and e f f e r e n t f i b e r s were pa tho log i c . These da ta s t r o n g l y imp ly t h a t neuropathy i n the sympathet ic t runk o f the d i a b e t i c Chinese hamster may be a c r i t i c a l f a c t o r under l y ing g a s t r o i n t e s t i n a l dys func t ion .

N I H g r a n t AM-21933-02. Supported by Upjohn Company g r a n t and

W . J . DDUGHERTY and S. I . COLEMAN, Department o f Anatomy, Medical U n i v e r s i t y o f South Caro l ina , Char leston, SC 29403. E f f e c t s o f hypophysectomy and growth hormone o r thy rox ine replacement on the occurrence and c a l c i f i a b i l - it.y o f m a t r i x ves i c les i n r a t t i b i a l epiphyses.

Young female Long-Evans r a t s were hypophy- sectomized o r hypophysectomized p l u s thy ro - para thyro idec tomized, h e l d f o r 14 days, and then i n j e c t e d w i t h normal s a l i n e , growth hormone, o r L - thy rox ine tw ice d a i l y f o r 2 o r 4 days. Proximal t i b i a e were prepared f o r ana lys i s by l i g h t and e l e c t r o n microscopy a t t he te rm ina t ion o f these t rea tments . I n a d d i t i o n t o measuring whole body we igh ts and the w id ths o f t i b i a l ep iph s e s , t he number of

ma t r i x , the percentage o f m a t r i x ves i c les con ta in ing minera l c r y s t a l l i t e s and t o t a l serum Ca l e v e l s i n each o f t he c o n t r o l and exper imenta l groups o f animals were recorded. Hypophysectomi zed animals l o s t we igh t and showed a s i g n i f i c a n t decrease i n the w id ths o f t he epiphyseal p la tes . Serum Ca l e v e l s re - mained normal. Whi le the re a re approx imate ly 276 m a t r i x ves i c les p e r 100 urn2 i n bo th c o n t r o l and hypophysectomized animals, hypophysecto- mi red animals e x h i b i t e d 50% fewer m a t r i x ves i c les c o n t a i n i n g minera l c r y s t a l l i tes than c o n t r o l animals. Replacement o f 0.68 I . U . r a t growth hormone produced an i nc rease i n the number o f m a t r i x ves i c les w i t h c r y s t a l l i t e s t h a t was 124% g rea te r than i n hypophysectomized animals and 24% g r e a t e r than i n c o n t r o l s . Thy- rox ine replacement i n hypophysectomized animals r e s t o r e d the number o f m a t r i x ves i c les w i t h c r y s t a l l i t e s approx imate ly t o c o n t r o l l e v e l s . Thus, w h i l e the numbers o f m a t r i x ves i c les pe r u n i t area a re n o t a f f e c t e d by hypophysectomy o r hormone replacement, t he c a l c i f i a b i l i t y o f m a t r i x ves i c les i n v i v o i s a f f e c t e d by bo th growth hormone and thy rox ine .

m a t r i x ves i c les p e r 100 un 3 o f e x t r a c e l l u l a r

DYESS, *E r i c M.and W. D. Davenport, J r . , Department o f Anatomy, U n i v e r s i t y o f M i s s i s s i p p i Medical Center, Jackson, M iss i ss iDo i . E f f e c t s o f d i f f e r e n t fuchs ins on t h e a ldeh de- fuchs in method f o r t he demonst ra t ion _____ of e1;stic tissue. - -

A m o d i f i c a t i o n o f t he Cameron and S tee le Method f o r t he i d e n t i f i c a t i o n o f neurosecre tory substance (1959) was in t roduced by Davenport (1979) as a polychrome technique f o r t he i d e n t i - f i c a t i o n o f t i s s u e types. Th is m o d i f i c a t i o n was f u r t h e r discussed as a q u a l i t a t i v e technique by Davenport and B a l l (1979).

t he pr imary s t a i n , namely a ldehyde- fuchs in , which i s used f o r t he i d e n t i f i c a t i o n o f e l a s t i c t i ssue . As w i t h a l l o t h e r techniques employing aldehyde- fuchs in , a major concern has been which i s t he bes t f uchs in t o use and a lso , what i s t he probab le mechanism o f at tachment o f t he aldehyde- f u c h s i n complex t o the e l a s t i c t i s s u e . I n the present study, t h ree fuchs ins were used: r o s a n i l i n , p a r a r o s a n i l i n , and magenta 111 (new f u c h s i n 111). Q u a l i t a t i v e l y , t he paraldehyde- p a r a r o s a n i l i n was observed t o be the most s p e c i f i c f o r e l a s t i c t i s s u e as w e l l as g i v i n g the most i n tense s t a i n i n g reac t i on .

t he [mechanism o f a c t i o n o f t he paraldehyde- p a r a r o s a n i l i n i n a t tach ing t o the e l a s t i c t i ssue ; namely, t he i n t e g r i t y o f t he resonance s t r u c t u r e respons ib le f o r t he reac t iveness o f t he azo- methines as proposed by Buehner st (1979) seems t o be p o s i t i v e l y a f f e c t e d by the s p e c i f i c combina t ion o f p a r a r o s a n i l i n and paraldehyde. The more s t r u c t u r a l l y complex fuchs ins , r o s a n i l i n and new fuchs in 111, p o s s i b l y cause a d e s t a b i l i z a t i o n o f the carbonium i o n o r perhaps i n t roduce the problem o f s t e r i c hindrance i n t h e i n t e r a c t i o n o f t he azomethines w i t h t i s s u e aldehydes.

The s p e c i f i c component o f t h i s technique i s

These da ta a l s o prov ided some suppor t f o r

Th is work was supported by N I H Biomedical Support Grant f5-SO7-RR05386.

Biomechanics of Two Joint Muscles in the Human Lower Extremity. ROBERT M. FISK, JAMES P. WELLS, West Virginia School of Osteopathic Medicine

The biomechanical significance of two-~olnt muscles has remained an unanswered question in the literature. The two-joint muscles of the human thigh provide several interesting models for such a mechanical system because of their close association with single-joint muscles which have similar functional roles; further- more, these two-joint muscles normally parti- cipate in both stereotyped, phasic movements (during locomotion) and in special, non-phasic movements. A series of muscle function experi- ments with male and female subjects and repre- senting a range of body types were initiated to determine the mechanical role of these muscles in a variety of different movements. The experiments ernplayed radiotelemetered EMG, in conjunction with indwelling (fine wire) electrodes to determine muscle activity. Correlative kinematic data were recorded with standard video techniques. The subjects per- formed normal locomotor and postural activi- ties and, in addition, worked against various resistances to elicit differential activity and/or phasic patterning between one- and two- joint muscles. two-joint muscles have evolved to meet require ments necessary for moving three limb segments

It has been proposed that

s imul taneous ly dur ing phas i c locomotor ac t i - v i t i e s ; however, t h e two- jo in t muscles o f t h e t h igh are r a r e l y c a l l e d upon t o do t h i s dur ing locomotion. I n f a c t , t h e unique b i - peda l s t a n c e o f man and t h e important r o l e of i n e r t i a l fo rces i n human locomotion b r ing about a r e l a t i v e l y low l e v e l of a c t i v i t y in t h e s e muscles dur ing such phas ic movements. Our s tudy demonstrates t h a t t he f u n c t i o n a l d i f f e r e n c e s between one- and two- jo in t muscles appear dur ing non-locomotor of t h e lower l i m b . We conclude from t h e s e EMG da t a and from comparative morphological s t u d i e s t h a t t h e presence of two- jo in t muscles i n t h e human th igh i s due p r imar i ly t o mechanical needs i n t h e a n c e s t r a l t ypes .

S.D. Gaede and S.L. Q u a t t r o p a n i , Departments of Anatomy, Univers i ty of V i rg in i a , C h a r l o t t e s v i l l e , Vi rg in ia and Medical Col lege of V i r g i n i a , Richmond, V i rg in i a . Endothe l ia l mi tos i s i n the immature gonadotrophin-stimul a t ed r a t ovary .

The m i t o t i c a c t i v i t y o f vasclrlar endothelium i n t he ovary was examined i n normal and gonado- t roph in - s t imu la t ed immature r a t s us ing 1 i g h t microscopy and au toradiography. The l a b e l i n g of endo the l i a l nuc le i with t r i t i a t e d thymidine was u t i l i z e d a s an index of vascu la r growth. A n i - mals rece ived an i n t r a p e r i t o n e a l i n j e c t i o n of thymidine 20 minutes p r i o r t o r e t r o g r a d e a lde - hyde per fus ion v i a the a o r t a . Twenty-one day o ld r a t s were adminis te red a s i n g l e i n j e c t i o n of PMS. An i n j e c t i o n of HCG 56 hours l a t e r induced su- perovula t ion by 17 hours a f t e r t rea tment (meth- od of France and Pincus, ‘ 6 4 ) . Four groups were s t u d i e d : 1 ) 28 hours a f t e r t h e PMS i n j e c t i o n , 2 ) 4 hours , 3 ) 17 hours, and 4 ) 24 hours a f t e r HCG i n j e c t i o n . Twenty-one and twenty-four day o ld animals which had not rece ived i n j e c t i o n s of hormones were g iven s i m i l a r dosages of thymi- d i n e and examined as c o n t r o l s .

The percentages of l abe led endothel i a l c e l l s var ied according t o the reg ion o r t i s s u e and the s t a g e of d i f f e r e n t i a t i o n of t h a t reg ion o r t i s - sue. Stromal v e s s e l s were gene ra l ly l e s s heavily l abe led than theca l v e s s e l s . Thecal v e s s e l s su r - rounding l a r g e heal thy f o l l i c l e s were more heav- i l y l abe led than surrounding sma l l e r o r a t r e t i c f o l l i c l e s . The heav ie s t l a b e l i n g was seen i n the v e s s e l s surrounding rup tu red f o l l i c l e s and w i t h - i n developing corpora l u t e a found i n t h e PMS- H C G t r e a t e d o v a r i e s .

Labeling ind ices of t he endothelium in the PMS-treated o v a r i e s were s i g n i f i c a n t l y e l eva ted in a l l reg ions cmpared t o the con t ro l o v a r i e s . This r e s u l t was not a l t e r e d by t ime o r HCG t rea tment .

These r e s u l t s i n d i c a t e t h a t endothel i a l mito- s i s i s a s e l e c t i v e response occuring in spec ia - l i z e d reg ions o f t h e ovary . I t i s sugges ted t h a t the gonadotrophic s t imu l i promote t h e production of f a c t o r s which e l i c i t such a response .

GARRIS, David R., J. FAIR*, and D. S. WHITEHEAD*, Department o f Anatomy, East Carol ina Un ive rs i t y School o f Medicine, Greenvi l le, N. C. 27834. A l te ra t i ons i n u te r i ne blood f l ow dur ing deciduoma fonnat ion 7 n t h e pseudopregnant ra t .

The a l te ra t i ons i n u t e r i n e blood f l ow (UBF) occurring dur ing deciduoma (DT) formation i n t h e r a t were studied t o de l ineate t h e e x i s t i n g developmental re la t ionships between c e l l u l a r d i f f e r e n t i a t i o n and t i ssue vascularization. DT-formation was induced i n pseudopregnant (PSP: Day 0 = ovulat ion) r a t s on Day 4 by a k n i f e scratch along the antimesometrial border o f each u te r ine horn. Sham-operated animals served as con t ro l s (C). Between Days 4-15 o f PSP. ra t s were prepared f o r UBF measurements by surg ica l exposure o f each u te r ine artery. Blood f l ow was measured w i t h an e l e c t m a g n e t i c blood f l ow monitor and analyzed f o r mean f l ow r a t e (ml/min). u t e r i were subsequently removed, cleaned, b l o t t e d and weighed t o t h e nearest mg. Uter ine weight remained constant a t basal l e v e l s (127-167 mg/horn) i n contro l ra ts throughout PSP. I n contrast, ra ts subjected t o u te r i ne trauma exhib i ted a s i g n i f i c a n t increase (p < 0.001) i n u te r i ne weight (Day 9: 1440 v s 138 mg/horn; DT vs C) as a resu l t o f t h e DT fonnation between Days 5 and 9 o f PSP. S im i la r l y , UBF remained at constant basal l e v e l s (0.5-0.8 ml/min) i n contro l ra t s dur ing PSP. DT-bearing ra ts exhib i ted a dramatic r i s e i n UBF between Days 4-7, fol lowed by a p la teau i n UBF l e v e l s (3-4.5 m l / m i n ) on Days 7-9. Between Days 9 and 15, both u te r i ne weight and UBF i n DT r a t s f e l l t o basal leve ls . t h a t UBF increases i n response t o induced dec idua l i za t i on dur ing PSP and tha t an increase i n UBF preceeds t h e period o f maximal s t romal d i f f e r e n t i a t i o n . These resu l t s suggest t h a t an inTrease i n t i s s u e vascular izat ion i s mandatory f o r t h e support o f c e l l u l a r d i f f e r e n t i a t i o n i n the r a t uterus. (Supported i n par t by a Grant fm t h e North Carol i na United Way).

The

The resu l t s o f t h i s study i n d i c a t e

GASSLER”, John H . and Margaret L . KIRBY, De- partment o f Anatomy, Medical Col lege o f Georgia, Augus ta Georgia. E lec t r ica l record- i ng of embryonic c h i c k m o t i l i t y .

The s t u d y o f av i an embryonic m o t i l i t y t r a d i t i o n a l l y has used the technique of w i n - dowing eggs and d i r e c t l y observ ing movement. Shortcomings w h i c h may a f f e c t t h e r e l i a b i l i t y of t h i s technique are: i t involves a major mod i f i ca t ion o f the embryo‘s environment, i t i s i m p o s s i b l e t o observe t h e e n t i r e embryo a t a l l times, and i t is d i f f i c u l t t o main ta in a n o b j e c t i v e s tandard f o r movement among d i f - f e r e n t observers . T h i s r e p o r t p re sen t s t h e use of a mod i f i ca t ion of a n e l e c t r i c a l record- ing t e c h n i q u e ( Jackson and R u b e l , J . Comp. Phys . P s y c h . , 1978) t o q u a n t i t a t i v e l y d e s c r i b e m o t i l i t y d u r i n g t h e las t h a l f o f i ncuba t ion i n t h e normal ch ick embryo.

F e r t i l i z e d White Leghorn e g g s f r o m 11 t o 1 9 d a y s o f i ncuba t ion i n c l u s i v e l y w e r e candled t o de te rmine t h e p o s i t i o n of t he embryo. Re- cord ing e l ec t rodes were i n s e r t e d and s e c u r e d 180° a p a r t on the eggs. Eggs younger than 14 d a y s were windowed, and the amnion c u t , t o prevent amnionic con t r ac t ion a r t i f a c t . Eggs were r e tu rned t o t h e incubator and connected t o a p o l y g r a p h . A f t e r 45 m i n u t e s o f acc l ima- t i z a t i o n , 15 minute record ings of m o t i l i t y

296

were made from f i v e e g g s on e a c h day . Record- i n g s w e r e a n a l y z e d and t h e t i m e s p e n t i n m o t i l i t y was c a l c u l a t e d .

M o t i l i t y i n c r e a s e d to day 1 3 , p l a t e a u e d on d a y s 1 4 and 1 5 , and s t e a d i l y d e c l i n e d from day 1 6 onward. Random (Type I ) and s t a r t l e (Type 11) movements w e r e no ted t h r o u g h day 1 5 . On day 1 6 o c c a s i o n a l p r e h a t c h i n g (Type 111) b u r s t s o f a c t i v i t y were observed between Types I and I1 movements. From day 1 6 o n , Type 1 and I1 a c t i v i t y d e c r e a s e d a s t i m e s p e n t i n Type 111 m o t i l i t y p a t t e r n s i n c r e a s e d .

s e r v a t i o n s of embryonic c h i c k s made by o t h e r i n v e s t i g a t o r s . T h i s t e c h n i q u e p r o v i d e s a n o b j e c t i v e , a c c u r a t e method o f q u a n t i t a t i n g t o t a l somat ic m o t i l i t y i n a v i a n embryos. It c a n be used t o e v a l u a t e e f f e c t s of experimen- t a l p r o c e d u r e s o n embryonic a v i a n m o t i l i t y .

These f i n d i n g s c o r r e l a t e w i t h d i r e c t ob-

GULATI, A . K., L a b o r a t o r y of Neurochemis t ry , NINCDS, N a t i o n a l I n s t i t u t e s o f H e a l t h , Bethesda Maryland and Department o f Anatomy, West V i r - g i n i a U n i v e r s i t y , West V i r g i n i a . Macromolecular changes i n t h e n e u r a l r e t i n a d u r i n g r-egenera- t i o n of a l e n s .

r e t i n a s t i m u l a t e s l e n s r e g e n e r a t i o n i n newts . The p r e s e n t s t u d y was d e s i g n e d t o i n v e s t i g a t e macromolecular (RNA and p r o t e i n ) changes i n t h e n e u r a l r t i n a a f t e r l e n removal . Incor - p o r a t i o n of 'H-uridine and H- leuc ine was used as a n i n d e x t o e v a l u a t e RNA and p r o t e i n syn- t h e s i s .

An i n i t i a l i n c r e a s e i n 3H-uridine u p t a k e was s e e n o n e t o t h r e e days a f t e r l e n t e c t o m y . T h i s was f o l l o w e d by g r e a t e r i n c o r p o r a t i o n o f

H- leuc ine between day 5 t o 15. To f u r t h e r c h a r a c t e r i z e p r o t e i n changes , s o l u b l e pro- t e i n s of n e u r a l retina from normal eyes and e y e s 1 2 d a y s a f t e r l e n t e c t o m y were a n a l y z e d by SDS ( 0 . 2 % ) polyacry lamide (7.5%) g e l e l e c t r o - p h o r e s i s . Coomassie b l u e s t a i n i n g r e s o l v e d a b o u t 30 bands , among t h e s e t h e r e were 1 6 prominent bands w i t h a p p a r e n t m o l e c u l a r w e i g h t s r a n g i n g from 18 ,500 t o 185,000. The banding p a t t e r n o f t h e n e u r a l r e t i n a w a s s i m i - l a r i n normal e y e s and i n e y e s 1 2 d a y s a f t e r l e n t e c t o m y . S y n t h e s i s o f s o l u b l e p r o t e i n s i n n e u r a l r e t i n a were also s t u d i e d by s l i c i n g j i s c g e l s and by a u t o r a d i o g r a p h y on s l a b g e l s .

i n t r a p e r i t o n e a l l y t h r e e h o u r s p r i o r to t i s s u e p r e p a r a t i o n f o r g e l s l i c i n g and a u t o r a d i o g r a p h y r e s p e c t i v e l y . Both d i s c and s l a b g e l s re- v e a l e d a b o u t 11 r a d i o a c t i v e b a n d s . The p a t t e r n of s y n t h e s i s of t h e s o l u b l e p r o t e i n s i n n e u r a l r e t i n a by b o t h e l e c t r o p h o r e t i c pro- c e d u r e s was q u i t e s imilar . L i q u i d s c i n t i l l a - t i o n c o u n t i n g on g e l s l i c e s showed some d i f f e r - e n c e s i n t h e n e u r a l r e t i n a o b t a i n e d from normal e y e s as compared t o l e n t e c t o m i z e d e y e s . How- e v e r , such d i f f e r e n c e s were n o t s e e n w i t h a u t o r a d i o g r a p h y . The p r e s e n t r e s u l t s demon- s t r a t e s i g n i f i c a n t changes i n RNA and p r o t e i n s y n t h e s i s i n n e u r a l r e t i n a d u r i n g l e n s regen- e r a t i o n . These changes may h e i n d i r e c t l y re- l a t e d t o t h e p r o d u c t i o n of n e u r a l r e t i n a l f a c - t o r , a l t h o u g h t h e p r e c i s e r e l a t i o n s h i p needs f u r t h e r i n v e s t i g a t i o n . E l e c t r o p h o r e s i s of s o l u b l e p r o t e i n s i n d i c a t e s t h a t n e u r a l r e t i n a l f a c t o r ( i f a s o l u b l e p r o t e i n ) is p r e s e n t i n normal, u n s t i m u l a t e d r e t i n a .

There i s e x t e n s i v e e v i d e n c e t h a t n e u r a l

3

H-leuc ine and 5S-methionine were i n j e c t e d

HIGHTOWER, James A . . Department of Anatomy, School of Medic ine , U n i v e r s i t y of South Caro- l i n a , Columbia, South C a r o l i n a . The e f f e c t s of s h a r k serum on DNA r e p l i c a t i o n i n murine lymphocytes .

t i c p r o p e r t i e s , h u t t h e mechanism(s) by which i t i n d u c e s tumor r e m i s s i o n is ( a r e ) unknown. However, d u r i n g t h e p a s t s e v e r a l y e a r s a number o f i n t e r e s t i n g r e p o r t s h a v e encouraged f u r t h e r s t u d y of t h i s q u e s t i o n . One i n v e s t i g a t i o n , f o r example, h a s demonst ra ted t h a t n u r s e s h a r k serum p o s s e s s e s a l e c t i n - l i k e molecule c a l l e d f r u c t o s a n - s p e c i f i c p r o t e i n (FSP). The f u n c t i o n of t h i s p r o t e i n i n v i v o and i n v i t r o is un- c e r t a i n , b u t w o r k e r s have demonst ra ted t h a t o t h e r ( p l a n t ) l e c t i n s c a n (1) be c y t o t o x i c f o r c e r t a i n tumor c e l l s , ( 2 ) a c t i v a t e immunocompe- t e n t c e l l s and ( 3 ) s t i m u l a t e t h e r e t i c u l o e n d o - t h e l i a l sys tem. Whether FSP p o s s e s s e s any of t h e s e b i o l o g i c a l p r o p e r t i e s is n o t known. Hence, t h i s i n v i t r o s t u d y w a s u n d e r t a k e n t o d e t e r m i n e t h e r o l e of whole n u r s e s h a r k serum and FSP on a c t i v a t i o n o f irmnunocompetent c e l l s a s a s s a y e d by DNA r e p l i c a t i o n i n mur ine thymic and s p l e n i c lymphocytes. R e s u l t s i n d i c a t e t h a t growth promot ing a g e n t s , even though l e s s e f - f e c t i v e t h a n t h o s e i n f e t a l c a l f s e r a , a r e pre- s e n t i n t h e s e r a of n u r s e s h a r k s . The a b i l i t y of b o t h mammalian and c h o n d r i c h t h i a n s e r a t o s u p p o r t mur ine s p l e n i c and thymic lymphocyte p r o l i f e r a t i o n induced by c o n c a n a v a l i n A st imu- l a t i o n s u g g e s t s t h a t t h e mechanism by which serum promotes growth of immunocompetent c e l l s a r o s e e a r l y p h y l o g e n e t i c a l l y and h a s been con- s e r v e d . FSP was p r e c i p i t a t e d by t r e a t i n g n u r s e s h a r k serum w i t h a n o p t i m a l amount of i c v a n P-6. The p r e c i p i t a t e d complexes were s o l u b i l i z e d , then chromatographed on Bio-gel P-150. P r o t e i n c o n t e n t h e r e was done v i a t h e Lowry a s s a y u s i n g bovine serum a lbumin (BSA) a s a s t a n d a r d . D i f f e r i n g l o t s of s h a r k serum y i e l d e d 90-18Opg FSP/ml serum. P r e l i m i n a r y d a t a s u g g e s t t h a t FSP is m i t o g e n i c f o r mur ine s p l e n i c lymphocytes , b u t n o t thymic lymphocytes.

j lhole s h a r k serum d e m o n s t r a t e s a n t i n e o p l a s -

Hin ton , David E . , E l i z a b e t h R . Walker, and John H. S t r o h l . Depar tments of Anatomy and Chemistry, West V i r g i n i a U n i v e r s i t y , Morgantown. West V i r g i n i a . Manganese-induced a l t e r a t i o n i - n - g i l l o f f a t h e a d minnow (Pimephales p r o m e l a s , Raf in- e s q u e ) : A l i g h t and e l e c t r o n m i c r o s c o p i c s t u d y .

V i r g i n i a c o n t a i n e d h igh l e v e l s of manganese ( M A ? maximum 83 mg/L) which were n o t s i g n i f i c a n t l y reduced by t r e a t m e n t w i t h a l k a l i n e s o l u t i o n s ( i . e . , s o d a a s h . l i m e s t o n e , l i m e , or c a u s t i c s o d a ) . L e v e l s of 3 ppm M*+ have been r e c o r d e d i n t h e Monongahela R i v e r and c o n c e n t r a t i o n s o f 10-20ipm a r e n o t uncommon i n ambient waterways o f c o a l - p r o d u c i n g r e g i o n s . S i n c e l i t t l e i n f o r m a t i o n was a v a i l a b l e on t i s s u e e f f e c t s o f Mn++ i n t e l e o s t s , t h i s a c u t e s t u d y (96 h r s ) w a s per formed t o de- t e r m i n e s t r u c t u r a l e f f e c t s o f Mn++ O n g i l l s of t h e f a t h e a d minnow. Twenty randomly s e l e c t e d f i s h ( 5 p e r each of 4 g l a s s a q u a r i a ) were p l a c e d i n w a t e r C o n t a i n i n g Mn++ as MnS04.H20 a t a f i n a l c o n c e n t r a t i o n o f 0 , 10, 50, o r 7 5 ppm. Mrt+con- c e n t r a t i o n a t 96 h r s was w i t h i n 10% of t h e i n i - t i a l c o n c e n t r a t i o n . Following f i x a t i o n (4% g l u - t a r a l d e h y d e i n 200 mOSm phosphate b u f f e r ) tis- s u e s were p r o c e s s e d f o r s c a n n i n g (SEMI andtrans-

- U n t r e a t e d w a t e r from s t r i p mine sites i n West

miss ion e l e c t r o n microscopy a s w e l l as h igh re- s o l u t i o n l i g h t microscopy (HRLM). SEM of g i l l s from a l l f i s h exposed t o Mn++ showed a l t e r a t i o n which i n c l u d e d : f o c a l absence o f secondary l a - m e l l a e ; d i s r u p t i o n o f m i c r o r i d g e p a t t e r n on p r i - mary and s x o n d a r y l a m e l l a e ; and h i g h a m p l i t u d e s w e l l i n g o f i n d i v i d u a l c e l l s on secondary l a - m e l l a e . Changes on buccopharyngea l s u r f a c e of g i l l a r c h p s i n c l u d e d d i s r u p t i o n of m i c r o r i d g e p a t t e r n and a p p a r e n t s w e l l i n g o f t a s t e buds . HRLM r e v e a l e d rounded, p a l e c e l l s wi th one or more l a r g e v a c u o l e s which formed b u l g e s v i s i b l e a t s u r f a c e s o f pr imary and secondary l a m e l l a e . I n a d d i t i o n , f o c i o f pr imary l a m e l l a e showed edema and c e l l d e g e n e r a t i o n i n secondary lam- e l l a r s i t e s which produced a smoother o u t e r con- t o u r on pr imary l a m e l l a e wi th no s u r f a c e i n d i c a - t i o n o f normal secondary l a m e l l a r p a t t e r n . T h i s novel l e s i o n , a p p a r e n t l y unique t o Mn++, may b e u s e f u l on f i e l d e v a l u a t i o n of f e r a l f i s h e s ex- posed t? mine w a t e r s . (Suppor ted by O f f i c e of Water Research and Tech- n o l o g y , U.S. Department o f I n t , o r i o r . P r o j e c t A-037-W.Va.).

H o l t , R . K . and G . S . S o h a l . Department o f Anatomy, Medica l C o l l e g e of C e o r e i a , Augus ta ,

t o r s have been p o s t u l a t e d t o d e s c r i b e t h e manner i n which numerous d i f f e r e n t i a t e d c h a r a c t e r i s t i c s of a d u l t s k e l e t a l musc le a r e m a i n t a i n e d . The r o l e o f i n n e r v a t i o n on t h e embryonic development o f s k e l e t a l musc le , how- e v e r , i s p o o r l y documented. The p r e s e n t s t u d y was under taken t o d e t e r m i n e t h e r e l a t i v e i m - p o r t a n c e of musc le a c t i v i t y and n e u r n t r o p h i c f a c t o r s on t h e d i f f e r e n t i a t i o n and main tenance of s k e l e t a l musc le . Normal, u n i n n e r v a t e d and p a r a l y z e d s u p e r i o r o b l i q u e musc les of w h i t e Peking duck embryos were u l t r a s t r u c t u r a l l y e v a l u a t e d throughout development w i t h t h e assumpt ion t h a t normal musc le d e v e l o p s under che i n f l u e n c e o f b o t h nerve-evoked musc le a c t i v i t y and t r o p h i c f a c t o r s , t h e u n i n n e r - v a t e d under n e i t h e r , and t h e p a r a l y z e d o n l y under t h e t r o p h i c f a c t o r s r e l e a s e d by t h e p r e s y n a p t i c n e r v e t e r m i n a l s . For embryos w i t h u n i n n e r v a t e d musc les t h e t r o c h l e a r n u c l e u s was d e s t r o y e d on day 7 of i n c u b a t i o n , 3 d a y s p r i o r t o normal i n n e r v a t i o n . P a r a l y s i s was produced i n a n o t h e r group by d a i l y a p p l i c a t i o n of t h e p o s t s y n a p t i c b l o c k i n g a g e n t a -bungaro toxin t o t h e v a s c u l a r i z e d c h o r i o a l l a n t o i c membrane from day 10 onwards. Both t h e p a r a l y z e d and unin- n e r v a t e d groups d i f f e r e d from normal i n t h a t development was r e t a r d e d . I n b o t h groups f u s i o n o f m y o b l a s t s t o form myotubes was d e l a y e d , myof i laments and 2 l i n e s were o l t e n d i s o r g a n i z e d , and l i p i d i n c l u s i o n s , f r e e r ibosomes , and c e l l d e a t h were f r e q u e n t l y s e e n . C e l l s c o u l d n o t d i f f e r e n t i a t e p a s t t h e myotube s t a g e i n u n i n n e r v a t e d musc le whereas many were a b l e t o p r o g r e s s t o t h e myof iber s t a g e i n p a r a l y z e d muscle. Reginning on day 21 o r 2 2 a mass ive wave o f c e l l d e a t h fo l lowed by c o n n e c t i v e t i s s u e rep lacement o c c u r r e d i n u n i n n e r v a t e d musc les h u t n o t i n p a r a l y z e d musc les . These o b s e r v a t i o n s s u g g e s t t h a t b o t h musc le a c t i v i t y and t r o p h i c f a c t o r s a r e neces-

s a r y f o r t h e normal development o f t h e musc le . Trophic f a c t o r s a p p e a r t o p l a y a more c r u c i a l r o l e t h a n musc le a c t i v i t y . g r a n t from t h e ? luscular Dys t rophy A s s o c i a t i o n and by a g r a n t from NIH).

(Suppor ted by a

Kinney, F. C l e v e l a n d , Department of Anatomy U n i v e r s i t y of Alabama i n Birmingham. BirminP-

V I 0

ham, Alabama. C i r c l i n g Behavior and Patho- l o g i c a l S l e e p i n t h e C a t .

P a t h o l o g i c a l s l e e p and c i r c l i n e b e h a v i o r

V I 0

ham, Alabama. C i r c l i n g Behavior and Patho- l o g i c a l S l e e p i n t h e C a t .

P a t h o l o g i c a l s " ~~

have been observed i n a s e r i e s o f e x p e r i m e n t s f o l l o w i n g t h e a b l a t i o n o f o r i r r i t a t i o n of d i f f e r e n t l e v e l s of t h e c e n t r a l n e r v o u s sys tem i n t h e c a t . I n a s e r i e s of b i l a t e r a l s t e r e o - t a x i c , e l e c t r o l y t i c l e s i o n s of t h e p o s t e r i o r hypotha lamus , immedia te ly d o r s a l and l a t e r a l t o t h e mamil la ry b o d i e s , p a t h o l o g i c a l s l e e p ( n a r c o l e p s y ) was o b s e r v e d . In o n e an imal a c a t a l e p t i c l i k e s t a t e coupled w i t h n a r c o l e p s y was s t r i k i n g l y a p p a r e n t . I n a n o t h e r an imal d e c e r e b r a t e r i g i d i t y and coma r e s u l t e d from a b i l a t e r a l s t e r e o t a x i c , e l e c t r o l y t i c l e s i o n o f t h e p o s t e r i o r hypotha lamic a r e a which i n c i - d e n t a l l y involved t h e b a s i l a r a r t e r y . T h i s c a t was u n r e s p o n s i v e t o p a i n and l a c k e d a c o r - n e a l r e s p o n s e . Fol lowing p e r i o d s o f p a t h o l o - g i c a l s l e e p , c i r c l i n g b e h a v i o r was observed from b i l a t e r a l l e s i o n s of t h e p o s t e r i o r hypo- tha lamus which ex tended i n t o t h e o v e r l y i n g v e n t r a l tha lamus .

b e h a v i o r h a s been observed from i r r i t a t i v e l e s i o n s of t h e f r o n t a l and i n s u l a r c o r t i c a l a r e a s i n t h e c a t , t h e d i r e c t i o n o f r o t a t i o n be- i n g dependent upon t h e p a r t i c u l a r c o r t i c a l a r e a i n v o l v e d . P e n i c i l l i n f i l l e d p e l l e t s p l a c e d over t h e head o f t h e c a u d a t e n u c l e u s have a l s o r e - s u l t e d i n s e i z u r e s d u r i n g which t h e a n i m a l s c i r c l e d toward t h e s i d e o f t h e i r r i t a t i o n . Of- t e n t h e s e i z u r e s became so v i o l e n t t h a t t h e a- n i m a l s f e l l o v e r backward. Immedia te ly fo l low- i n g t h e s e s e i z u r e s s l e e p was observed which l a s t e d f o r a s h o r t p e r i o d of two t o t h r e e min- u t e s .

s e r v e d from i r r i t a t i o n of v a r i o u s c o r - t i c a l a r e a s , from i r r i t a t i v e l e s i o n s of t h e head of t h e c a u d a t e n u c l e u s and f o l l o w i n g de- s t r u c t i v e l e s i o n s which involved t h e p o s t e r i o r hypotha lamus and t h e v e n t r a l tha lamus . S l e e p h a s been observed f o l l o w i n g s t i m u l a t i o n ( i r r i - t a t i o n ) o f t h e head of t h e c a u d a t e n u c l e u s and f o l l o w i n g d e s t r u c t i v e l e s i o n s o f t h e p o s t e r i o r hypo tha lamus .

Using p e n i c i l l i n a s a n i r r i t a n t , c i r c l i n g

I n summary, c i r c l i n g b e h a v i o r h a s been ob-

L a n t z , R . C l a r k and David E . H i n t o n , Department of Anatomy, West V i r g i n i a l l n i v e r s i t y , Morgantow W.V. A l t e r a t i o n o f lung s t . r u c t u r e by p a r t i c u - l a t e e f f l u e n t s from f l u i d i z e d bed c o a l combus- __ t i o n : A rnorphometric s t u d x .

l y deve loped p r o c e s s f o r u t i l i z a t i o n of s u l f u r - c o n t a i n i n g c o a l , is b e i n g developed f o r cumrner- c i a 1 a p p l i c a t i o n . P a r t i c u l a t e e f f l u e n t ( f l y & ) from t h i s p r o c e s s had d i f f e r e n t p h y s i c a l and chemica l p r o p e r t i e s t h a n c o n v e n t i o n a l f l y a s h . We a r e c o n d u c t i n g s t u d i e s on t h e pulmonary tox- i c i t y o f FBC p a r t i c u l a t e s and p r e s e n t i n i t i a l f i n d i n g s i n t h i s r e p o r t . Baghouse f i l t e r - c o l -

F l u i d i z e d bed coa l combust ion ( F B C ) , a r e c e n t -

l e c t e d f l y a s h from t h e MERC 18" a t m o s p h e r i c FBC b u r n i n g Montana Rosebud sub-b i tuminous c o a l was suspended i n s a l i n e ( 1 5 m g / m l ) and i n t r a - t r a c h e a l l y i n s t i l l e d i n t o male S y r i a n g o l d e n h a m s t e r s ( 3 mg f l y a s h i n 0 . 2 m l s u s p e n s i o n v o l - ume). C o n t r o l s r e c e i v e d e q u a l volumes of s a l i n e s o l u t i o n a l o n e . for 4 wks fo l lowed by A w k s p o s t exposure p r i o r t o k i l l i n g . Airways and l u n g s were f i x e d & - s i t u by i n t r a t r a c h e a l p e r f u s i o n of 3% g l u t a r a l - dehyde i n 0.1 m phosphate b u f f e r a t 20 cm HzO p r e s s u r e for 1 h r . T i s s u e s were embedded i n Epon and s e m i t h i n s e c t i o n s ( 1 ~ ) were s t a i n e d w i t h t o l u i d i n e b l u e for h i g h r e s o l u t i o n l i g h t microscopy. S t a n d a r d morphometric t e c h n i q u e s were used t o d e t e r m i n e volume d e n s i t i e s o f a l - v e o l a r d u c t s and s a c s and parenchymal t i s s u e . Two f i e l d s p e r s e c t i o n , one s e c t i o n p e r l o b e were s e l e c t e d a t random from p r o c e s s e d t i s s u e s . T o t a l f i e l d s a n a l y z e d were 32 for c o n t r o l s and 28 f o r e x p e r i m e n t a l a n i m a l s . N o s i g n i f i c a n t d i f f e r e n c e s were found between volume d e n s i t y of a l v e o l i o r a l v e o l a r d u c t s and s a c s i n c o n t r o l and e x p e r i m e n t a l a n i m a l s . However, volume den- s i t y o f parenchymal t i s s u e ( V v t ) w a s s i g n i f i c a - n t l y i n c r e a s e d i n e x p e r i m e n t a l a n i m a l s ( V v con- t ro l=17.8%+6.8 ; exper imenta l=22.4+8.9 P<O . 6 5 ) , T h i s may be i n d i c a t i v e of f i b r o s i s , a l v e o l a r pneumonocyte p r o l i f e r a t i o n o r b o t h . F u r t h e r t e s t i n g is [under way t o d e t e r m i n e t h e n a t u r e bf t h e i n c r e a s e i n parenchymal t i s s u e volume den- si t y . Suppor ted by Depart.ment o f Energy c o n t r a c t number DE-AT21-79 MC 1123A.

Exposures were g i v e n once/wk

MARK, Marvin R . , Cathy Pennington and Mohamed Sharawy. S u r f a c e F e a t u r e s of t h e Kat P a r o t i d Gland. D e n t i s t r y , Au$usta, Georg ia .

g l a n d was s t u d i e d i n t h e s c a n n i n g e l e c t r o n m i - c r o s c o p e f o l l o w i n g e i t h e r m i c r o d i s s e c t i o n o r enzymat ic d i g e s t i o n w i t h c o l l a g e n s e and o t h e r enzymes. Examination of d i s s e c t e d f r e s h t is- s u e conf i rmed t h e o r g a n i z a t i o n of t h e g land a s c o n c e p t u a l i z e d by c l a s s i c a l h i s t o l o g i c a l methods, v i z . , a n e l a b o r a t e sys tem of branch- i n g d u c t s w i t h g r a p e - l i k e t e r m i n a l a c i n i . The number of c e l l s composing a n a c i n u s v a r i e d w i d e l y from a s few a s 6 t o a s many a s about 20. I n t i s s u e p r e p a r e d by d i s s e c t i o n a l o n e , a c i n a r c e l l s were covered by a f e l t w o r k of d e l i c a t e c o l l a g e n f i b e r s . The f i b e r s tended t o f i l l i n t h e s p a c e between t h e b a s a l s u r f a c e s o f ad- j a c e n t c e l l s . Acinar c e l l s p r e s e n t e d s e v e r a l d i s t i n c t i v e s u r f a c e f e a t u r e s . The c e l l s had a t e x t u r e d s u r f a c e t h a t was o r g a n i z e d i n p a r a l l e l KOWS g i v i n g a s c a l l o p e d a p p e a r a n c e . P a r a l l e l t o t h e s e rows a c e l l was o f t e n seen t o he d i v i d e d i n t o hemispheres by a s i n g l e c l e f t . ?lost c e l l s were a l s o p u n c t u a t e d by s e v e r a l c r a t e r s on t h e o r d e r of 1 wn. A t high- e r m a g n i f i c a t i o n a l a r g e number of v e r y s m a l l p o r e l i k e s t r u c t u r e s was a l s o s e e n .

The i n t e r l o b u l a r d u c t s c o u l d n o t be i d e n t i f i e d w i t h c e r t a i n t y by s u r f a c e mor- phology a l o n e . In g e n e r a l , however, d u c t s had a much smoother s u r f a c e t h a n t h e a c i n a r c e l l s . O c c a s i o n a l l y a d u c t was s e e n unas- s o c i a t e d w i t h a c i n a r c e l l s . t e c m i n a t i n e i n

X e d i c a l C o l l e g e of Georgia School of

The s u r f a c e morphology of t h e r a t p a r o t i d

a t u f t o f f i n g e r - l i k e p r o j e c t i o n s . Smal l a r t e r i o l e s were o f t e n i d e n t i f i a b l e by t h e p r e - sence of p e r i c y t e s d i s t r i b u t e d a t r e g u l a r

enzymat ic t r e a t m e n t a c i n a r c e l l s a p p e a r e d a u i t e d i f f e r e n t from t h e i r u n t r e a t e d c o u n t e r -

L I E B , Richard J. , Robert L . JORDAN and J . S t u a r t OGLESBY, Department o f Anatomy, Medical Co l lege o f V i r g i n i a , V i r g i n i a Commonwealth i n t e r v a l s a l o n g t h e s u r f a c e . Fol lowing U n i v e r s i t y , Richmond, V A 23298. E f f e c t s o f pu l sed 27.12 MHz e lec t romagnet ic r a d i a t i o n on prenata l development i n the r a t .

descr ibed the t e r a t o l o g i c a l e f f e c t s o f non- i o n i z i n g rad io f requency r a d i a t i o n i n the r a t (Ann. N . Y . Acad. Sc i . , 247~367 , 1975; Tera to logy , E : 3 6 A , 1 9 7 r These e f fec ts have been a t t r i b u t e d t o thermal i n s u l t caused by the i r r a d i a t i o n . The present i n v e s t i g a t i o n u t i l i z e d a pu lsed r a d i a t i o n source which d i d no t produce apprec iab le temperature e l e v a t i o n i n the exper imenta l animals. A t o t a l o f 110 pregnant Sprague-Dawley r a t s were d i v i d e d i n t o two c o n t r o l (na ive and sham i r r a d i a t e d ) and n ine exper imental groups. The sham c o n t r o l and the exper imental animals were i r r a d i a t e d f o r 20 minlday on days 5 t o 15 o f ges ta t i on w i t h a dose o f 0 (sham c o n t r o l ) t o 4.9 mW/gm o f non ion i z ing i r r a d i a t i o n . Con t ro l and exper i - mental animals were examined f o r bo th s k e l e t a l and v i s c e r a l de fec ts . Ana lys is o f t e r a t o l o g i c a l data by means o f t he Chi-square d i s t r i b u t i o n w i t h Yates' c o r r e c t i o n f o r c o n t i n u i t y revea led no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between c o n t r o l and exper imenta l animals. The r e s u l t s o f t h i s s tudy suppor t t he hypothesis o f p rev ious i n v e s t i g a t o r s t h a t ma1 fo rmat ions observed i n o f f s p r i n g o f animals t r e a t e d w i t h 27.12 MHz non ion iz ing i r r a d i a t i o n a re the r e s u l t o f thermal i n s u l t t o the deve lop ing embryo and n o t a " f requency window" e f f e c t .

Supported, i n p a r t , by an A.D. W i l l i ams Summer Research Fe l lowsh io .

Recent r e p o r t s i n the l i t e r a t u r e have p a r t s . Blebs 0.5 t o 1 .0 Um were p r e s e n t on t h e m a j o r i t y of t h e t r e a t e d c e l l s . The c r a t e r s a l s o became more n o t i c a b l e , b u t were l o c a l i z e d between :he b l e b s . The d i s t r i b u t i o n of b l e b s appeared q u i t e s i m i l a r t o t h a t s e e n i n t r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of s i m i - l a r l y t r e a t e d t i s s u e . These o b s e r v a t i o n s sug- g e s t t h a t enzymat ic d i g e s t i o n might c r e a t e c e l l s u r f a c e a r t i f a c t s .

ElcDONALD*, Alexander J . , Department of Anatomy, U n i v e r s i t y o f South C a r o l i n a School of Medic ine , Columbia, South C a r o l i n a . ( I n t r o d u c e d by Wil l iam L. P o t e a t ) C y t o a r c h i t e c t u r e of t h e b a s o l a t e r a l amygdala: a G o l g i s t u d y i n t h e ra t .

The amygdala, a t e l e n c e p h a l i c n u c l e a r com- p l e x involved i n b e h a v i o r and emot ion , h a s been t h e s u b j e c t of numerous a n a t o m i c a l , b i o c h e m i c a l , p h a r m a c o l o g i c a l , p h y s i o l o g i c a l , and b e h a v i o r a l i n v e s t i g a t i o n s . S i n c e t h e rat i s t h e a n i m a l most o f t e n u t i l i z e d i n s u c h e x p e r i m e n t s , i t is of i n t e r e s t t o d e t e r m i n e t h e c y t o a r c h i t e c t u r e of t h e amygdala i n t h i s s p e c i e s . T h i s s t u d y is based on o b s e r v a t i o n s of t h e b a s o l a t e r a l p o r t i o n of t h e amygdala i n twenty rat b r a i n s s t a i n e d w i t h t h e Golgi-Kopsch t e c h n i q u e and s i x b r a i n s s t a i n e d w i t h t h e r a p i d Golg i t e c h n i q u e . Three major n e u r o n a l c l a s s e s a r e r e c o g n i z e d i n t h e b a s o l a t e r a l amygdhla. C l a s s I c e l l s , t h e main p r o j e c t i o n n e u r o n s , a r e pyramidal OK s t e l l a t e n e u r o n s whose d e n d r i t e s d i s p l a y a dense c o v e r i n g

of sp ines . In many cases the axon h i l l o c k and/or i n i t i a l segment may a l s o have sp ines . Axons g ive o f f a f e w beaded c o l l a t e r a l s and course i n the d i r e c t i o n of t he main e f f e r e n t pathways of the amygdala. Class I1 neurons a r e cha rac t e r i zed by sp ine-sparse d e n d r i t e s and a moderate t o dense l o c a l axonal a r b o r i z a t i o n . ’ These neurons vary i n s i z e , d e n d r i t i c branching p a t t e r n , and axonal morphology. Thei r axons g ive o f f numerous beaded c o l l a t e r a l s which a rbo r i ze and con tac t d e n d r i t e s of c l a s s I neurons. The axonal c o l l a t e r a l s of one v a r i e t y of c l a s s I1 neuron, t he “amygdaloid chandel ie r c e l l “ , d i sp l ay c l u s t e r e d v a r i c o s i - t i e s t h a t appear t o con tac t axon i n i t i a l seg- ments of c l a s s I neurons. Class I11 neurons a r e neurogl i form c e l l s t h a t have 7-9 s h o r t , t o r tu - ous , sp ine-sparse d e n d r i t e s t h a t branch exten- s i v e l y . Axons of Class I11 neurons g ive r ise t o numerous c o l l a t e r a l s which branch p ro fuse ly t o form a dense l o c a l axonal a r b o r i z a t i o n . Both d e n d r i t e s and axonal c o l l a t e r a l s con tac t dend- r i t e s of c l a s s I neurons. The r e s u l t s of t h i s s tudy i n d i c a t e t h a t the c y t o a r c h i t e c t u r e of the r a t b a s o l a t e r a l amygdala i s b a s i c a l l y similar t o t h a t seen i n o the r mammals. Future s t u d i e s w i l l i n v e s t i g a t e v a r i o u s u l t r a s t r u c t u r a l , hodologi- c a l , and neurochemical a s p e c t s of t he bas i c c e l l types found i n t h e b a s o l a t e r a l amygdala. (Sup- por ted by the Un ive r s i ty of South Caro l ina Grant 11040-F411).

MCKINNEY, R . V. , D. E . STEFLIK, J . L. BOSHELL, R. BAKER and E . E . SINGH. Department o f Ora l Pathology, Medical Co l lege o f Georgia, Augusta and U n i v e r s i t y o f L o u i s v i l l e , Kentucky.

umns i n s k i n and the repo r ted presence o f spe- c i f i c c e l l columns i n the f i l i f o m p a p i l l a e o f mouse tongue (Hume and Pot ten , J . C e l l Sc i . 2 2 : 24, 1976) has prov ided an impetus t o i n v e s t i g a t e the s t r u c t u r a l o rgan iza t i on o f spec ia l i zed ep i - t h e l i a such as the p a p i l l a e o f t he tongue. The purpose o f t h i s study was t o examine t h e c e l l u - l a r o rgan iza t i on o f t he tongue f i l i f o r m p a p i l l a e o f cow, dog and monkey i n o rder t o c o r r e l a t e the p a p i l l a e morpho log ica l development. Dorsal tongue fran Rhesus monkey, cow and dog was f i x e d i n 10% neu t ra l bu f fe red fo rma l in , embedded i n p a r a f f i n , c u t s e r i a l l y a t 5 pm and s ta ined w i t h H & E. For scanning e l e c t r o n microscopy (SEM), a d d i t i o n a l t i ssues were f i x e d i n Karnovsky’s paraformaldehyde-glutaraldehyde and p o s t - f i x e d i n 1% osmic ac id . C o r r e l a t i o n o f SEM and the s e r i a l sec t ions o f monkey tongue showed the presence o f two major d i f f e r e n t i a t i n g . c e l l l i n e s compris ing the a n t e r i o r ( s o f t k e r a t i n ) and pos- t e r i o r (hard k e r a t i n ) p o r t i o n s o f t h e p a p i l l a e . A t h i r d c e l l l i n e cu lmina ted i n the i n t e r p a p i l - l a r y area. p l e x p a p i l l a r y s t r u c t u r e w i t h two major c e l l l i n e s , bo th w i th recogn izab le boundar ies, con- s t i t u t i n g the pr imary f i l i f o r m p r o j e c t i o n . Two l a t e r a l c e l l l l n e s gave r i s e t o secondary p ro - j e c t i o n s and 5-7 c e l l l i n e s c o n s t i t u t e d sma l le r t e r t i a r y p r o j e c t i o n s i n dog tongue. species the f i l i f o r m p a p i l l a cons is ted o f cen- t r a l l y p laced v i a b l e e p i t h e l i a l c e l l s and k e r a t i n i z a t i o n occur red as these c e l l s migra ted v e r t i c a l l y and l a t e r a l l y . The cow f i l i f o r m p a p i l l a e was unique among species s tud ied i n our

Dog p a p i l l a e e x h i b i t e d a r a t h e r corn-

I n bo th

l a b o r a t o r y i n t h a t t he ex te rna l p a p i l l a r y p ro- j e c t i o n was f u l l y d i f f e r e n t i a t e d hard k e r a t i n analogous t o the h a i r co r tex . The hard k e r a t i n apparent ly d i f f e r e n t i a t e s f rom a dominant pos- t e r i o r c e l l l i n e and i t i s p a r t i a l l y ensheathed by a t h i n l a y e r o f s o f t k e r a t i n . The obvious species v a r i a t i o n i n tongue f i l i f o m p a p i l l a e c e l l l i n e s and the r e s u l t i n g s i g n i f i c a n t l y d i f - f e r e n t k e r a t i n i z a t i o n pa t te rns and p a p i l l a r y morphology may suggest an e v o l u t i o n a r y p a t t e r n o f development f o r spec ia l i zed use.

MONG, F .S .F . , A.P. KAYS, and A. MIRANDA, Dept. of Anatomy, Medical Col lege of V i r g i n i a , Richmond, V a . , and Dept. of Pa thology, Columbia Medical School, N Y , NY. Crea t ine phospho- k inase (CPK) isozyme t r a n s i t i o n i n regene- r a t i n g f r e e muscle t r a n s p l a n t s of mice.

CPK i n a d u l t s k e l e t a l muscle i s almost exc lus ive ly MM isoz jme, a dimer of two iden- t i c a l subun i t s . I n i t i a l l y , i n embryonic muscle, on ly t h e b r a i n form of CPK (BB) i s p resen t . But a s muscle developed t h e r e i s a rise of t o t a l CPK a c t i v i t y i n a g radua l t r a n - s i t i o n from BB t o MM. A hybr id form of CPK (MB) i s a l s o de t ec t ed dur ing t h i s pe r iod . The purpose of t h i s i n v e s t i g a t i o n w a s t o de te rmine whether a s i m i l a r t r a n s i t i o n i n CPK isozymes occurred i n r egene ra t ing t r a n s p l a n t s . F ree muscle a u t o g r a f t i n a d u l t male mice(1CR s t r a i n ) w e r e performed wi th or without i n j e c t i o n of marcaine, a myotoxin t h a t l e a d t o nec ros i s and even tua l r egene ra t ion of muscle f i b e r s . Trans- p l a n t s and so leus muscle from sham opera ted c o n t r o l were removed on day 1 ,3 ,5 ,7 ,9 ,15 & 25 af ter surgery and prepared f o r h i s to logy o r e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e membrane. I n e a r l y t r a n s p l a n t s t r e a t e d wi th marcaine most f i b e r s were n e c r o t i c bu t i n un t r ea t ed t r a n s - p l a n t s , muscle f i b e r s a t t h e per iphery o f t e n remained i n t a c t . The CPK isozymes changed as t h e r egene ra t ion of muscle p rogres s morphologi- c a l l y . The f e t a l BB and MB isozymes appeared t r a n s i e n t l y on day 3 5 7 By da t h e CPK w a s almost exc lus ive iy ’ac id t , an$ ?he regene- r a t i n g muscle f i b e r s reached an in t e rmed ia t e s t a g e of morphological d i f f e r e n t i a t i o n . The BB & MB isozymes seemed p a r t i c u l a r l y prominent i n marcaine i n j e c t e d t r a n s p l a n t s . Our r e s u l t s resembled t h o s e r epor t ed p rev ious ly for r egene ra t ing a d u l t muscle grown i n t i s s u e c u l t u r e s and sugges t t h a t r egene ra t ing muscles i n v ivo r e c a p i t u l a t e t h e ontogeny of muscle b iochemica l ly and morphologica l ly .

ODOR, D . L . , P. GADDUM-ROSSE, and R . J . BLANDAU. Department of Anatomy, Un ive r s i ty of South Caro l ina , Columbia, South Caro l ina and Depart- ment of B io log ica l S t r u c t u r e , Un ive r s i ty of Washington, S e a t t l e , Washington. Sec re to ry c e l l s of t h e oviduct of t h e p i g - t a i l e d monkey, Macaca nemest r ina .

The l i g h t and e l e c t r o n microscopic s t ruc - t u r e of t he s e c r e t o r y c e l l s of the ov iduc ta l ep i the l ium of the p ig - t a i l ed monkey has been s tud ied du r ing d i f f e r e n t s t a g e s of the menstru- a l cyc le and a f t e r ovariectomy wi th subsequent e s t rogen t rea tment . During the e a r l y f o l l i c u - l a r phase the ep i the l ium is recover ing from the d e c i l i a t i o n and s e c r e t o r y ce l l a t rophy t h a t occurs dur ing the l a t e l u t e a l s t a g e . Occas iona l f i m b r i a l and a f a i r number of ampullar and i s thmic cel ls e x h i b i t a f e w e l e c t r o n dense , homogeneous s e c r e t o r y

granules a p i c a l l y . During the l a t e preovula- t o ry and e a r l y pos tovu la to ry pe r iods t h e s e c r e t o r y c e l l s t r u c t u r e v a r i e s apprec iab ly i n d i f f e r e n t animals and i n d i f f e r e n t samples from the same animal. Usual ly f i m b r i a l c e l l s have a p i c a l p ro t rus ions con ta in ing no o r a few smal l homogeneous s e c r e t o r y granules . The cytoplasm is crowded wi th Golgi complex e lements , g ranu la r endoplasmic re t icu lum p r o f i l e s o f t e n in t ima te ly a s soc ia t ed wi th mi tochondr ia , and v a r i a b l e numbers of polysomes and glycogen g ranu les . Secre tory granules and lucen t vacuoles are more abundant i n ampullar and i s thmic than i n f i m b r i a l c e l l s . The granules a r e of two types : 1) those having an e l e c t r o n dense , homogeneous mat r ix and 2 ) those possess ing l ame l l a r s t r u c t u r e s wi th in moderately dense ma t r i ces . The lamel lae course i n p a r a l l e l a r r a y s and e x h i b i t a p e r i o d i c i t y . Homogeneous g ranu le s predominate i n the preovula tory phase and l ame l l a r granules i n the pos tovu la to ry pe r iod . Two types of vacuoles occur. During the l a t e l u t e a l phase and a f t e r ovariectomy i n a l l o v i d u c t a l segments on ly r a r e o r no s e c r e t o r y granules occur and o rgane l l e s involved i n s e c r e t i o n are usua l ly poorly developed. I n ovariectomized-estradiol t r e a t e d monkeys some a r e a s of a l l t h r e e segments may be we l l s t imu la t ed whi le o t h e r s have no g ranu le s and minimally developed o rgane l l e s . (Supported by subcon t rac t s from the Un ive r s i ty of Washington's USPHS g ran t HD 03752.)

OWERS, Noel 0. Department of Anatomy, Xedical College of V i rg in i a , V i r g i n i a Commonwealth Un ive r s i ty , Richmond, V i rg in i a 23298. A p o s s i b l e c o r r e l a t i o n between opac i ty i n a human ca t a rac tous l e n s and t h e presence of a c i d p ro teases .

from p a t i e n t s , who were o therwise normal and hea l thy . Two were cha rac t e r i zed a s be ing nuc lea r c a t a r a c t s and one as a pos t subcapsular c a t a r a c t based on s l i t - l amp obse rva t ion . A f t e r t h e i r removal, t h e l e n s e s were f rozen i n Tissue Tek O . C . T . compound and 8-16 li s e c t i o n s were c u t i n a c r y o s t a t maintained between -10 and -3OOC. g e l a t i n membrane s u b s t r a t e con ta in ing I n d i a ink and suppor ted on a g l a s s s l i d e , p rev ious ly i n - s o l u b i l i z e d by t r ea tmen t wi th g lu ta ra ldehyde ( O w e r s , 1970 J. Ce l l B io l . 47:152a, h e r s and

Three human ca t a rac tous l e n s were obta ined

The s e c t i o n s were p laced on a

Blandau, 1971 i n The Biolo of t h e Blas toc st Chicago Unive r s i ty P res s , g. 2 0 7 - 2 2 d ' s e c t i o n was submerged under a t h i n l a y e r of b u f f e r and t h e s l i d e s were incubated i n a humid chamber a t 23, 37, 40 and 4a0c f o r about 20 hours . A few drops of w a r m g l y c e r o l j e l l y were app l i ed and a g l a s s c o v e r s l i p was p laced over t h e j e l l y . Cont ro l s l i d e s were prepared by hea t ing t h e t i s s u e s e c t i o n i n water at 90°C f o r 10 minutes, followed by b u f f e r t rea tment t h e same a s f o r exper imenta l s l i d e s . The bu f fe r s used w e r e 30-50 nS+ KC1-HC1 uH 1 and 2.

t h e pos t subcapsular ca t a rac tous l e n s , d iges- t i o n occurred mostly a s s o c i a t e d wi th t h e pos- t e r i o r subcapsular t i s s u e s a l s o a t a c i d pH's. Neu t ra l p ro t eases were ub iqu i tous . Plaximw? d i g e s t i o n occurred at 37 and bO°C, minimum o r none a t 23 and 4 8 O C . done to determine whether t h e opaque t i s s u e i t s e l f causes t h e d i g e s t i o n of t h e membrane. Pre l iminary trials i n d i c a t e t h a t t h e a r e a s of d i g e s t i o n could be q u a n t i t a t e d us ing an Opt ronics drum scanning microdens i tometer (Model C4100).

Fur the r work needs t o be

PEPPLER, R . , T . KWASIGROCH and M . HOUGLAND, Departments of Anatomy and O b s t e t r i c s and Gyn- ecology, Quillen-Dishner Col lege of Medicine, East Tennessee S t a t e Un ive r s i ty , Johnson C i ty , Tennessee. Cross-sec t iona l anatomy: An easy - and inexpens ive % prepa re teaching spec i - mens.

With t h e advent of computerized a x i a l tomo- graphy (CAT) scans , g r o s s anatomy cour ses must now expose medical s t u d e n t s t o c ros s - sec t iona l anatomy. Commercial sources f o r such t each ing specimens a r e non-exis ten t and academic sources a v a i l a b l e w i l l supply specimens f o r as l i t t l e a s $100/sec t ion up t o $9-10,00O/complete body. These c o s t s a r e p r o h i b i t i v e t o many depar tments . Recent ly , we prepared 40 c ross - sec t ions which we used f o r a CME seminar and u l t i m a t e l y , which w e ' l l use i n our teaching programs. A t h i n , male cadaver w a s s e l e c t e d and c i r c u m f e r e n t i a l measurements i n t h e head, neck, thorax , abdomen, p e l v i s and e x t r e m i t i e s were obta ined . Subse- quen t ly , t h i s embalmed body was f rozen a t -2O'C. f o r two weeks u n t i l s ec t ioned . Bids were ob- t a ined from g l a s s companies f o r the cons t ruc t ion of 40 r ec t angu la r boxes accord ing t o our mea- surements. The boxes were made from ?I;'' p l a t e g l a s s , had an i n s i d e depth of 1" and a 4," ho le was d r i l l e d in one s i d e . The i n s i d e d iameters of t he boxes va r i ed from 7%" x 1 2 " t o 1%' x 24". F ive s i d e s of t h e boxes were s e a l e d wi th GE s i l - icone c l e a r rubber cau lk (GE 2567-012) by the g l a s s company. The specimen was removed from t h e f r e e z e r and sec t ioned i n 1" increments us- ing a commercial band saw. Represen ta t ive sec- t i o n s were r in sed in running water and placed i n t o the appropr i a t e s i z e d g l a s s boxes which conta ined J o r e s I1 f l u i d a s a p re se rva t ive . A cork ( s i z e 0) covered wi th pa ra f i lm was i n s e r t - ed i n t o the ?' hole . Sec t ions were allowed t o thaw overn ight and t h e p re se rva t ive w a s changed t h e fo l lowing day i n o rde r t o g e t r i d of excess d e b r i s . A bead of t he GE s i l i c o n e cau lk was placed around t h e top edges of t h e f d u r s i d e s and the g l a s s t op was pos i t i oned . Af t e r s u f f i - c i e n t d ry ing t ime, t he boxes were edged wi th y' f i r corner molding which was then sea l ed wi th a low g l o s s tung o i l varn ish . T o t a l investment f o r t he 40 specimens was $1200 o r $30 / sec t ion not count ing our expenses in ob ta in ing the ca- daver . Self-assembly of t he g l a s s boxes would reduce t h i s c o s t cons iderably .

-

phospha te -c i t r i c pH 3 , ve rona l acetate-HC1 pH 4 , 5 , 6 , and Tris-HCl pH 7 .5 con ta in inp e i t h e r 10 mM C a and 1 f i j Mg o r 10 d4 EDTA. POOLE, M. C . , H . W. B U R D E N , I . E . LAWRENCE, The a r e a under t h e t i s s u e where d i g e s t i o n of J r . , and C. A. HODSON*, Department o f Anatomy t h e membrane occurred appeared as l i q h t spo t s and Department o f Obstetr ics and Gynecology, a g a i n s t a dark background. In t h e two nuc lea r School o f Medicine, East Carolina University, c a t a r a c t o u s l e n s e s , d i g e s t i o n of t h e membrane Greenville, N. C. 27834. Suppression o f occurred i n t h e nuc lea r zone a t a c i d pHs. I n Gonadotropin Secretion by Hyperprol actinemia.

301

Hyperprol a c t i nemi a (excess ive pro1 a c t i n ) i s known t o suppress gonadotropin sec re t i on , and impa i r rep roduc t i ve f u n c t i o n i n women. The f o l l o w i n g s tudy i n v e s t i g a t e s t h e suppression o f s t i mu 1 a ted gonadot ropes f o l l o w i n g ovar iectomy i n r a t s bear ing a p r o l a c t i n s e c r e t i n g tumor (Fu r th MtTW15). group o f c y c l i n g female Wis ta r -Fur th r a t s were implanted subcutaneously w i t h the M t T W l S tumor. A f te r 35 days o f tumor growth, t he p r o l a c t i n l e v e l s were e leva ted (26.7 t 3.8 ng/ml i n d ies t rous tumor f r e e animals vs. 4162 +_ 297 ng/ml i n tumor bea r ing r a t s ) , and the animals were a c y c l i c . These r a t s were then ovar iec tomized and f o l l o w i n g 7 days the p i t u i t a r i e s were removed f o r e l e c t r o n microscopy and the serum assayed f o r LH ( l u t e i n i z i n g hormone) and FSH ( f o l l i c l e s t i m u l a t i n g hormone). An a d d i t i o n a l group o f tumor f r e e 7 day ovar iec tomized r a t s were used as con t ro l s . The concent ra t ions o f bo th LH and FSH were suppreksed i n the tumor bear ing ovar iec tomized animals as compared t o t h e 7 day ovar iec tomized tumor f r e e r a t s (FSH, 868 i 109 ngiml i n tumor bear ing vs. 2589 +_ 155 ng/ml i n tumor f ree ; LH, 40.1 +_ 10.7 ng/ml i n tumor bear ing vs. 377 +_ 24 ng/ml i n tumor f r e e ) . E lec t ron microscopy revea led t h a t gonadotropes i n the ovar iec tomized tumor f r e e animals were l a r g e c e l l s , w i t h an ex tens ive network o f d i l a t e d R E R , and a l a r g e Golgi complex. The gonadotropes i n t h e MtTW15 bear ing animals were smal le r , w i t h decreased v e s i c u l a r R E R , and smal le r Go lg i complexes. Furthermore, t he re was an apparent decrease i n the volume o f mi tochondr ia . These s tud ies suggest t h a t hyperpro l a c t i nemia suppresses bo th the syn the t i c and sec re to ry mechanisms o f t he gonadot ropes.

A

. !u:i t troyzni, S teven >. DeDartment of Anatomy, l e d i c a l Co1lei.e of V i r g i n i a , Richmond, . i r g i n i a , 23298. ATPase i n t h e R e t e and

Cystadenomas o f t h e Guinea P i g Ovary. Serous cystadenomas form s p o n t a n e o u s l y i n

a l l g u i n e a p i g o v a r i e s as a c o r r e l a t e of age and r e s u l t o f d i l a t i o n o f t h e r e t e o v a r i i . CJ-sts r e a c h a d i a m e t e r of 5 cm and a r e l i n e d w i t h a s imple e p i t h e l i u m o f c i l i a t e d and n o n c i l i a t e d b u t no s e c r e t o r y c e l l s . I n a n e f f o r t to d e t e r n i n e t h e method of d i l a t i o n , c y s t i c and p r e - c y s t i c t i s s u e s have been e x m i n e d f o r .ATPase a c t i v i t y . O v a r i e s from 12 and 26 day o l d and a d u l t a n i m a l s were a ldehyde-f ixed . Vibratome s e c t i o n s (100 u )

ng a n i m a l s c o n t a i n i n g n o n d i l a t e d and

I t s were i n c u b a t e d a t 37' f o r 20 min. d i l a t e d r e t e , and c y s t f ragments

i n a ne6iis c o n t a i n i n g 1 mM ATP, 100 mM N a C l , 1 5 d 4 KC1, 10 mN I4gSO,,: 3.6 mM Pb ( N O ) and 3% s u c r o s e i n 0.1 I 4 T r i s b u f f e r ( p H 7353. C o n t r o l i n c u b a t i o n s i n c l u d e d e l i m i n a t i n g t h e s u b s t r a t e o r v a r i o u s c o f a c t o r s . Fol lowing i n c u b a t i o n t i s s u e s were washed i n b u f f e r , f i x e d i n osmium and p r o c e s s e d f o r e l e c t r o n microscopy.

Cyst o r r e t e e p i t h e l i u m of a l l a g e s e x h i b i t e d r e a c t i o n Froduct w i t h i n t h e i n t e r c e l l u l a r s p a c e . I n t h i S r e e i o n smooth s u r f a c e d c a v e o l a e which were more common i n a d u l t s t h a n immatures a l s o were p o s i t i v e f o r phospi ia tase . The r e a c t i o n p r o d u c t was

l i m i t e d t o t h e s e r e g i o n s , w a s dependent on t h e p r e s e n c e o f ATP and w a s n o t formed when f i -g lycerophosphate was u s e d i n p l a c e of ATP. '%en 'la, K and Xg were e l i m i n a t e d , t h e r e w a s no r e a c t i o n . When o n l y Na and K o r 14g was e l i m i n a t e d , however, r e a c t i o n p r o d u c t , a l t h o u g h n o t as prominent , was s t i l l d e t e c t e d i n some p r e p a r a t i o n s .

P r e v i o u s s t u d i e s have i n d i c a t e d t h a t t h e e p i t h e l i u n o f t h e c y s t i c and n o n c y s t i c r e t e i s permeable t o t r a c e r s such as lan thanum and h o r o e r a d i s h p e r o x i d a s e and e x h i b i t s no e x o c r i n e s e c r e t o r y a c t i v i t y . The p r e s e n t s t u d y i n d i c a t e s t h a t t h e l a t e r a l c e l l membranes have a n A'I'Pase a c t i v i t y which i s i o n dependent . It is s u g g e s t e d t h a t t h i s may f a c i l i t a t e a pum?ing sys tem f o r the d i l a t i o n o f t h e r e t e . ( S d p p o r t e d by NI l i Grant AG 0 0 6 5 2 ) .

ROSENQUIST, Thomas and Donald STEWART, Depar t - ment of Anatomy, Medical C o l l e g e o f G e o r g i a , Augus ta , G e o r g i a . Measurement o f T i s s u e D i s u l f i d e Groups by A l c i a n Blue 8GX and Atomic A b s o r p t i o n Spec t rophotometry .

R e c e n t l y BEck (1978) h a s r e p o r t e d a p r o c e d u r e f o r h i s t o c h e m i c a l d e m o n s t r a t i o n o f d i s u l f i d e groups i n t i s s u e p r o t e i n s by c o n v e r s i o n o f c y s t i n e t o S - s u l f o c y s t e i n e and subsequent s t a i n i n g o f t h e r e s u l t a n t s u l f a t e s by t h e c r i - t i c a l e l e c t r o l y t e c o n c e n t r a t i o n method f o r A l c i a n b l u e 86X. I n t h i s s t u d y , k i d n e y s were o b t a i n e d from mice and f i x e d i n a s o l u t i o n of 10% f o r m a l i n b u f f e r e d w i t h 0.5M phosphate , pH 7 .0 (NBF) o r i n a s o l u t i o n of 95 p a r t s e t h a n o l / 1 5 p a r t s f o r m a l i n 1 5 p a r t s g l a c i a l a c e t i c a c i d (AFA). Fol lowing p a r a f f i n embedding, s e c t i o n s o f 1 0 pm were o b t a i n e d and were s t a i n e d i n a 0.52 s o l u t i o n of A l c i a n b l u e 8GX ( P o l y s c i e n c e s , I n c . ) ; o r were s t a i n e d f o l l o w i n g t h i o s u l f a t i o n . S t a i n e d and u n s t a i n e d c o n t r o l s e c t i o n s were o b s e r v e d , t h e n were removed from t h e s l i d e s and weighed t o t h e n e a r e s t mg. Pooled s e c t i o n s were d i s s o l v e d i n 35% n i t r i c a c i d and t i le amount of A l c i a n i c copper was measured. T i s s u e f i x e d i n 10% NBF c o n t a i n e d 1 . 1 5 ug/Cu/mg t i s s u e w i t h o u t t h i o s u l f a t i o n and 1 . 9 9 pgCu/mg t i s s u e a f t e r t h i o s u l f a t i o n ; t h e r e f o r e 0.84 ugCu/mg were due t o t h i o s u l f a t i o n . T i s s u e f i x - ed i n AFA c o n t a i n e d 1 . 3 5 pgCu/mg t i s s u e w i t h o u t t h i o s u l f a t i o n and 3 .25 pgCuImg t i s s u e a f t e r t h i o s u l f a t i o n ; t h e r e f o r e 1 .90 iigCu/mg were due t o t h i o s u l f a t i o n . No copper was d e t e c t e d i n u n s t a i n e d samples . Y i c r o s c o p i c o b s e r v a t i o n showed t h a t most of t h e v i s u a l i z a b l e n a t i v e s u l f a t e s were i d t h e basement membrane o f Bowman's c a p s u l e , whereas f o l l o w i n g s u l f a t i o n A l c i a n b l u e was v i s i b l e i n g l o m e r u l a r , t u b u l a r and x a s c u l a r b a s a l laminae a s w e l l : a s shown by Bock. We concluded t h a t : 1 ) X a t i v e s u l f a t e s a r e p r e s e n t i n q u a n t i t i e s e a s i l y d e t e c t a b l e b y AAS b u t d e t e c t a b l e less e a s i l y o p t i c a l l y and t h e y a r e found p r i n c i p a l l y i n t h e b a s a l lamina o f Borjman's c a p s u l e ; 2 ) AFA p r e s e r v e s more n a t i v e s u l f a t e s a s w e l l a s d i s u l f i d e s ( e . g . , i n Type I V c o l l a g e n ) t h a n d o e s 10% NBF. It should b e p o s s i b l e t o c a l c u l a t e t h e number o f d i s u l f i d e s p e r u n i t weight of t i s s u e by measur ing A l c i a n i c copper w i t h AAS a f t e r t h e s t a i n i n g p r o c e d u r e of B:ck i f one u s e s a t y p i - c a l A l c i a n b l u e 8GX which c o n t a i n s 1 copper atom p e r molecule .

Rudeen, P. Kevin, Department o f Anatomy, Bowman Gray School o f Medicine, Winston-Salem, Nor th Caro l ina . Alcohol- induced increase o f serum p r o l a c t i n i n male r a t s .

A lcoho l has been shown t o induce a fem in i ra - t i o n e f f e c t i n ch ron ic a l c o h o l i c males. Associ- a ted w i t h t h i s fem in i za t i on i s an inc rease i n plasma p r o l a c t i n . Th i s s tudy was made t o inves- t i g a t e the e f f e c t s o f an acu te a d m i n i s t r a t i o n o f a l coho l on serum p r o l a c t i n i n male r a t s . S i x t y - f o u r a d u l t male Sprague-Dawley r a t s were used i n t h i s study. The animals were a l lowed food and water ad 1 i bi tum f o r 2 weeks p r i o r t o e x p e r i m e n t a t i o n T - O n t a y o f t he exper iment, t he animals were p laced randomly i n t o groups which rece ived e i t h e r an i n t r a p e r i t o n e a l i n j e c - t i o n o f a l coho l (2.5 gm a l coho l / kg body we igh t ) o r an equ iva len t volume o f i s o t o n i c s 'a l ine . Fo l l ow ing the i n j e c t i o n , animals were k i l l e d a t i n t e r v a l s o f up t o 120 minutes; b lood was c o l l e c t e d and the serum was harvested f o r t he eva lua t i on o f p r o l a c t i n , l u t e i n i z i n g hormone (LH), and a l coho l . The r e s u l t s i n d i c a t e t h a t serum p r o l a c t i n inc reases above c o n t r o l l e v e l s w i t h i n 10 minutes o f t he i n j e c t i o n o f a l coho l . Th i s inc rease i s t r a n s i e n t , s ince serum p ro lac - t i n l e v e l s r e t u r n t o c o n t r o l l e v e l s w i t h i n an hour. Serum LH i s depressed below c o n t r o l l e v e l s w i t h i n 10 minutes, bu t r e t u r n s t o n o r - mal l e v e l s w i t h i n 2 h r s a f t e r t he i n j e c t i o n o f a l coho l . Serum a lcoho l l e v e l s a re increased t o about 225 mg% w i t h i n 1 0 minutes a f t e r t he i n j e c t i o n and a re s t i l l e leva ted above c o n t r o l l e v e l s 2 hrs a f t e r t he i n j e c t i o n . These data con f i rm o the r repo r t s showing reduced serum L H a f t e r a lcoho l admin i s t ra t i on and i n d i c a t e t h a t an acu te i n j e c t i o n o f a lcoho l w i l l induce a concomitant r i s e i n serum p r o l a c t i n . T h i s sug- ges ts t h a t a l coho l o r an a l coho l me tabo l i t e may a f f e c t p r o l a c t i n sec re t i on by i n f l u e n c i n g t h e p i t u i t a r y o r th rough i n h i b i t i o n o f hypo- tha lamic p r o l a c t i n i n h i b i t i n g f a c t o r . Add i t i on - a l research i s con t inu ing t o d iscount the s t r e s s f u l e f f e c t s o f a l coho l i n j e c t i o n as a p o s s i b i l i t y i n enhancing the serum p r o l a c t i n l e v e l s seen under cond i t i ons o f t h i s exper iment. Th is r e p o r t i n d i c a t e s t h a t some o f t he e f f e c t s o f a l coho l on the hypothalamo-pituitary-gonadal a x i s a re i nvo l ved w i t h the r e g u l a t i o n o f p ro - l a c t i n sec re t i on . (Supported by a g r a n t f rom the Nor th Caro l i na A lcoho l ism Research A u t h o r i t y . )

SCHIEBLER, T.H., ReTZEL, H., Department of

Anatomy, University of Wiirzburg, Wiirzburg/ FRG. The prenatal and early postnatal development of the glial cells of the rat

median eminence. The development of the glial cells of the median eminence ( M E ) , including the supra- ependymal cells, was investigated from em- bryonic day (ED) 14 through postnatal day (PD) 7, and pituicyte development from ED 12 through ED 17. The anlage of the ME and neurohypophysis shows a neuroepithelial-like

structure at ED 12. From ED 13 to 15, the

cells of both regions start to differentiate, accompanied by the formation of multiple cell layers. At the ultrastructural level, only one cell type occurs in the anlage of the ME

and neurohypophysis. A$ the start of ED 16, gliohlasts of the cligodendrocyte and astro-

cyte series migrate laterally (from the arcuate nucleus) into the ME. Also at this time the first distinctive structural fea-

tures appear in the neurohypophyseal anlage, whose cells later develop into pituicytes.

Starting at ED 18, tanycytes and astrocytic tanycytes form in the ME from local glial

cells, and somewhat later cligodendroblasts and astrohlasts form from immigrant glio- blasts of the arcuate nucleus. Owing to their common features, the pituicytes, tany-

cytes and astrocytic tanycytes apparently represent different forms of differentiation of the same parent cell type. Microglial and supraependymal cells are first seen at ED 12. Both cell types are apparently of meso- dermal origin.

Schneider", Peter J . and J. David Deck, Depart- ment of Anatomy, University of Virginia, Char- lottesville, Virginia. Turnover measurements of cardiovascular connective tissue components & -~ in vitro labeling.

Connective tissues of the heart valve are reg- ularly subjected to the stresses of cyclic valve operation. We have previously shown by autora- diography that the tissues of normal rat valves respond with a turnover of protein and ground substance, respectively higher in leaflet and leaflet attachment, apparently associated in each case with the regions of greatest tension and flexion. We have now found that similar evidences of tissue turnover can he obtained from excised rat valves labeled in vitro. !hen studied using liquid scintillation methods, valve tissues exposed to a 1.5 hour pulse label of tritiated-proline in vitro readily showed evidence of marked-precursor uptake, demonstra- ting the continued viability of the tissues. In addition, autoradiographic comparison of tis- sues labeled in vitro and in viva has produced the same patterns of labeling in both cases, namely higher labeling of proteins in leaflet than elsewhere. These observations indicate that tissue excision and short-term in vitro culture do not alter the pattern of protein synthesis in valvular tissues. We now expect to apply this technique to valves of larger an- imals where experimental alteration of struc- ture is more feasible. Changed structure re- flected in how these valves function may result in different turnover patterns and indicate the

regions most affected by the altered stresses. The in vitro labeling method can thus strength- en our evidence for stress-related tissue turn- over. Supported by NIH grant HL 16935.

SCHUSTER, G. S . , 6.6. SINGH, J . F . ERBLAND and D. E . STEFLIK, School o f D e n t i s t r y , Med ica l Co l leqe o f Georgia, Augusta. Growth and C h a r a c t e r i z a t i o n o f Hamster C h Z k T u c k E p i t h e l i u m I n V i t r o .

V a r i o u s c m e techn iques have been employed t o s tudy o r a l e p i t h e l i a b u t most r e s u l t e d i n l i m i t e d o r no ou tgrowth o f an e p i - t h e l i a l c e l l sheet. The o b j e c t i v e o f t h i s i n - v e s t i g a t i o n was t o deve lop a method t o ach ieve sus ta ined outgrowths o f e p i t h e l i a l c e l l s f rom hamster cheek pouch e x p l a n t s and d e s c r i b e t h e c e l l s ' ma jor c h a r a c t e r i s t i c s . Exp lan ts o f ham- s t e r cheek pouches were s u r g i c a l l y exc ised, washed i n T r i s b u f f e r e d s a l i n e and p laced i n .1% t r y p s i n f o r v a r y i n g t i m e p e r i o d s up t o 60 min- u t e s , t h e n m e c h a n i c a l l y scraped t o remove l o o s e connect ive t i s s u e . 1 x 2 mm p ieces o f t i s s u e were p laced on .22 Urn, .45 um, .8 um M i l l i p o r e f i l t e r s o r on p o l y s t y r e n e cover s l i p s . A l l were covered wi th a p l a s t i c cover s l i p , f e d w i t h MEM supplemented w i t h 102 ( v / v ) f e t a l bov ine serum and incubated a t 37OC i n 95% A i r - 5% C02 f o r up t o 15 days. r e p r e s e n t a t i v e samples each day i n 10% n e u t r a l b u f f e r e d f o r m a l i n and process ing them by p a r a f - f i n techniques f o l l o w e d by s t a i n i n g w i t h hema- t o x y l i n and eos in . A d d i t i o n a l c u l t u r e s were used t o c h a r a c t e r i z e t h e n a t u r e o f c e l l u l a r o u t - growths employing scanning e l e c t r o n m i c r o s c o p i c (SEM) and t r a n s m i s s i o n e l e c t r o n mic roscop ic (TEM) techn iques . P r e f e r e n t i a l e p i t h e l i a l c e l l ou tgrowth was seen on .22 pm and .45 urn M i l l i p o r e f i l t e r s w h i l e l i t t l e o r no ou tgrowth was e v i d e n t on .8 f i l t e r s . F i b r o b l a s t s were t h e predominant c e l l t y p e when t h e p l a s t i c cover s l i p was used as a g rowth sur face . I n genera l , immersion o f e x p l a n t s i n .l% t r y p s i n f o r 10-15 minutes was deemed e s s e n t i a l f o r i n i t i a t i o n o f c e l l u l a r ou tgrowth . 5EM s t u d i e s demonstrated po lygona l c e l l s o rgan ized i n sheets . The con- s t i t u e n t c e l l s were s i m i l a r i n shape and s i z e and covered w i t h m i c r o v i l l i . were o f t e n seen connect ing a d j a c e n t e p i t h e l i a l c e l l s w i t h occas iona l t h i c k e n i n g s probab ly r e p r e s e n t i n g i n t e r c e l l u l a r j u n c t i o n s . Obser- v a t i o n o f the c e l l u l a r ou tgrowth by TEM showed the presence o f abundant t o n o f i l a m e n t s and desmosomes, c o n f i r m i n g t h e e p i t h e l i a l n a t u r e o f t h e c o n s t i t u e n t c e l l s . Supported by DE 05449 frm N I D R .

Growth was mon i to red by f i x i n g

The m i c r o v i l l i

SCHWEISTHAL, M.R. and COLE, T . B . , JR., Department o t Anatomy, Oral Roberts Uni- versity, Tulsa, Oklahoma. A case of achalasia: gross anatomical -~ findix.

Achalasia is defined as a dilatation of the esophagus due to the inability o f the lower esophageal sphincter to relax arid to the absence of peristalsis in the body of

~ ~ _ _ _ _ _ _ _ _ _ _

the esophagus in response to swallowing. This postmortem study was done on an 85- year-old male who had a diagnosis of severe achalasid. Examination of the esophagus revealed that it was greatly enlarged between the upper and lower esophageal sphincters. The lumen of the esophagus as it passed through the diaphragm was so small that it would not accept an attempt to pass the index finger into the stomach. Three important anatomical features were observed: one, the criropharyngeus muscles were well defined; two, a distinct trans- verse ridge was found to project into the esophageal lumen dlrectly helow the upper esophageal opening; three, there was a well-defined extension of the dilated esophagus located posterior to the larynx. Muscle fibers from the cricopharyngeus were found to rontrihute to b o t h thr upper esophageal sphincter and the transverse ridge located helow the sphincter. Four hundred and ten grams of solid food ma- terial was found in the esophagus, but none was found 111 the respiratory tract. I t is concluded that the upper esophageal sphinc- ter wds functional and played an important role o t longevity i n this rase. The ana- t o m i c a l findings from this study w ~ l l be disriisscd wiLh regard t o somr of Lhe other cases reported i n the literature.

SICKLES, Dale W. and Roger E. McLENDON, Depart- ment of Anatomy, Medical College of Georgia, Augusta, Georgia. Metabolic Heterogeneity of

~~~ ~~

Rat Lumbosacral Motoneurons. Skeletal muscle fibers are known to differ in activity of enzymes involved in energy produc- tion and contraction. It has been suggested that motoneurons also exhibit a heterogeneity in activity of these metabolic enzymes. How- ever, Campa and Engel (Neurology 20: 559, 1970) found no difference in enzyme activities of a-motoneurons. More recent cytophotometric studies suggest that motoneurons are not iden- tical (Penny et al., J Neurol Sci 26: 179, 1975) . The current investigation examines quantitatively the activities of certain enzymes in the a-motoneuron pool of the rat lumbosacral cord with the photographic den- sitometry and atomic absorption spectrophoto- metry technique of Troyer and Rosenquist (J Histochem Cytochem 23: 941, 1975) . Rats were killed by decapitation; the spinal cords re- moved by rapid injections of saline into sac- ral vertebral column and quenched in liquid nitrogen. Enzyme histochemical techniques for the demonstration of succinic dehydrogenase (SDH) NADH dehydrogenase, menadione-linked a- glycero phosphate dehydrogenase, (MaGPDH) acid phosphatase, phosphorylase and Mg+Z-ac- tivated ATPase activities were performed on 10 p frozen sections taken through the long axis of anterior horn of lumbosacral cord. Photo- graphs of the motoneurons were taken on Kodak SO-115 film; the area corresponding to the neuron cytoplasm was cut out, weighed and the silver eluted o f f the negative with 2 ml of 35% nitric acid. The quantity of silver was determined with a Perkin-Elmer atomic absorp- tion spectrophotometer. The data indicates that a spectrum of activities exist in the rat

304

spinal cord motoneurons, similar to that pre- banks Zoo loq ica l Park, Columbia, South Caro l i na . viously reported in rat tibialis anterior muscle fibers. The activities of Mg+2-acti- vated ATPase, MaGPDH and SDH were uniformly negative. The greatest differences were ob- served with the NADH-D, phosphorylase and acid phosphatase techniques. The significance of the findings in reference to neuromuscular organization and selectivity of motoneurons to neuromuscular diseases will be discussed. Supported by BRSG Grant from N L H #2507RR05365- 19.

SINGH, B. B., J . L. BOSHELL, D. E. STEFLIK, S . L. ZUNT, R. V . MCKINNEY and D . L. SCHAFFNER. Department o f Ora l Pathology, Medical Co l lege o f Georgia, Augusta and Ind iana U n i v e r s i t y , I n d i a n a p o l i s . Observat ions on the Kera tohya l i n Granules o f Tongue F i l i f o r m P a p i l l a e fran G e r b i l , Cow, Spec ios is Monkey, P i g and Rabb i t .

shown t h a t t h e tongue f i l i f o r m p a p i l l a e o f t he hamster, r a t , mouse and guinea p i g con ta in bo th b a s o p h i l i c k e r a t o h y a l i n g ranu les (BKHG) and eos inoph i l i c kera tohya l i n granu les (EKHG) which a re i nvo l ved i n tongue k e r a t i n p roduc t i on (Anat. Record 178:464, 1974). The dog tongue i n t e r e s t - i n g l y e x h i b i t e d on ly EKHG i n t h e f i l i f o r m p a p i l l a e . cern ing species v a r i a t i o n i n g ranu le morphology we began a comparat ive s tudy o f tongue kera to- h y a l i n g ranu les . The tongues o f g e r b i l , cow, spec ios i s monkey, p i g and r a b b i t were f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n o r 80% methanol f o r l i g h t microscopy and 3% g lu ta ra ldehyde and 1% Os04 f o r e l e c t r o n microscopy. chemical s tud ies p a r a f f i n sec t i ons were c u t a t 5pm and s ta ined w i t h t h e f o l l o w i n g : H & E f o r genera l morphology and KHG i d e n t i f i c a t i o n ; H a r r i s ' hematoxyl in t o i d e n t i f y t he BKHG; eos in o n l y t o i d e n t i f y EKHG; Pau ly ' s reagent t o iden- t i f y h i s t i d i n e and t y r o s i n e con ta in ing p r o t e i n s i n the KHG; and, t he Sakaguchi ox ine r e a c t i o n f o r a rg in ine . L i g h t mic roscop ic observa t ions revea led t h a t t he g e r b i l and cow tongue p a p i l l a conta ined EKHG as w e l l as a sma l le r number o f BKHG; spec ios i s monkey conta ined p r i m a r i l y EKHG and occas iona l BKHG observed i n s e r i a l sec t i ons ; and, t he r a b b i t tongue granu les s ta ined p r e f e r - e n t i a l l y w i t h hematoxy l in b u t a l s o reac ted m i l d l y w i t h eos in . reac ted s t r o n g l y w i t h P a u l y ' s reagent and the EKHG w i t h the Sakaguchi ox ine r e a c t i o n suggest- i n g the presence o f a s p e c i f i c p r o t e i n mo ie ty f o r t he r e s p e c t i v e granu les . U l t r a s t r u c t u r a l l y t he BKHG and EKHG e x h i b i t e d suborgane l le v a r i - a t i o n s i n t h e i r u l t r a s t r u c t u r e w i t h the l a t t e r e x h i b i t i n g a l e s s e r e l e c t r o n d e n s i t y . types o f g ranu les were assoc ia ted w i t h r ibosanes and/or tonof i laments . These p r e l i m i n a r y h i s t o - chemi c a l and morpho log ica l observa t ions s t r o n g l y suggest a species v a r i a b i l i t y i n the na tu re o f t h e tongue f i l i f o r m p a p i l l a KHG which m igh t i n t u r n r e f l e c t phy logenet ic development o f t he tongue p a p i l l a e .

Prev ious s tud ies frm our l a b o r a t o r i e s have

Because o f these observa t ions con-

For h i s t o -

H is tochemica l l y t he BKHG

Both

STEFLIK, 0. E. , 6 . B. SINGH, J. R. SMITH and R. V. MCKINNEY. Department o f Ora l Pathology, Medical Co l lege o f Georgia, Augusta and R ive r -

A Cor re la t iGe TEM, SEM and LM Study o f Capybara LHydrochoerus hydrochoerus) F i l i f o r m P a p i l l a e .

The comparat ive morphology and e p i t h e l i a l d i f f e r e n t i a t i o n of the tongue f i l i f o n n p a p i l l a e of va r ious species has been o f r e c e n t i n t e r e s t i n our l abo ra to ry . v e s t i g a t i o n was t h e examinat ion o f t he f i l i f o n n p a p i l l a e o f t he capybara w i t h p a r t i c u l a r mpha- s i s on i t s ke ra tohya l i n (KHG) granu le popula- t i o n . The capybara resembles a g i a n t guinea p i g , i s t h e w o r l d ' s l a r g e s t l i v i n g roden t and weighs approx imate ly 100 l b s . samples of va r ious s i zes were exc ised frm an a d u l t capybara post-mortem and r o u t i n e l y p ro - cessed f o r scanning e l e c t r o n microscopy (SEM), t ransmiss ion e l e c t r o n microscopy (TEM) 'and l i g h t microscopy (LM). By SEM the do rsa l su r face of t h e capybara tongue showed approx imate ly 16 p o s t e r i o r curved con ica l p a p i l l a e pe r square m i l l i m e t e r . p layed desquamation o f f l a t t e n e d squamous c e l l s which e x h i b i t e d rn ic ro r idges . H i s t o l o g i c a l l y t he convex a n t e r i o r p o r t i o n o f t he p a p i l l a appeared t o a r i s e frm granu la r c e l l s w h i l e the concave p o s t e r i o r p o r t i o n was c m p r i s e d p r e d m i n a n t l y o f agranu lar c e l l s . The g ranu la r c e l l s d i sp layed a mixed popu la t i on o f bo th e o s i n o p h i l i c and baso- p h i l i c g ranu les w i t h the e o s i n o p h i l i c group be ing dominant. Basoph i l i c g ranu les appeared t o e x i s t p r i m a r i l y a t t he base o f t h e p a p i l l a . U l t r a s t r u c t u r a l l y , t he KHG's were assoc ia ted w i t h tono f i l amen ts and/or r i b o s m e s . These granu les were arranged randan ly th roughout the c e l l ' s cytoplasm b u t occas iona l l y appeared t o form a necklace type arrangement surrounding the c e n t r a l l y l oca ted nucleus. KHG's o f t he capy- bara appeared t o con ta in a suborgane l le s t r u c - t u r e c o n s i s t i n g o f va r ious amounts o f dense homogeneous depos i ts (DHD). These DHO were con- t a i n e d w i t h i n the normal f i n e g ranu la r m a t r i x o f t he KHG and v a r i e d i n s i z e and shape. Th is s tudy suggests a species s p e c i f i c e p i t h e l i a l d i f f e r e n t i a t i o n p a t t e r n i n capybara tongue w i t h d i s t i n c t r e g i o n a l o r i e n t a t i o n o f b a s o p h i l i c and e o s i n o p h i l i c KHG's. Fu r the r , t he KHG's con ta in va r ious degrees o f OHD and d i s p l a y d i s s i m i l a r morphologies. The compos i t ion o f these KHG's i s c u r r e n t l y under study.

The o b j e c t i v e o f t h i s i n -

For t h i s study,

The sur face o f t he p a p i l l a e d i s -

TERRACIO, L. and DOUGLAS, W.H.J., Department of Anatomy, University of South Carolina, Colu- mbia, SC. and Tufts University, Boston, MA. (Introduced by R . J . Weymouth). Densitometric Evaluation of Growth in Primary Cultures of Rat Ventral Prostate Epithelial Cells.

The use of a photovoltic cell densirometer to assess clonal growth of cell lines is well established. Densitometry has not been success- fully applied to primary culture systems, es- pecially those that are established from cellu- lar aggregates. for establishing primary cultures in 24-well culture vessels and evaluating the effects of serum, hormones, and other factors o n cell growth using densirometry. Aggregates of rat ventral prostate epithelial cells were prepared by a collagenase digestion and selectSve at- tachment procedure (Terracio % &., Proc. EMSA 37:138-139, 1979). The epithelial aggregates were uniformly distributed into 24-well culture vessels containing F12K culture medium supple-

This study describes a method

mented w i t h v a r i o u s c o n c e n t r a t i o n s of t h e f o l - lowing s u b s t a n c e s : f e t a l bovine serum, h o r s e serum, t e s t o s t e r o n e , d i h y d r o t e s t o s t e r o n e , es - t r i d i o l , h y d r o c o r t i s o n e , z i n c , t r a n s f e r r i n , cy- s t e i n e , g l u t a m i n e , se len ium and a s c o r b i c a c i d . The c u l t u r e s were m a i n t a i n e d a t 37% i n a n a t - mosphere of 5% CO2 i n a i r , and t h e media and supplements r e p l e n i s h e d every o t h e r day. A f t e r seven days i n v i t r o t h e c u l t u r e s were washed w i t h PBS, f i x e d i n 1 .0% g l u t a r a l d e h y d e and s t a i n e d w i t h 0 .5% c r y s t a l v i o l e t f o r 2 min. The e f f e c t o f each supplement on c e l l growth was e v a l u a t e d u s i n g a p h o t o v o l t i c c e l l d e n s i t o m e t e r des igned t o r e a d t h e t o t a l c u l t u r e s u r f a c e of a 16 nun w e l l . A d e c r e a s e i n t r a n s m i t t e d l i g h t when compared t o a c o n t r o l c u l t u r e i n d i c a t e d a n i n c r e a s e i n c e l l growth by a supplement. In- c r e a s i n g c o n c e n t r a t i o n s o f b o t h h o r s e and f e t a l bovine serum r e s u l t e d i n a n i n c r e a s e i n c e l l growth. T e s t o s t e r o n e , e s t r i d i o l , z i n c , hydro- c o r t i s o n e , t r a n s f e r r i n , and a s c o r b i c a c i d each had a s t i m u l a t o r y e f f e c t on c e l l growth. Cy- s t e i n e , g l u t a m i n e , and d i h y d r o t e s t o s t e r o n e had no e f f e c t on c e l l growth w h i l e se len ium was i n - h i b i t o r y t o c e l l growth. T h i s d a t a compares f a v o r a b l y w i t h t h a t produced by o t h e r methods s u c h a s , o r n i t h i n e d e c a r b o x y l a s e p r o d u c t i o n . These r e s u l t s i n d i c a t e t h a t d e n s i t o m e t r y is a u s e f u l method €or d e t e r m i n i n g t h e e f f e c t of supplements on c e l l growth. (Suppor ted by Grant KA26063 from N C I ) .

___

THOMAS*, D. A . , Department o f Anatcmy, west V i r g i n i a U n i v e r s i t y , Morgantown, W e s t V i r g i n i a . (Sponsored by J. L. C u l b e r s o n ) . Motor u n i t s i n s e l e c t e d h i n d l i m b m u s c l e s o f t h e opossum, D i d e l p h i s v i r g i n i a n a .

T h i s is a p r e l i m i n a r y s t u d y o f n e u r a n u s c u l a r i n n e r v a t i o n i n t w o a n t a g o n i s t i c h i n d l i m b musc les , t h e m e d i a l g a s t r o c n e m i u s (FIG) , a n a n k l e f l e x o r , and t h e t i b i a l i s a n t e r i o r (TA), a n a n k l e e x t e n s o r . The i n n e r v a t i o n , motor u n i t s i z e a n d f i b e r t y p e s of t h e s e m u s c l e s w e r e s t u d i e d . D i s s e c t i o n of t h e lumbosacral p l e x u s r e v e a l e d t h a t t h e s e m u s c l e s a r e s u p p l i e d by b r a n c h e s of t h e s c i a t i c n e r v e , which arises from (L ) L - S2. r e a c h t h e head o f MG and m u l t i p l e b r a n c h e s e n t e r t h e head o f TA. Nerves t o t h e s e m u s c l e s w e r e osmium s t a i n e d , epoxy embedded, s e c t i o n e d and f i b e r s were counted and measured. Mean n e r v e f i b e r c o u n t was 227 f o r MG and 3 5 1 f o r TA. D e a f f e r e n t a t i o n of t h e s e n e r v e s w a s accompl ished by removing d o r s a l root g a n g l i a L5-S2. E f f e r e n t n e r v e f i b e r s were counted i n p e r i p h e r a l n e r v e s a f t e r a 45-day s u r v i v a l p e r i o d . The MG and TA have also been i n j e c t e d w i t h h o r s e r a d i s h p e r o x i d a s e t o l o c a l i z e t h e i r motoneuron pools. E a r l y r e s u l t s demonst ra ted motoneurons i n L and L6 o f t h e v e n t r a l - l a t e r a l horn . from normal a n i m a l s w e r e embedded i n p a r a f f i n , s e c t i o n e d , s t a i n e d w i t h H & E and used for t o t a l f i b e r c o u n t s . A d j a c e n t b l o c k s were removed, f r o z e n s e c t i o n e d and r e a c t e d f o r a l k a l i n e a d e n o s i n e t r i p h o s p h a t a s e . The MG h a s a 1:l r a t i o o f f a s t to s l o w f i b e r s ; a 211 r a t i o of f a s t t o s l o w f i b e r s e x i s t s i n TA. Cross- s e c t i o n a l areas o f musc le f i b e r s w e r e measured u s i n g a n o p t i c a l image a n a l y z i n g sys tem (MOP/AM 01). The mean c r o s s - s e c t i o n a l area o f a TA s l o w f i b e r w a s 3935 IJ‘ and t h e mean c r o s s -

4 One or t w o d e s c e n d i n g s c i a t i c branches5

Muscle?

s e c t i o n a l area of a TA f a s t f i b e r w a s 3751 u z ; t h e r e w a s no s i g n i f i c a n t d i f f e r e n c e i n s i z e between t h e t w o f i b e r t y p e s . The mean cross- s e c t i o n a l area of a MG s l o w f i b e r was 6179 u z and 6498 1-1’ f o r a MG f a s t f i b e r ; there was n o s i g n i f i c a n t d i f f e r e n c e i n s i z e between t h e t w o f i b e r t y p e s . The f a s t f i b e r s o f t h e MG w e r e s i g n i f i c a n t l y l a r g e r t h a n t h e f a s t f i b e r s of t h e TA ( P < 0 . 0 2 ) ; t h e s l o w f i b e r s o f MG, however, were n o t s i g n i f i c a n t l y l a r g e r t h a n s l o w f i b e r s of TA (0.05<p<0.10). (Suppor ted i n p a r t by NSF g r a n t BNS-7924172).

TILLMAN, L a r r y J . , Bruce R. Parks, Ben H. Douglas and John E. Rawson, Departments o f Anatomy and P e d i a t r i c s , U n i v e r s i t y o f M i s s i s s i p p i Medical Center , Jackson, M i s s i s s i p p i .

v e r t i n g enzyme i n h i b i t o r C a p t o p r i l (Squ ibb) , (D-2-methyl-3-mercaptopropanoyl -L-pro1 i ne) was a d m i n i s t e r e d t o t h r e e groups o f animals made h y p e r t e n s i v e by uninephrectomy, d a i l y i n j e c t i o n s of 5 ug/lOO g body d-a ldos terone (Sigma) and s u b s t i t u t i o n o f 0.9% NaCl f o r t h e d r i n k i n g w a t e r . nonpregnant animals d u r i n g t h e developmental phase o f h y p e r t e n s i o n and t o nonpregnant animals w i t h e s t a b l i s h e d h y p e r t e n s i o n ( f o u r weeks o f t r e a t m e n t ) . Treatment w i t h C a p t o p r i l p revented f u r t h e r inc reases i n b lood pressure i n the animals d u r i n g t h e developmental phase o f hyper- t e n s i o n , and reduced t h e b l o o d pressure t o normotensive l e v e l s i n t h e animals w i t h e s t a b l i s h e d hyper tens ion .

The pregnant and nonpregnant animals r e c e i v - i n g C a p t o p r i l d u r i n g t h e developmental phase o f h y p e r t e n s i o n demonstrated s i m i l a r u l t r a s t r u c - t u r a l changes w i t h no s i g n i f i c a n t d i f f e r e n c e s . G lomeru l i i n these animals showed l o c a l f o o t process f u s i o n and areas of s l i g h t basement membrane t h i c k e n i n g . E n d o t h e l i a l and e p i t h e l i a l c e l l s o c c a s i o n a l l y appeared en la rged. I n con- t r a s t , animals t r e a t e d w i t h C a p t o p r i l f o l l o w i n g e s t a b l i s h e d h y p e r t e n s i o n demonstrated d r a s t i c changes i n g lomeru la r a r c h i t e c t u r e . There was marked basement membrane t h i c k e n i n g w i t h redu- p l i c a t i o n and s p l i t t i n g . E p i t h e l i a l f o o t p ro- cess f u s i o n inc reased. E a r l y s u b e n d o t h e l i a l e l e c t r o n d e n s i t i e s and s u b e p i t h e l i a l humps were observed. U1 t r a s t r u c t u r a l r e s u l t s suggest t h a t C a p t o p r i l i s e f f e c t i v e i n p r o t e c t i n g g lomeru la r a r c h i t e c t u r e w h i l e a t t e n u a t i n g deve lop ing hyper - t e n s i o n . A l though i t i s e f f e c t i v e i n reduc ing e s t a b l i s h e d hyper tens ion , C a p t o p r i l does n o t appear t o r e v e r s e a l r e a d y e x i s t i n g h i s t o p a t h o - l o g i c a l damage t o t h e k idney .

C a p t o p r i l was g i v e n t o p regnant and

T r i l l J.J.“, J.M. B a r r e t t , and J . A . T i s c h f i e l d ” Department of Anatomy, Xedica l C o l l e g e of Georgia , Augusta, Georg ia L l i m i n a t i o n o f Mvcoplasma Contaminants i n Mammalian C e l l C u l t u r e s .

Normal cells have adenine phosphroibosyl- transferase (APRe) activity which converts adenine to adenylate. One can select APRT deficient mutants (APRT-) with toxic adenine analogs such as 2,6-diaminopurine (DAP) and 2-fluoroadenine (FA). Several APRT- mouse (L) and Chinese hamster ovary (CHO) cell clones were selected for resistance to DAP (DAPR) and FA (FAR). tants in alanosine-adenine medium (AA) . Test- ing of two putative revertants revealed low levels of APRT activity, yet there was a para- doxical growth increase with 0.5 to 5 uglml FA, and cell death in AA. These latter two observations are inconsistent with the APRT' phenotype. Microbiological examination in- dicated contamination with an anaerobic myco- plasma.

Initial testing (APRT activity values 2 .05>: wild-type' growth in DAP & FA; death in AA) of the DAPk CHO cells indicated that they were APRT-. Subsequent analyses (11.4% of wild-type adenine incorporation and 1.7 - 2.9% of wild-type APRT activity) however, indicated some APRT activity. ?licrobiological and bio- chemical testing revealed a mycoplasma of bovine origin.

To eliminate these mycoplasma, we devised a protocol involoving alternating passage of cells between antibiotic-free medium and me- dium containing 25 u g l m l tetracycline, 100 uglml spectinomycin and 60 ug/ml tylocin. To date, both microbiological and biochemical examination reveal no recurrence of the myco- plasma in treated cells. These tests are further substantiated by SEM examination of treated vs. untreated cells. Adenine incor- poration and APRT activity assays (5 1% and - c .05% of wild-type, respectively) also in- dicate that the treated cells are now within the range of other APRT- mycoplasma-free cell lines. Elvcoplasma may cause artifacts or misinterpretations in studies of purine meta- bolism and prenatal diagnosis. SEM examina- tion for this contaminant is extremely sen- sitive, rapid and definitive. (Supported by NIH Crdnt GM-26917)

iiTe then selected for APRT' rever-

Way, Charles F . , Department of Anatomy, Medical University o f South Carolina, Charleston, S.C. 2 9 4 0 3 Immunohistochem- ical comparison o f lysozyme activity in the livers of glucan-treated and glucan- hydrocortisone-treated nice.

Administration of the polyglucose glucan to rodents confers hyperfunction- a 1 status upon the reticuloendothelial (RE) system with hypertrophy o f the ma- jor RE o r g a n s . Livers in these animals exhibit increased macrophage numbers with prominent granuloma formation. Ly- sozyme (LZM), which is secreted by mac- rophages, is elevated in the s e r a of these animals. In this study, the liv- ers of glucan-treated mice were examined for LZM activity using an immunoglobulin enzyme bridge technique. As hepatic macrophages are thought by many t o ' d e - rive from circulating monocytes, we ex- amined the a b i l i t y d glucan t o enhance macrophage numbers in mice rendered mon-

ocytopenic by hydrocortisone acetate (HAc), and examined these tissues for LZM activity. Male C 5 7 B l mice were ad- ministered ~ l u c a n (.U5mg/mouse, i.p.) for 5 days. Controls received 5% dex- trose. Blood samples were taken at 2'4 hr intervals for WBC counts. Yonocytes were detected by esterase cytochemistry. Other mice were given a single subcutan- eous injection of 15ng !!Ac. Forty-eight hr later, monocytes were no longer de- tectable in the blood and elucan treat- ment (.45mg/mouse f o r 5 days) was begun. Twenty-four hr after the final glucan injection, tissues were excised and pre- pared for LM. Four micrometer sections were immunostained for LZM. Livers in glucan-treated mice showed LZ?1 positive cells scattered in sinusoids and in granulomas. Dextrose controls were L Z i l negative. Glucan failed to enhance mac- rophage numbers in monocytopenic mice, These hepatic tissues were also LZM neg- ative and organ weights were normal. These results indicate that glucan en- hances hepatic macrophage numbers by by recruitment o f circulating nonocytes into the sinusoids which differentiate into resident macrophages.

WEAVER, Macon M . and Bruce R. Parks. of Indomethacin 0" development recovery of offspring of DCA/NaCl pregnant rats.

Effect - -

In prior reports we have shown that prostaglandin synthesis inhibition by indome- thacin (I) may affect the severity of desoxy- corticosterone acetate (DCA)/salt induced renal lesions in the pregnant rat and her offspring. This study further Characterizes the injury and recovery of the offspring of the DCA-I rats. Sprague-Dawley rats with blood pressure (BP) ranging from 110-123 mm Hg and weighing from 200-22> gm were used. Animals were uninephrectomized and following recovery each received 4 mg/Kg/da (OK 28 mg/Kg/wk DCA and 0.9% saline for drinking water. When BP stabilized (163 to 201 rmn Hg) the animals were bred. Weights and BP were determined weekly. All animals were killed at intervals for gross and histological evaluation. Thirty rats served as hypertensive controls; 15 were given 3 mg/Kg/da indomethacin seven to 10 days prior to delivery. Some rats and their offspring from both groups were killed at term or allowed to recover. were killed at intervals up to weaning. Offspring treated transplacentally with indomethacin showed an increase in fetal morbidity and mortallty and cannibalism occurred more often. These findings support the concept that prostaglandins may influence BP of pregnant UCA/NaCl hypertensive rats and the survival of offspring.

Offspring

A Biomechanical Investigation of Selected Locomotor Modes in the Green Monkey (C. a. sabaeus). JAMES P. WELLS, ROBERT M. FISK, West Virginia School of Osteopathic Medicine.

motor modes habitually employed by Cercopithecus in the natural environment; horizontal and vertical leaping. In an attempt to view holistically the positional behavior of this species, these two modes were studied in the context of both their behavioral associations in the natural substrate and the biomechanics of the loco- motor event. Techniques employed include the use of high speed cinematography with kinematic and kinetic analysis and the use of telemetered electromyography.

The initial positional behavioral phase of the investigation was carried out on 'the island of St. Kits in the British West Indies This study showed vertical leaping to be highly correlated with entry into the trees while horizontal leaping was associated with branch diameter and orientation. The kine- matic portion of the study provided segmental displacements, velocities and accelerations. These data formed the basis for the kinetic analysis along with the information obtained on segment mass distribution by dissection. The inertial force versus muscle contribution in leaping was found to be much larger than expected. Finally, telemetered EMG of lower extremity muscle groups provided a view of the phasic patterns of muscle activity during leaping.

This investigation focuses upon two loco-

WELSH,* Marcia G . , Department of Anatomy, Uni- v e r s i t y o f South Caro l ina , School o f Medicine, Columbia, South Caro l ina . ( In t roduced by D.L. Odor) . The b lood-bra in b a r r i e r , CSF-brain b a r r i e r and p i n e a l conc re t i ons : The i r r e l a - t i o n s h i p i n the p inea l g land o f t he Mongolian g e r b i l (Meriones ungu icu la tus ) .

The poss ib le ex is tence o f e i t h e r a b lood- b r a i n b a r r i e r (BBB) and/or a CSF-brain b a r r i e r i n the p inea l g land o f t he g e r b i l was examined u l t r a s t r u c t u r a l l y us ing the t r a c e r s microperox- idase (MP) and horserad ish peroxidase (HRP). Gerb i l s were i n j e c t e d w i t h e i t h e r MP or HRP in- t ravenous ly ( I V ) o r they rece ived i n t r a v e n t r i - c u l a r ( IVT) i n j e c t i o n s o f HRP. Male g e r b i l s which had been main ta ined i n a c o n t r o l l e d l i g h t environment (LO 14:lO) were anes the t ized w i t h pen tobarb i ta l . For the I V i n j e c t i o n s the rena l a r t e r i e s were t i e d o f f t o p revent r a p i d c l e a r - ance o f t he t race rs . MP and HRP (bo th 15mg/ml s a l i n e ) were i n j e c t e d i n t o the i n f e r i o r vena cava. For the I V T i n j e c t i o n s , HRP (3mg/0.02ml s a l i n e ) was i n j e c t e d us ing an i n f l o w cannula p laced s t e r e o t a x i c a l l y i n t o the l a t e r a l v e n t r i - c l e . The t r a c e r s were a l lowed t o c i r c u l a t e f o r 15 o r 60 minutes. Fo l low ing f i x a t i o n , t he t i s - sue was incubated f o r p e r o x i d a t i c a c t i v i t y as repo r ted by Graham and Karnovsky ( J . Histochem. Cytochem. 1966, 3, 291) f o k HRP and by Simon- escu gal. (J. C e l l B i o l . 1975, %, 586) f o r MP. The t i s s u e was p o s t f i x e d i n 2% osmium t e t r o x i d e fo l l owed by r o u t i n e p repara t i on f o r e l e c t r o n microscopy. The I V i n j e c t i o n s r e s u l t e d i n bo th MP and HRP t r a n s p o r t th rough the endo-

t h e l i a l c e l l s o f t he g land by v e s i c u l a r t rans - p o r t and through the i n t e r e n d o t h e l i a l j unc t i ons . Fo l l ow ing the IVT i n j e c t i o n s , t he t r a c e r was demonstrated i n the subarachnoid space as w e l l as i n the g l i a l c e l l s assoc ia ted w i t h the p e r i - phery o f t he gland. I n bo th cases ( IVT and IV ) , t he r e a c t i o n produc ts were l oca ted w i t h i n the ex tens ive pe r i vascu la r reg ion as w e l l as i n bo th g l i a l c e l l s and p inea locy tes . There a l s o appeared t o be s p e c i f i c l o c a l i z a t i o n i n the r e g i o n o f t he vacuolated p inea locy tes and t h e p inea l concre t ions . The r e s u l t s o f t h i s study demonstrate t h a t n e i t h e r a BBB no r a CSF-brain b a r r i e r e x i s t s i n the p inea l g land o f t he ger- b i l . I n a d d i t i o n , t he techniques employed i n t h i s i n v e s t i g a t i o n may be a va luab le means f o r demonst ra t ing s i t e s o f sec re t i on and o f s to rage o f t he p inea l sec re to ry p roduc ts . (Supported by USC Grant #11040 E128).

Zuchelkowski*, Edwin M., and I.antz,R. Clark, DeparLment of Anatomy, West Virginia Universit~y, Morgantown, West Vjrginia. (Sponsored by David E. Hinton) Mucous cells in epidermis of control and acid-stressed (pH 4.0) brown bullhead cat- fish (Tctalurus nebiilosus): 4 morphometric - - ~ -~~ ~ ~

study. Mining and utilizdtion of coal affect quality

of surface waters, rendering them less suitable for supporting aquatic l i f e . Previous quelita- tive studies in ot.her species of teleosts tmve suggested an increase in epidermal mucosubstanc- es following exposure to severe acid stress. This increase was attributed to hypertrophy of mucous cells in epidermis of acid-exposed brook trout (Daye and Garside, Can. J . Zool., 54:2140- 2155, 1976). Previous study in o u r laboratory demonstrated that hyperplasia but. not hypertro- phy (of murous cells occurred in brown bullhead epidermis at a sublethal pH level (5.7-6.15). The purpose of the present study was to deter- mine thr effect of a lethal pli level (3.8-4.7) on epidermal mucous cells of the brown bullhead. Randomly selccLed young, hatchery-reared fish (n=9), obtained from a commercial supplier, were housed for 5 days in a 10 gallon aquarium in which water pH was adjusted via addition of sul- furic acid to the p H range 3.8-4.2. Control fish (n=9) were maintained in a tank containing no sulfuric acid (pH 6.8-7.0). Following ex- posure, epidermis from the anterior abdominal wall was prucessed for histochemical analysis using a combined AfI-PAS technique. A total of 144 microscopic fields were evaluated in each group of fish, using standard morphometric pro- cedures. Average cell daLa indicated no change in diameter or volume of individual mucous cells jn acid stressed f i s h . Howevrr, mucous cell voj . ume density (MCVV) and number density (MCN,) were significantly increased (pC.01) i n the acid stressed group. These results drmonstrate that even at lethal pH levels, hyperplasia but not hypertrophy of mucous cells occurs in the epi- dermis of the brown bullhead. T t i l s flnding suggests that the brown bullhead epidermis may be a useful model for studies involving t h e mechanism of riucous cell hyperplasia. Supported by WaLer Research Institlite, WVU, as part o f i'roject A-037-W.Va.