CHARACTERIZATION OF THE ASCENDING MICTURITION …1986). the organizing center for the coordination of bladder function The basic micturition reflex pathway was thought to involve the

CHARACTERIZATION OF THE ASCENDING MICTURITION PATHWAY TO THE PERIAQUEDUCTAL GRAY

Myto Duong

Submiüed in partial fiWhnent of the rqukmen& for the d e p of Master of Science

Dalhousie University Halifax, Nova Scotia

September, 1997

O Copyright by Myto Duong, 1997

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I would like to dedicate this thesis to Arron,

and my family for their unconditional love and suppon

TABLE OF CONTENTS

Page Number

Table of Contents ........................... ... ....................................... v ...

List of Figures ............ ..., ...................................................... vil1

List of Tables ......................................................................... x

............................................................................... Abstract xi ...

Ab breviations ....................................................................... ..xi11

Acknowledgements .................................................................. xvi

................................................................. INTRODUCTION. . - 1

Bladder Overview ............ .. ................................................ 1

Efferent Pathways ............................................................... 2

The Role of Barrington's Nucleus in Micturition .............. ......... 3

M-region and L-region of the Pontine Micturition Center .................. .5

Other Connections to and from Barrington's Nucleus ....................... 6

B ulbospinal Pathway for Micturition ......... .. ........................... 7

Suprapontine Influences on the Michirition Reflex Pathway .............. -8

Afferent Pathways ........... .,, ...................... ., ... 10

Bladder Afferent Terminais in the Sacral Spinal Cord ...................... 11

Spinobulbar Pathway for Micturition ......... ........... . . . 1 2

The Periaqueductal Gray (PAG) ................ .... ..................... 14

Evidence for PAG Involvement in Micturition ........... .,,, .............. 16

Researc h Proposal

.Rationale ................................................................ 17

-Airns and Surnmary of Experimental Preparation .................. 18

MATERIALS AND METHODS ................................................. 21

Neuroanatomical Experiments:

............................. Asep tic surgicd preparation for tracer injection 21

.............................................. PAG retrograde tracer injections 21

Bladder anterograde tracer injections ......................................... 22

.................................................................... Post-surger y -22

................................. Experimen ta1 design: survival days (staged) 22

.................................. Surgical preparation for bladder distension 24

.............................................................. Bladder distension 24

..................... .............. Perfusion and tissue collection ....... 25

Sectioning the PAG and spinal cord .................... ... .............. 25

......................................................... Immunohistochemistry 26

................................................................... Data Handling 27

NeurophysiologicaI Experiments:

...................................... Surgical preparation .......... ...... 27

Mapping of the PAG - extracellular field potentials ......... .. ........... 29

Mapping of the Spinal Cord - single units ................................... 31

Histology ........................................................................ 33 . . ................................................................... Statistical Test 34

RES ULTS ............ - .................. ........ 35 .... Neuroanatomical Experiments:

....................... ............ PAG retrograde tracer injections .... 35

Primary afferent terminal distribution in spinal cord ........................ 37

c-Fos induction by bladder distension ........................................ 37

Neurophysiologieal Experiments:

PAG extracellular field potentials ............... ... ....................... 50

Spinal Cord Units ............................................................... 63

Spinal neurons antidromicdy activated from the PAG ............ 66 Convergence of afferent input ......................................... 73 Effects of PAG stimulation on spinal neuron activity .............. 74

DISCUSSION ........................................................................ -78

Neuroanatomical Experiments:

c-Fos protein induction by bladder distension ............................... 85

Neurophysiological Experiments:

PAG extracellular field po ten tials ........... .... ......................... 88

............................................................. Spinal cord units -90

................................ ............................ Conclusion .. 97

........ Significance .. ...... .. ...................................................... 97

APPENDICES .......................................................................... 99

REFERE NCES ......................................................................... 103

Figure 17: Distribution of Lumbosacral Neurons Activated by PAG ............. 68 Figure 18: Sacral Spinal Neuron Which Rojects to the PAG Conveys PLN

Input .......................................................................... 70

Figure 19: Spinal Neuron, Which Receives SFP Input, Projects to PAG ....... 72

Figure 20: PAG Orthodromically Activated Spinal Neuron ........................ 75

Figure 21: Distribution of Spinal First and Higher Order Intemewons in

Sacral Spinal Cord .......................................................... 76

Figure 22: PAG and PLN Interactions ............................................... 77

LIST OF TABLES

Page Number

Table 1: Tracer Injections ............................................................. 23

.............................................................. Table 2: HRP CeU Counts 39

Table 3: Sum of c-Fos hmunoreactive Cells in Each Spinal Cord

................................................................... Segment 45

Table 4: Sum of c-Fos Immunoreactive Cells in Different Zones of Each

Spinal Cord Segments ................................................... 48

Table 5A: Median Latencies for Different Components of the Multiphasic Field

Potential Responses .............. .. .............................. 53

Table 5B: Median CVs for Different Components of the Multiphasic Field

Potential Responses ........... .. ............................... 54

Table 6A: Median Latencies for the 2nd Component of the Field Potentiai

........ Repsonse for Ipsilateral and Contralaterd PLN Stimulation 55

Table 6B: Median CVs for the 2nd Component of the Field Potenriai

........ Repsonse for Ipsilateral and Contralateral PLN Stimulation 56

Table 7: Counts of PAG Antidromicdy and Orthodromically Activated

Neurons in the Sacral Cord ........................................... 67

ABSTRACT

The micturition reflex pathway is a supraspinai pathway. There is recent

anatomiciai evidence to suggest that the periaqueductal gray is involved in the ascending

limb of this reflex We propose to determine whether the periaqueductal gray conveys

bladder-relateci information and to characterive the (type of) information that is king

conveyed.

Injections of antemgrade tracer into the bladder wall revealed bladder eerent

terminah in S2 of the sacral spinal cord. These aEferent tenniLlals were within the

vicinity of spinal neurons retrogradely labelled from the periaqueductal gray. Bladder

distension induced c-fos exp-on in neurons located within the regions where

retmgradely labelled neurons were located. This provides some support for the

transmission of bladder inputs via axons of tbese periaqueductal gray -projecting

neurons.

Stimulation of pelvic nerves evoked maximum field potentials in dorsolateml and

ventrolaterai regions of caudal periaqueductal gray. Since the regions activated by pelvic

nerve stimulation differed from those activated by seasory pudendal and superficial

perineal stimulation, the possibilty of sWc pathways for different nerve inputs exists.

However, these fhdings are not conclusive.

Extmcellular single unit studies were undertaken to characte& the inputs

received by newons projecting to the periaqueductal gray. Seventeen c e h were

identiiïed which projected directly nom the sacral spinal cord to the periaqueductal gray,

and only one of these received pelvic nerve input. Although the specifïty of this pathway

was not detennined because this neuron was lost before the other nerves could be tested,

it was observed that at least a portion of this pathway conveyed pelvic nerve

information. This demonstrates that the periaqueductal gray is hvolved in the

transmission of bladder inputs. However, the fact that only 4 out of the 17 cells with

direct projections to the periaqueductal gray received inputs h our test ne- implies

that the major part of this pathway is not dhctly related to lower urinary tract functions.

ABBREVIATIONS

AP

CCF

CD

Qn

cm 5 0

CV

DAB

DC

hr

H R P

Hz

im.

i.v.

kg

kHz

L

L-PL

LDH

L W

M

m/sec

mA

caudai cutaneous femoral

cord dorsum

centimeter

centimeter of water

conduction velocity

diaminobemdine

dorsal commissure

hour

horseradish peroxidase

Hertz

in tramuscular

in travenous

kilogm

kilohertz

lit=

left pelvic

lateral dorsal horn

lateral ventral hom

molarity

meters per second

milliamperes

xiii

MDH

mg

min

ML

mL

mm

H g

MOhms

mPUD

mSec

NaCl

NaNO,

4 p.0,

PAG

PL

PLN

P m

PUDN

R-PL

sec

SFP

SPN

SPUD

WGA-HRP

w

medial dorsal hom

milligram

minutes

mediolateral

rnilliliter

millimeter

millimeter of mercury

megaohms

motor pudendd

milli,ceconds

sodium chloride

sodium nitrite

oxm=n

oral

periasueducd gray

pelvic

peivic nerve

pudendd

pudendal nerve

right pelvic

seconds

superficial perineal

sacral parasympathetic nucleus

sensory pudendal

wheat gem agglutinin horseraàish pemxidase

microamperes

xiv

CLL

CLm

SC

IC

NRM

Lm'

N.Aug.

CAq

CPed

FL

mimliter

micrometer

superior coUiculus

inferior coIlicuIus

nucleus raphe magnus

latedorsal tegmental nucleus

nucleus augustus

cerebral aqueduc t

cerebek peduncle

fasciculus longitudinus

ACKNOWLEDGEMENTS

1 would lilie to take this opportuni~ to achowledge J e d e r Martin for allowing

me to use data c o k t e d during her rotation shidy with Dr. Downie. Some of these data

included 3 neurophysiologicai a h a l studies and 1 neuroanatornical (conml animai in

the c-fos induction) study.

1 would Iike to thank Dr. Huan-Ji Du and Scott Pmnych for their technicd

expertise. They were both instrumental in developing my understanding of the ber

aspects involved in my neurophysiological and neuroanatomical saidies.

To Dr. Kanie Semba and lab members, a sincere thank you, for the use of theV

microscope and cornputer softwares, and helpfd instnictions. 1 would also like to thank

Dr. Gary Ailen for his ideas, constructive cnticisms, and teaching.

On a personal level, 1 would like to thaak my good fi5ends Karen, Cindy, Mke,

Mostafa, Vidya, Leslie, Isabel, and the Sears family for their support, helpfid advices,

and companionship.

And las& but most importantly, 1 would iike to express my appreciation and

gratitude to my supenisor, Dr. John Downie for his patience and understanding. His

enthusiasm for science and vast scientinc knowledge have been inspiratiod. 1 am also

grateful for the many hours he devoted to reading and editing my work and training me.

xv i

INTRODUCTION

Bladder Overview

The urinary bladder is a distensible hoDow organ cornpriseci of a smooth muscle

layer, the detmor muscle. lying between outer serous and inner mucous coats. Aithough

the bladder detrusor is anatomically continuous, it is neuromorphologically divisible into

body (the storage chamber) and base (inlet and outlet) regions. The junction of bledder and

urethra is referred to as the bladder neck (intemal sphincter). The two main functions of the

bladder are to store and to periodically eliminate urine (Elbadawi, 1996).

Urine is produced in the kidney and fiows to the bladder via the ureters. As the

bladder fills, the iension of the bladder wali adjusts to its capacity. so that there is minimal

change in the bladder pressure. Pudendal neme (l?UDN) activity keeps the extemal

sphincter closed Continued bladder f i lhg results in a progressive inmase in the afferent

nerve activity and a gradud increase in sensation. Fit there is a sensation of fullness and

then the desire to void.

The temis "micturition" or 'iirination" refer to the periodic cornplete emptying of the

bladder (involving coordinated bladder contraction and urethral sphincter relaxation). When

the bladder is full, mechanoceptors (stretch receptors) in the bladder neck and detrusor are

activated and impulses are sent to the sacral spinal cord via the pelvic nerve (PLN).

Anerent information from the bladder and the urethra reaches Barrington's nucleus

(Bmington. 19 15) or the M-region in the dorsolaîeral pontine tegmentum (Holstege et al.

1986). the organizing center for the coordination of bladder function The basic micturition

reflex pathway was thought to involve the propagation of information h m the bladder to

the sacml spinal cord to the Barrington enter, and then back to the sacral spinal cord to

elicit a bladder response.

If mictuxition does not occur, continueci bladder fîiiing leads eventually to a feeling

of urgency followed by pain and severe discodort. The sensation of bladder W g . of

conscious desire to void, and of painful distension are mediated by aEerait fibers in the

PLN (Nathan and Smith, 1951).

Efferent Pathways

In order for the bladder and mthra to serve their functions (to store mine without

leakage at Iow pressures and to expel urine periodically through a relaxed outlet), the neural

events in the autonomie and somatic systems must be cooniinated. These processes are

controlled by the interplay of sympathetic and parasympathetic neurons and the somatic

motor neurons. A failure to coordinate these events results in increased postvoid residud

urine with high resting pressures in the bladder. Los of rend function occurs when high

intravesical pressures are transmitted to the upper urinary tract. Other manifestations

include irritative voiding or urinary incontinence (Chai and Steers, 1996).

The preganglionic neurons controlling the bladder are found in the intermediolateral

region of the sacral spinal cord and their axons project to the blad&r via the PLN. When

the bladder is empty the parasympathetic preganglionic neurons are quiescent but they are

activated by bladder distension via stretch receptors at micturition threshold (Vaughan and

Satchell, 1994).

Low levels of activity in the sensory afferents that mpond to bladder distension

will activate sympathetic preganglionic neurons in the lumbar spinal cord which resdt in:

(a) inhibition of parasympathetic activity in the pelvic ganglion via alpha-adrenoceptors

(dffiroat and Kawatani, 1985). (b) relaxation of the detnisor via beta-adrenoceptors

(Nergardh and Boreus, 1972; Levin and Wein, 1979; Edvardsen and Setekleiv, 1968;

Awad et al, 1974). and (c) excitation of the intemal urethral sphincter muscle via alpha-

adrenoceptors (Slack and Downie, 1983). Therefore, during bladder f i h g , sympathetic

3 activity promotes continence by producing: (a) direct bladder wall relaxation, (b) indirect

blad&r relaxation by inhibithg parasympathetic activity, and (c) by direct contraction of the

intanal sphincter.

The PUDN innervates the extemal urethral sphincter, a muscle that forms part of

the pelvic fioor musculature, which is reguiated by the somatic motor system (Fedirchuk

and Shefchyk, 1991). There are projections h m the dorsolateral pontine tegmentum to

PUD motoneurons innervating the extemal urethral sphincter (Holstege et al, 1986;

Griffths et al, 1990; Dhg et al, 1995). located in the ventrolateral region of Ooufs nucleus

(Thor et al, 1989). Projections h m the dorsornediai part of Onuf's nucleus innemate the

anal sphincter (Thor et al. 1989). These motoneurons are activated at low levels of bladder

distension. At high leveis of distension, supraspinal newons that inhibit the nring of both

the sympathetic and the sornatic motor nemm are activated and result in the removal of the

sympathetic inhibitory effects on the parasympathetic system to elicit bladder contraction

and expulsion of urine.

The Role of Barrington's Nucleus in Micturition

Micnintion is mediated by activation of the sacral parasympathetic efferent pathway

to the bladder and reciprocal inhibition of the somatic pathway to the urethral sphincter.

Electrical stimulation in the rosaal brainstem in the region of the dorsolateml pontine

reticular formation elicits detnisor contractions and firing in the parasympathetic efferent

pathways to the bladder (Lalley et aI, 1972; DeGroat, 1975; McMahon and Spillane, 1982).

w h e m lesion in this area produces irreversible depression of bladder reflexes

(Barrington, 1925; Kum, 1965). Patients with spinal cord injuries above the sacrai cord,

have diniculty emptying their bladders because of uncwrdinated actions of the bladder and

sphincter (detnisor-sphincter dyssynergia) (Blaivas et al, 198 1). Patients with neurologie

lesions rostral to the pons, do not experience this disorder, indicating a coordinating center

4

Iocated in the pontine tegmentum (Blaivas, 1982; Holstege, l m ) . Brain lesioning studies

in the cat demonstrated that neurons in the brainstem at the level of the inferior coUcuIus

have an essential role in the control of the parasympathetic component of micturition (Tang

and Ruch, 1956). IatercollicuIar decerebration facilitates micturition by eliminating

inhibitory inputs h m higher centers (Tang and Ruch, 1956). However, micturition is

abohhed by transections below the coilicdi (Tang and Ruch, 1956).

In the cat, bilateral lesions in the rostral pons in the region of the locus c d e u s

(Barrington, 1925; Griffiths et ai, 1990) abolish micturition, whüe electrical stimulation of

these sites aiggers bladder contractions and micturition (Kuru, 1965; McMahon and

Spillane, 1982; dffiroat, 1975). These observations led to the concept of a

spinobulbospinal micturïtion reflex pathway that involves a center in the rostral brainstern

(pontine mictufition center).

The micturition reflex center has been shown to be located in the dorsohteral

pontine tegmental region around the locus coeruleus (Barrington, 1925; Kuru, 1965) but

lesion studies to the locus coenileus demonstrated that micturition reflex œnter in the pons

did not include the locus coenileus region in rat (Satoh et al, 1978). Lesion to an area

rostral to the locus coedeus region in the rat (iaterodorsal tegmental nucleus) did resdt in

urinary disorder (Satoh et al, 1978). In the cat the pontine micturition center has b e n

identiaed with the locus coenrleus (deGroat, 1975), the locus coenileus alpha (Sugaya et

al, 1987) or with a distinct population of neurons located ventromedial to the locus

coedeus adjacent ?O the mesencephalic tract of the trigeminal neme (Tan and Hoistege.

1987)-

Barrington's nucleus, a pontine nucleus ventromedial to the locus coenileus, has

been implicated in micturition (Barrington, 192 1, 1925). Using an anterograde tracer

(Holstege et al, 1986; Blok and Holstege, 1997). this nucleus has been shown to project to

the intermediolateral column of the lumbosacral spinal cord in the region of preganglionic

neurons that innervate the pelvic viscera. Furthermore, retrograde tracers injected in the

intermediolatera1 column of the lumbosarral cord labeled neurons in the area corresponding

to Barrington's nucleus (Hida and Shimini, 1982; Holstege et al. 1986; Loewy et al.

1979). Bladder injection with pseudorabies virus (tran~ynaptic tracer) also labeled these

neurons. implicating an involvement in micturition regdation (Nadelhaft and Vera, 1995).

Barrington's nucleus was desctibed in this snidy in the rat as an oval area 0.5 mm X 0.2

mm X 0.25 mm (dorsoventral X mediolateral X rostmcaudal dimensions) centered on a

point 1 mm lateral to the midliae and 0.5 mm below the border of the fourth ventricle

(Nadelhaft and Vera, 1995).

Elecnical stimulation of Barrington's nucleus in rats and cats resulted in bladder

contraction and increased bladder postgangiionic nerve activity (Noto et al, 199 1; Kabat et

al, 1936). Single unit recording in this pontine region identined neurons that responded to

changes in bladder pressure moto et al, 1989). Sugaya et al (1987) elicited micturition by

injecting carbachol to the poniine micturition center. Micturition cm be inhibited by lesions

(Barrington, 1925) or opiate administration to Barrington's nucleus (Wiette et al, 1988).

M-region and L-region of the Pontine Micturion Center

It has been observed that the dorsornedial part of the donolateral pontine

tegrnenaun (M-region) projects preferentially to the spinal parasympathetc nucleus while

the ventrolateral part of the dorsdateral pontine tegmentum (L-region) projects

preferentially to the nucleus of ûnuf (Holstege et al, 1986). The M-region &O overlaps

Bacrington's nucleus and is therefore regarded as the micturition center, while the L-region

is thought to be the control center for the storage of urine. Electrical stimulation of the L-

region elicited a prompt increase in the pelvic floor electmmyogram and urethral pressure

but had little effect on the intravesical pressure (HoIstege et al, 1986; Kun, and Yamamoto,

1964). Electrical stimulation of the pootine micturition refiex center in the cat inhibited

6 extemal urethral sphincter activity, and elicited an increase in intravesicai presmre

(Holstege et al, 1986; K m and Yamamoto, 1964). Bilateral lesions in the M-region result

in a long penod of urinary retention, depression of detnisor activity and an increase in

bladder capacity (Grifnths et al, 1990). Bilaterai lesions of the Lregion result in an

inability to retaidstore urine, reduction in bladder capacity, and premature urine expulsion

by detmsor hyperactivity accompainied by urethral relaxation (Grfiths et al, 1990).

During the filhg phase, the L-region exerts a continuous excitatory effect on the nucleus of

Onuf to elicit contraction of the extemal urethral sphincter. mictuxition, the M-region

excites, via a direct pathway, the sacral parasympathetic motoneurom, while iahibiting the

Lregion to allow mictuntion to occur (HoIstege et al, 1986).

Other Connections to and Crom Barrington's Nucleus

Numerous regions such as the forebrain lirnbic structures which have been

implicated in fiuid balance or blood pressure reguiation (Valentino et al, 1994).

neuroendocrllie hinction and reproductive behaviour (Rizvi et al, 1994), project to

Barrington's nucleus.

Besides the sacral parasympathetic nucleus, Barrington's nucleus projects to other

regions such as the dorsal motor nucleus of the vagus nerve, rostral ventrolateral medulla

(Ruggiero and Reis, 1987), locus coedeus (Viuard et al, 1995) and the paravenmcular

thalamic nucleus (ûtake and Nakamura, 1996). Inhibition of reflex biadder contraction by

opioid receptor stimulation in the pontine micturition center results in concomitant inhibition

of increases in blood pressure, hem rate, tidal volume and respiratory rate n o m d y

associated with increases in vesical pressure, but no inhibition of basal cardioresphtory

parameters (WiUette et al, 1988). The inhibition of blood pressure increases associated with

bladder contraction suggests that Barrington's nucleus, although not an integrai participant

in the cardiovascular r d e x pathway, may modulate neurons responsible for

cardiorespiratory functions (ie; rostral ventdateral rnedulla, an area essential for

cardiorespiratory regdation) (Ruggiem and Reis, 1987).

Barrhgton's nucleus pmjects to the paraventricular thalarnic nucleus. This nucleus

provides a "limbic" component of basal ganglia-thaIamocortical circuits implicaîed in the

neurocopitive, emotional or visceral concomitants of bebaviours (Otake and Nakarnura,

19%). This subset of neurons in Banington's nucleus which projects to the paraventricular

thalamic nucleus also projects to the sacral parasympathetic nucleus via axon cohterals in

rats (Otake and Nakamura, 19%). Retrograde double labelling aiso demonstrated fibers

h m Barrington's center projecMg to L6 of the spinal cord, tbalamic midline, locus

c o d e u s , subcoeruleus nucleus and sublaîerodorsal nucleus in rat (Otake and Nakamura,

1996).

Bulbospinai Pathway for Micturition

The neurons in the L- and M-regions send their axons to the sacral spinal cord via

specinc tracts. Kum (1965) lesioned areas which could elicit bladder motility upon

stimulation. His study on the locations of lesioned descending tracts resulted in

identification of three descending tracts: the laterai, ventral and medial retidospinal tracts

(Kuru, 1965). In cars, bladkr efferent fibers have also been shown to be located in the

dorsolateral funiculus (Kerr and Alexander, 1964) and lateral huuculus (McMahon and

Momison, 1982a; Fedirchuk and Shefchyk, 1991). Fedirchuk and Shefchyk (1991) found

that axons from the pontine micturition center travel in the lateral reticulospinal tract

occupying a ventral location at the œ ~ c a l levei but shiftng to a dorsolateral position at

thoracic and lumbar leveis. Axonal tracing studies demonstrated that this pathway pmjects

dVectly to the sacral parasympathetic nucleus and to lamina I on the lateral edge of the

dorsal hom Wolstege et al, 1986). an area bat contains dendntic projections fmm the

sacrai preganglionic neurons and pelvic atTerent terminais (Nadelhaft et al, 1980; Morgan et

8 al, 1981; deGroat, 1986). Although, the site of tennination of descendhg projections from

the pontine micturition enter are optimally located to reguiate reflex mechanisnu at the

spinal level, it is not known if they make monosynaptic connections with the preganglionic

neurons or segmental interneurons (Yoshimura et al, 199ûa,b).

McMahon and Morrison (1982a) proposed that the descendhg excitatory pathway

h m the pont+ micnintion center is gated in the spinal cord by afferent input b r n the

lower urinary tract However, pontine mictinition center stimulation and stimulation of the

descendhg axons before and after transeciion of the lumbosacral dorsal mots revealed that

the excitatory action of the descendhg pathway was independent of facilitaion h m

primary afferent input (Shefchyk. 1989; Kruse and deGroat, 1992).

Suprapontine Innuences on the Michirition Reflex Pathway

Several discrete regions of the pons and medulla initiate and coordinate lower

urinary tract function (Barrington, 192 1, 1925; dffiroat, 1975; deGroat and Ryall, 1969;

Kum, 1965; Satoh et al, 1978; Tang and Ruch, 1956). In &als and humans with an

intact neuraxis, bladder distension is a primary stimulus for initiating a michrition reflex

involving a supraspinal pathway. Superimposeci on this reflex mechanism is input from

faciliatory and inhibitory centers in the brainstem, and pontine-mesenphalic reticular

formation.

For example, bilateml pamcentrai gyrus lesions cause inability to initiate rnictuntion

voluntarily (Torrens, 1995). Bilateral lesions of the superior frontal gyrus causes

incontinence in the fonn of coordinated voiding with loss of social awareness or sometimes

unconsciousness in the act of voiding (Andrew and Nathan. 1964).

Some studies suggest that the anterornedial amygdala has inhibitory influences on

micturition, and the posterolateral arnygdala and hippocampus are faditatory (Gjone, 1966;

Edvardsen and Ursin, 1968).

9 The hypothahmus has predominantly faditatory (dmIaterai) and predominately

iahibitory (ventromedial) areas relating to micturition (Torrens, 1995). The medial preoptic

area of the hypothalamus has been shown to be involved in the conml of male semal

behaviour and pede erection, But this site does not project to the spinal cord (Simerly and

Swanson, 1988). Instead, it projects heavily to the PAG (Rizvi et ai, 1992) and

Barrington's nucleus (Rizvi et ai, 1994). It is possible that these projections regulate

mictuntion reflexes during reproductive behaviour by rnoduiating the activity of the extemal

urethrai sphincter since pseudorabies Wus injection into the external urethrai sphincter

resulted in labelhg in the preoptic area (Nadelhaft and Vera, 1996) while pseudorabies

Wus injection into the bladder did not (Nadelhaft and Vera, 1995).

Electricai stimulation of the pontine micturition center (alpha-locus coeruleus

nucleus) in the rat causai bladder contractions which were inhibiteci by electncal

stimulation of the nucleus reticuiaris pontis oralis (Kimura et al, 1995). Therefore, it was

concluded that the nucleus reticularis pontis oralis has an inhibitory influence on the

functions of the micturi tion center (either via a direct inhibitory pmjec tion from the nucleus

reticulaRS pontis ora l . to the pontine micturition center, to the sacral cord, or via a direct

excitatory projection to the locus subcoedeus nucleus (a urine storage center).

Micturition can occur without the cerebellum (Mshizawa et al, I989), but

stimulation of cerebeiïar nuclei has been shown to have both facilitatory (Chambers, 1969)

and inhibitory effects (Bradley and Teague, 1969; Martner, 1975). The fact that

cerebellectomy had no effect on urethral ekctromyogram, but decreased the threshold

pressure and volume in the collecting phase, and also decreased the micturition pressure

(N'shizawa et al, 1989) suggests that during filling, the cerebellum inhibits michrition but

facilitates it during emptying (Tomns, 1995).

10 Afferent Pathways

The body of the bladder and bladder neck are innervated by parasympathedc pelvic

nerves and sympathetic hypogastnc nerves. The extemal sphincter is composed of sniated

muscle and is innervated by the PUDNs. The pelvic, hypogastric and PUDNs ail contain

sensory and motor fibers (afferent and efferent fibers).

The bladder wail ee ren t axons terminate as fke nerve endings within the muscle

fascicles and the perifascicular connective tissue of the smooth muscle and mucous layers

(Fletcher and Bradley, 1970). The feline PLN is composed of small myelinated A-delta

fibers and unmyelinated C-fibers. The srnail myehated biadder wall mechanoreceptor

fibers respond both to active contraction and passive distension (Bahns et ai. 1987).

The hypogastric afferent fibers f o m a bctionally homogenous population of

bladder wall mechanoreceptors, comprising equal numbers of myelinaîed A-delta and

unmyelinatted C-fibers in the cat (Bahns et ai, 1986).

W e the PLN afferent fibers innervate detnisor and bladder neck equally,

hypogastnc aEerent tenninations are largely restncted to the bladder base (Uemura et al.

1973). The PLN tenninals are more concentrated in the smooth muscle layers and the

hypogastric nerve afferent te& are more numerous in the mucosa and submucosa

(Uemura et al, 1974 and 1975).

The hypogastric afferents appear to have ongoing actvity when the bladder is

empty, unWre the PLN mechanoreceptor fibers which are silent when the bladâer is empty

(Bahns et al, 1987).

The function of the vast majority of the unmyelinated PLN bladder afferents

remains obscure (Janig and Koltzenburg, 1990). Some unmyelinated fibers are

chernosensitive, while others are mechanosensitive at high pressures (40-55 cmH20)

(Habler et al, 1990). These fibers are probably involved in mediating visceral pain (Janig

11 and Koltzenburg, 1990). Some llnmyelinated fibers can act as cold receptors (Wntter,

197 1) while others are involved in the micturition reflex in spinal cats (de Groat et al.

198 1).

Bladder Afferent Terminais in the Sacral Spinal Cord:

Flurorescent tracers injected into the bladder and urethra resulted in labelhg

prirnarily of S2 dorsal root ganglia (Downie et al, 1984). but bladder aerents also enter

the fzst and third sacral segments (Mawe et al, 1984; Momson, 1987). Application of

horse-radish peroxidase (HRP) to the PLN demonstrated that afferents entering the dorsal

hom of the spinal cord at Lissauer's tract and lamina 1, extend via the lateralcollaterd

pathway II to IV) toward the sacral parasympathetic nucleus in lamina W (Morgan

et al, 198 1) and via the mediai-collateral pathway to extend into the dorsal gray commissure

(lamina X) (deGroat, 1986). The relative density of afferent projections to the lateral

collateral pathway and medial collaterai pathway varies according to the orgaa Projections

to the lateral collateral pathway are prominent for colon, blad&r and uterine pathways,

whereas projections to the medial collaterd pathway and dorsal gray commissure are

relatively weak for colon afferents in cornparison to bladder and uterine pathways

(deGroat, 1986). Injection of different tracers into the bladder and large intestine

demonstrated that sunilar nurnbers of dorsal root ganglion c e k (mainly in S2) innervate the

bladder and large intestine. It was also noted that 46% of this population of neurons was

doubly labeiled and presumably provided an innervation to both organs. However, the

hinctional signincance of a f f e ~ n t newons with receptive fields in two different orgam is

unlmown (deGroa~ 1986; Kawatani et al, 1985). There is no evidence for branching

afferents innervating different regions of the lower urinary tract (Downie et al. 1984).

The functions of the pelvic viscera are closely linked to hinctions of various somatic

stmchires innervated by the PUDN such as the urethrd and anal sphincters (NadeIliaft and

12 Booth, 1984). Since the performance of excretory and sexual hinctions requires integration

of viscerai and somatic mechanisms. an overlap of PL and PUD afferent pathway might be

expected to and does occur at ceriain sites in the sacral spinal cord (üeyama, 1984. 1985).

PUD af5erent projections overlap with Msceral afferents in the lamina I and V of the laterai

coilateral pathway. the media1 collateral pathway, and in the dorsal gray conmisme- PUD

afferents atso project to lamiaa II and IV which do not receive significant nurnbers of

viscerd afferents (Roppolo et al. 1985). These projections represeat input from cutaaeous

afferents in the perineum (Brown and Fuch, 1975) whereas the projections to the lamina 1

and V of the lateral collateral pathway are Iikely to represent in part input b m pelvic flwr

muscle (Craig and Mense, 1983) and urethra (deGroat, 1986). Electmphysiological studies

have also shown a prominent convergence of viscerd and somatic aHerent inputs ont0

intemeurons and spinal tract neurons in lamina 1 and V and the dorsal commissure of the

lumbosacral spinal cord (deGroat et ai, 1981, McMahon and M o & u ~ , 1982a,c; Honda,

1985).

Spinobulbar Pathway for Micturition

Demy-Brown and Robertson (1933) proposed a local sacral reflex for rnictuntion,

mediated entirely by the PL and PUDNs. However, the micturition pathway has been

shown to involve a supraspinal component since electrophysiological saidies in cars and

rats indicate that the parasympathetic efferent outllow to the urinary bladder is activated by

a long latency supraspinal reflex pathway (KULU, 1965; deGroat and Ryall, 1969; deGroaf

1975). Electncal stimulation of afferents in the PLN elicits a long latency (120- 140 msec)

discharge measured on the bladder nerves. This corresponds to the sum of the ascending

latency to activation of neurons in Barrington's center (80 rnsec) and descendhg latency to

recording a discharge on the pelvic nerve when stimulahg in Barrington's center (40

13 msec) (MaUory et al, 1989; Nom et al. 1991). These hdings are consistent with the notion

of a supraspinial micturition reflex.

nie spinal pathways that transmit sensory information h m the visceral afferent

tenninations in the spinal cord to more rosaal structures can be found in the dorsal, lateral

and ventral columns. Primary afferent cokterals carrying touch and pressure sensation in

the urethra and hocuous sensations fkom the PL floor muscle are conveyed via the dorsal

column (Nathan, 1956). The dorsal column also has a synaptic path, the postsynaptic

dorsal column pathway which has been shown to convey visceral (colocectal not bladder)

and noxious cutaneous inputs to the nucleus gracilius (Al-Chaer et al, 1996). These

ipsilaeral dorsal column projections synapse in the grade nuclei in the medulla (Morgan et

al, 198 1, Ueyama et al, 1985). The lateral columns transmit information concerning

temperature sensation in the urethra, the sensation of bladder fullness and desire to

micturate, sexual sensations, and pain sensations from the bladder, ureihra, lower ureter,

and skin. This pathway is thought to be the spinothalamic tract (Nathan and Smith, 195 1;

Nathan, 1956). Electrophysiological studies in the cat have identined a tract in the dorsal

half of the lateral column near the surface of the spinal cord and a tract in the ventrai and

ventrolateml column which convey infornation fbm the bladder tension receptors (Kuni,

1965; McMahon and Momison, 1982a; Fedirchuk and Shefchyk, 199 1; Fields et al, 1970).

Fibers in the dorsdateral tract in the thoracic spinal cord form the primary pathway for

activating the supraspinal michnition reflex as stimulation of this tract evokes coordinated

rnicturîtion (Fedirchuk and S hefchyk, 199 1).

Spinal tract neurons presumed to conmbute to the ascending limb of the micturition

reflex pathway exhibit long latency discharges (30-40 rnsec) foliowing P M afferent

stimulation (McMahon and Morrison, 1982a). This latency raises possibility that at Ieast

one intemeumn is interposed between primary Serent and spinal tract neurons. Sacral

intemeurons responding at shorter latency (5 msec) have k e n reported (Morrison, 1987).

14 Interneu~ons responsive to bladder distension have been identined in both dorsai

c o ~ u r e and the intemediolateral regions of the sacral spinal cord (deGroat et ai, 1981;

McMahon and Morrison, 1982b; Honda, 1985).

In general, the basic rnicturition refiex pathway invoives: (1) activation of

mechanoreceptor in the bladder wail; (2) transmission of bladdex inputs to the sacral cord

via PLN; (3) termination of bladder afferents in the sacral cord (lamioa 1, II, IV, and X);

and (4) bladder impulses ascending in spinal columns to Barrington's nucleus where

micturition is elicited via descendhg tracts to the sacral puasympathetic nucleus, and

activation of efferent fibers in the PLN. The only problem is that there is a pauàty of sacral

spinal projections to Barrington's center @Blok et al, 1994). Therefore it has been proposed

that spinal projections tenninate in other brainstem area(s) before the information is

conveyed to Barrington's nucleus. This brainstem structure should be caudal to the

superior coiliculi (in accordance with lesion studies performed by Barrington, 192 1 and

Kuru, 1965) and have some connectivity with Barrington's nucleus.

There are many structures which are located caudal to the inferior colliculus and

receive numerous spinal projections. An example would be the parabrachial nucleus (Ma et

al, 1989). c-Fos saidies have implicated this nucleus in the processuig of visceral

nociceptive inputs h m the area of the sacral parasympathetic nucleus @ing et al, 1994).

Pseudorabies v ins injection into the bladder wall resulted in labelhg in Barrington's

nucleus, locus coeruleus, paraventricular nucleus of the hypothalamus, medial preoptic

nucleus, and the perhqueductal gray (PAG), but not in the parabrachial nucleus (Nadelhaft

et al, 1992).

The Periaqueductal Gray (PAG)

The PAG extends from the level of the posterior commissure and the oculomotor

nucleus to the level of the dorsal tegmental nucleus. There are different ways of dividing

15 the PAG into subdivisions. Beitz (1985) divided the PAG into dorsal, dorsolateraî,

ventroiateral, and medial subdivisions. The medial zone is the region that immediately

surrounds the cerebral aqueduct and the remaining regions are based on the dorsal-vend

dimensions. The PAG is organized in columns: dorsal, dorsolateral, ventral and

ventrolateral columns (Depaulis et al. 1992). Electrophysiological studies provideci

evidence for longitudinal columns arrangements of neurons for defernive reactions in the

rat PAG in the dorsolateral and ventrolateral subregions (Depaulis et al, 1992).

The PAG is involved in at least five major bctions. These include: pain

processing and modulation, vocalization. autonomie regdation (i; cardiovascular control),

fear and anxiety, and lordosis. AU of these fiinctions interact and the PAG is involved in

the integration of these p m e s s e s (Behbehani, 1995).

The PAG is an important site in ascending pain transmission (Magoun et al. 1937).

It receives afferents fmm nociceptive neurons in the spinal cord and sen& projections to

thalamic nuclei that pmess nociception (Yezierski and Schwarz, 1986; Yezierski. 1988).

The PAG is also a major cornponent in the descending pain inhibitory pathway (Reynolds,

1969; Mayer et al, 197 1). Activation of this pathway can inhibit nociceptive neurons in the

spinal dorsal hom.

The dorsal PAG is a major site for processing fear and anxiety (Bander and

Canive, 1988; Bander and Depaulis, 1988). It ioteracts with the amygdala which is the site

responsible for generating fear and anxiety (Bander et al, 1985; Rizvi et al, 1991). Lesion

of the PAG alters fear and aflxiety produced by stimulatiug the amygdala. Lesion of PAG

wiII result in mutism (Mehck et al, 1958; Jurgens and Pratt, 1979 ) while stimulation of

the PAG produces vocdization (Magoun et al, 1937; Jurgens and Pratt, 1979). The PAG is

also involved in lordosis (Sakuma and Pfaff, 1979).

16 Evidence for PAG Involvement in Micturition

Anatomical studies suggest that the PAG may be a major target of afferent input

from the s a d spinal cord (Blomqvist and Craig, 1991; Vanderhorst et al. 1996). Blok et

al (1995) found that there were more antemgrade iabeiled tenninals in the PAG than in

Barrington's nucleus after WGA-HRP injection into the lumbosacral cord Projections

from the lateral PAG to Barrington's nucleus has been established (Blok and Hoktege.

1994). Valentino et al (1994) demonstrated ushg a retrograde tracer, cholera toxb subunit

B, a projection h m ventdateral PAG and Banhgton's nucleus. The type of information

canïed by projections from spinal cord to the PAG and then PAG to Bamhgton's nucleus

is unlaiown. However, the injection of pseudorabies vim. a traasynaptic tracer, into the

bladder produced labelling in Barrington's nucleus and the PAG (Nadelhaft et al, 1992),

providing some evidence for PAG involvement in conveying bladder information. Further

support for PAG involvement in micturition can be found in electrophysiological studies.

Stimulation of the PAG produces bladder contraction (Kabat, 1936; Noto et al, 1991).

Noto et al (1991) demonstrated that PLN stimulation produced shorter latency negative

field poientials in the PAG (13-15 msec) than in laterd dorsal tegmentual nucleus

(Barrington's nucleus) (30-40 mec) in rats. The PAG may be a receiving area for

spinomesencephalic tracts carrying afferent information from the urinary bladder and

neurons in the PAG may in tum process and relay this information to the lateral dorsal

tegmental nucleus (Noto et al, 1991)-

Since the PAG is lcnown to be an inkgrative center for numemus autonomie

processes, and it does make the appropriate connections to elicit micturition, there is reason

to believe that the PAG is involved in the rnicturition reflex pathway.

Research Proposa1

Rationate

Little is known about the supraspinal control of micturition. h the p a s it was

accepted that Bankgton's nucleus was the controlling center for micturition but there are

several hdings which supports the clah that the PAG is also involved in the processes of

michuition:

(1) PAG stimulation can cause biadder contraction (Kabat, 1936);

(2) PAG is a major target of neurons pmjecting from the sacral spinal cord. (Blomqvkt and

Craig, 199 1 ; Vanderhorst et al. 1996).

(3) There is a substantial projection h m the PAG to Barrington's nucleus (Blok and

Holstege, 1995).

(4) PW stimulation in the rat evoked potentials with shorter latency in PAG than in

Barrington's nucleus (Noto et al, 199 1).

The mode1 that is presented by Blok and Holstege is as follows: bladder afferents

enter the sacral spinal cord, and bladder information is conveyed by spinal projecting

neurons to the PAG. Barrington's aucleus then receives bladder input from the PAG. The

descending limb of the basic micturition pathway consists of neurom projecting from

Barrington's nucleus to the sacral spinal cor& to activate pelvic pregangiionic neurons and

elicit bladder contraction (figure 1).

Although it has been demonsûated that there are spinal cord projections to the PAG,

it is not b o w n whether these projections are involved in bladder or bladder-related

functions. Therefore we will atternpt to detennine whether bladder-reiated information is

carried on this pathway. The hypotheses to be tested are: (1) the sacral spinal cord

projection to the PAG carries bladder-related information (pediaps in addition to other

information); (2) a component of this ascending pathway (Le. some individual neurons)

conveys bladder-s@c infornation.

Aims of Experimentai Preparations

Neuroanatornical and neurophysiological approaches will be used to locaiize.

identQ, and characterize bladder sensitive neurons projexting from the sacral spinal cord to

the PAG.

For the neumanatomical studies, three different techniques will be used to establish

the comectivity of neurons conveying bIadder input h m the sacral cord to the PAG:

(1) Retrograde m i n g techniques will be used to establish a direct projection h m the

sacral cord to the PAG.

(2) Induction of the imrnediate early gene, c-fos, by bladder distension @hier and

deGroat, 1992) will be used to identify spinal neurons related to bladder function.

Combining this technique with retrograde aacing wiU enable us to determine whether sacral

spinal ceUs projecting to the PAG are likeIy to carry bladder-related information.

(3) Antemgrade aacing techniques will be used to i d e n m the terminal field distribution of

bladder afferents in the sacrai spinal cord Although not definitive, the results of this type

of study may help in asssessing whether die comection between primary afferents and

projecting neurons is likely to be monosynaptic.

Extensive anterograde and retrograde tracing data were presented by Vauderhorst et

al (1996) while we were in the midst of our studies. Our neuroanatornical work was

abbreviated as a result.

Although, it was asssurned for a long t h e , 3 a t the ascending limb of the

micturition reflex probably synapsed directly in Barrington's nucleus. littIe

neurophysiological work has addressed this issue and the question of the routing of bladder

af£erent information. The characteristics of the neurons carrying bladder afferent inputs also

have not been adequately described.

Figure 1 : The Basic Michrition Reflex Pathway. It was thought this pathway involved

Barrington's nucleus with an aEerent and efferent limb h m and to the sacral spinal cord

(A). Recently, Holstege et al (1994) proposed an involvement of the PAG in the basic

micturition rdex pathway (B). SC, superior collicdus; IC, infenor coiliculus; CAq,

cerebral aqueduct; CPed, middle cerebeiiar peduncle; N. Aug, nucleus augustus.

For the nemphysiological studies, the four -c objectives are:

(1) to elicit field potentials in PAG with PLN stimulation.

(2) to construct a 3-D map of bladder-sensitive ce& located in the PAG.

(3) to characterize the cells in the sacral spinal cord that pmject to the PAG.

(4) to determine whether any cells projecting h m the sacral spinal cord to the PAG carry

PLN information exclusively. SPUD, SFP and CCF nerves were also stimulated to test the

specincity of this ascending pathway.

These objectives will be approached by using extracellular field potential recordings

in the PAG and extracellular single unit recordings in the sacral spinal cord

MATERIALS AND MEXHODS

Neuroanatomicd Experiments

Aseptic surgical preparation for tracer injection

Ten adult cats of either sex, weighing 3.1-5.2 kg, were used for the

neuroanatomical tracing studies. The animais were sedated with a mixture of 2 mglkg

acepromazhe (Atrovet@), 20 mglkg meperidine HCI (Demerol9). and 0.12 mgkg atropine

(im). and anesthetued with 28 mgkg thiopental (i-v.). After tracheal intubation. the cat's

head was shaved and placed in a stereotaxic apparatus. Body temperature was monitored

h g a rectal pmbe and maintained at 37OC using a heating pad layered with blankets.

Durhg the aseptic surgical procedures, reflex statu, heart rate (using an esophageal

stethoscope), and pulse pressure were rnonitored. Anesthesia was maintained with 1 5 2 %

halothane (Fluothane) in Oz (total flow rate of 1.5 Umin). The scalp was scrubbed with

4% chlorhexidine gluconate (Steri-Stat), followed by alcohol (isopropyl) and then a 10%

provioduie solution. A midline scalp incision was made and the muscle was dissected to

expose one side of the skull. Using a hand-held electric dRU @remel. WI), a small

craniotomy was performed to expose a s m d area of the braui. The dura was then opened.

PAG retrograde tracer injections

A glas micropipette secured with dental wax on a Hamilton syringe which was

placed in a stereotmic holder was used to inject tracers. Two to three percent fluoro-gold

(Fluorocbrome Inc.) (n=7), 5% wheat gem agglutinin horseradish peroxidase (WGA-

HRP) (Sigma Inc.) (n=3), or acidic 10% biotin de- (Molecular Probes Inc.) (n= 1) was

22 injected into various regions of the PAG (table 1). Some of these animais were ais0 used in

the anterograde tracer and bladder distension studies.

Bladder anterograde tracer injections

A midline abdominal incision was made to expose the bladder. HRP (2.5-1546) or

WGA-HRP (5%) was injecwi at 5-6 injection sites at the base of the right detrusor and

trigone. Injections were made below the serosa without puncturing the bladder. The total

volume injected into the bladder wall rauged fkom 5-44 pL. Seven animals were used in

this part of the study.

Pos t-surgery

Following tracer injection, the head and abdomen were closed in two layers.

Morphine (0.075 mgkg, Lm.) and cefazolin sodium (20 mgkg, i-m.) were given after the

surgery and again 6 hours later. Supplernental doses of morphine were given as required

during the mt pst-operative day. Cefazolin sodium was administered via intramuxular

injection on the fmt day. Cefadroxil monohydrate (Cefa-Tabs@, 22 mgkg, p.o.) was

given twice a day for the next 3-7 days for animals with long survival times.

Experimental design: survival (staged)

Originally the PAG and blad&r injections were staged (n=2) since different tracers

are transported at different rates. PAG was injected with fluoro-gold two weeks prior to

bladder injection with 5% WGA-HRP. The cat was allowed to recover and a week later,

the bladder distension experiment was performed. In later experiments (n=3), where

WGA-HRP and/or Biotin-Dextran were used for the retrograde studies, the s d v a l time

was shortened to 1 week.

23

Table 1 : Tracer Injection Plans.

Various tracers and tracer volumes were injected into the PAG (retrograde tracers) and the bladder (anterograde tracers). For cases #773 and W85. the injection rimes for the different tracer injections were staged

Case #

retrograde anterograde


re trograde anterograde


retrograde

retrograde


retrograde an terograde


Tracer

2 % fluoro-gold 2.5% HRP

5% WGA-HRP 5% HRP

2% fiuoro-gold 5% WGA-HRP 15%

2% fluoro-gold 5% WGA-HRP 15% HRP

5% WGA-HRP

2% fluoro-gold 5% WGA-EIRP

3% fluoro-gold 5% WGA-HRP

10 % biotin dextran 2% fluoro-gold

Total Volume In jected

6OnL 10

80 I l . 5w 270 nL 7& 37 CLL 360 nL 8W 44 CrL 360 nL

180 nL 180 ni,

70 nL 10 w 73 nL 3w

240 nL 90 nL

24 Surgical preparation for bladder distension

Ketamine (25 mg/@ im.) was used for induction of anesthesia Halothane (14%)

in 1:l nitmgen in O2 at a total flow rate of 2 Umin was useci during the surgicai period The

trachea, carotid artery* and femoral vein were cannalated for amficial respiration, blood

pressure measurement and fluid and dmg administration, respectively. A continuous i.v.

drip (approximately 30 dropdmh) of 5% dextrose in Eünger's solution was maintained

during surgical and experimental periods. Through a rnidline abdominal incision. a

pediairic feeding tube was placed in the dorne of the bladder and secureci with a purse-

string suture. Nitrous oxide and halothane were discontinued at the end of the surgery and

anesthesia was maintained with chloralose (50 mgkg, iv.). Supplemental doses of

chloralose were given as required based on withdrawai responses to toe pad or interdigital

skin pinch.

BIadder distension

The bladder was i d ' with sterile saline using a motor-dnven syringe (Sage

Instruments, mode1 355) at a rate of 15 hourly diuresis units (1 hourly d i m i s unit = 1.1

ml/kg/hr). Bladder pressure was rnonitored on a side arm of the filling line with a pressure

transducer (Micron Instruments) comected to a Gould chart recorder. An abrupt increase in

bladder pressure during bladder nIling was taken to indicate a micturition contraction

because voiduig of fluid through the urethra may not occur rehbly under chloralose

anesthesia (Rudy et al. 1991). The blad&r was drained before the peak of the micaintion

contraction or during any sustained in- in bladder pressure contraction D avoid over-

distension of the bladder. Over a period of two houn. the bladder was repeatedly distended

in 5 animals, or continuousIy drained in one control cat (case W75 1). This control cat did

not receive any injections of either ~trograde or antemgrade tracers.

25 Perfusion and tissue collection

In the studies in which fluoro-gold was used as the retrograde tracer and the bladder

was distendeci, the animals were transcardially perfused with 1.0 L of a chilled pre-rllise

solution of NaNO, (0.1% sodium nitrite in phosphate buffered saline, pH 7.4). and h e d

with 1.5-2.0 L of 4% paraformaldehyde in phosphate buffer (pH 7.4). For the studies in

which 5% WGA-HRP was used for the PAG injection, the animals were transcardially

perfûsed with NaNo,, and fmed with 1.00% padormaldehyde with 1 .Z% glutaraldehyde

in phosphate buffer (pH 7.4).

After Iaminectomy, the spinal cord specimens were coilected as blocks: TlGT11,

L5-L6, L7-S 1, and S2Co. In 2 experiments, the spinai cord specimens were coilected as

individual segments (L7-S3). The midbrain was excised following craniotomy. The dorsal

mot ganglia and bIad&r were also coilected. The tissues were post-fixed in 4%

paraformddehyde at 4 OC for 2-3 days, and then transferred into a 20% sucrose solution in

phosphate buffer, pH 7.4. for another 1-2 days for cyroprotection.

Sectioning the PAG and spinal cord

'Ine midbmin and spinal cord segments were marked with knife cut on the left

ventral quadrant and placed on a fro~en stage. Frozen sections of 40 pm thickness were cut

with a sliding sledge microtome into four series. One of the four series was collected in

serial order whüe the other three series were collected in a non-serial order in phosphate

buffer saline solutions. The sections collected in a non-serial manner were then processed

for immunohistochemistry by the batch method.

For the fluoro-gold injections, the PAG and spinal cord serial sections were floated

out onto slides, coverslipped, and viewed under an Olympus microscope with ultraviolet

illumination Viewing of c-fos protein, biotin dextran and WGA-HRP transport Ui the

26 spinal cord or their injection sites in the PAG, requkd M e r processing (see next

section). Reacted sections were then fke floated onto gelatin subbed slides and allowed to

air dry for 24 hrs. The sections were dehydrated by subsequent rimes through an

increasing alcohol series, foilowed by two rimes in xylene. The slides were coverslipped

using EnteIlan coverslipping medium.

Immunohistochemistry

c-Fos reaction. To localite the neurms in the spinal cord which were activaîed by

bladder distension, one series of the spinal cord sections used a polyclond sheep anti-Fos

prirnary antibody (Cambridge Research Biochemicals, 1:1000) directed against the c-fos

protein. Sections were M e c processeci and stained according to the avidin-biotin

technique using biotinylated rabbit anti-sheep secondary antibody (Dimensions Lab Inc.,

1 : 1000 dilution). The chromagen, diaminobenzadine @AB)-nickel or DAB-brown, was

used to react with 0.34 hydrogen peroxide to stain and allow visii=iIi7ation of the antibody

bound-nuclear Fos protein product The control expriment (#75 1) and three other

experiments used DAB-brown for c-fos visuahation while two experiments used the

DAB-nickel method. Light microscopie analysis of the slides was perfonned. (See

Appendix for further details)

Retmgmde and a n t e r u g r . îracer imm~7~0histochern~cal reactiomr One series of the

spinal cord sections (antemgrade and retrograde tracing studies) was reacted in the same

manner as c-fos, but using a TMB reaction.

The WGA-HEW Iabelling was viewed under polarized Iight using an Olympus

microscope. Light microscopy was used to visualize the retrograde transport of biotin-

dextmn. For retrograde tracer injections, the PAG was also reacted to determine the

27 location of the injection site and the de- of spreadldiffusion of the tracer. (See Appendix

for M e r detatls.

Data handling

Fos-immunoreactive ce& were counted in designated zones of the different spinal

cord segments (L7-S3). The cord was divided into 5 major divisions: medial dorsal hom;

laterai dorsal horn; dorsai commissure; sacral parasympathetic nucleus; ventral hom. These

zones were created by setting the horizontal divison at the apex of the dorsal white matter

and at the ventral border of the central canal, These horizontal divisions were then bisected

with vertical lines to create the quadrants in the dorsal hom. Areas below the central canal

were considered to be the ventral hom zone. The number of c-fos positive ce& in a zone or

spioal segment were expressed as a percent of the total number of fos immunoreactive cells

in an experiment. The percentages for the five bladder distension experiments were then

averageû.

For the tracer studies, the sections. the injection sites and the labelled t e h a i s or

neurons were plotted using an Olympus microscope, Scope Ploüer 1.51 (Scott Pronych.

Dalhousie University) software and a Macintosh cornputer. In the retrograde tracing

experiments, WGA-HRP labelled c e b were counted Erom the 20 sections with the most

labelhg in each spinal cord segment

Neurophysiological Experiments

Surgical preparation

Twenty adult cats (2.9-5.7 kg) of either sex were anesthetized with ketamine

(25mglkg. i n ) . The tmchea was then cmulated for &cial respiration and delivery of

28

1-596 halothane in 1:l nitrous oxide in 9 (total flow rate of Wmia). The right femoral

vein and left cmtid artery were cannulated for the adminiseation of 5% dextrose in

Ringer's solution and dmg and for blood pressure monitoring, respectively. A catheter for

recordhg bladder pressure was inserted into the urînary bladder tbrough an incision in the

urethra. 1-2 cm below the bladder ne&. The pressures were measured using pressure

transducers (Micron Instruments Inc.) connected to a chan recorder (Gould Inc.).

Bladder-directed branches of the right and left PWs were exposed through a

midline abdominal incision and fieed of comective tissues. Silver foil electrodes, 2x10 mm

with a 2x2 mm area of the electrode bared, were used to stimulate the PLNs. The

electrodes were secured onto the uretbta with 5-0 polyester suture (Ethibond Ethicon

Su- Ltd). The nerves were placed onto the bard regions on the foil ekctrodes which

were then fol&d over the nerves. The electrodes and the nerves were insulated from the

surrounding tissues with PlastibaseB (Squibb Canada Inc.), or KwikCast (World

Precision Instruments). The abdomen was then closed in two layers.

With the cat prone, the sensory (SPUD) and motor branches (mPUD) of the

pudendal nerve, the superficial p e ~ e a l (SFP) nerve, and the caudal cutaneous femoral

(CCF) nerve were dissected on the left side. When possible, the motor branch of the

pudendal nerve was separated into anal and urethral branches. Inmuiectomy was carried

out nom LAS2 to expose L7 and more caudal spinal cord segments and their spinal roots.

After the surgical preparations, the halothane anesthesia was replaced by chloralose

(50 mglkg. i.v), and supplemental doses of chloralose (each 10-20 mg&, i.v.) were given

a s required based on a withdrawal response to pinching of the interdigital skin or toe pads.

Gallarnine triethiodide (20 mg initially and supplemented as quired, iv.) was used to

irnmobilize the animal during the recordhg session. Assessrnent of the anesthetic level

29 under these conditions was based on cardiovascular response to pinching interdigital skin

or toe pads. Body temperature was maintahed at 35-38 OC with a heating pad and lamp.

With the completion of the lamiiiectomy. the cat was transferred from the surgical

table into a stereotaxic hune (David Kopf Instruments, Mode1 1404) and a spinal unit on a

vibration isolation table (Technical Manufkchiring Company. M i m g series 63-500).

Bilateral pneumothorax was performed to demase respiratory movements. SPUD. m m .

SFP. and CCF were mounted on bipolar platinum iridium hook electrodes submerged in a

pool of heated mineral oil fonned by tyhg skin fiaps.

The dura was excised and refiected nom the spinal cord. and the cord was covered

by a heated mineral oil pool. Using a cartilage that nias across L741 junction as a

landmark, the nght S 1 and S2 dorsal mots were identified and traced to their spinal

segments. A silver bail rexording electrode was placed near the SI-S2 junction to record the

cord dorsum p0tentia.L

Access to the brainstem was achieved by craniotomy. In most experiments part of

the cortex and the bony tentorium were removed so that surface landmarks could be used

for more accurate placement of the electrodes. Dexamethasone (2 ml, Lv.) was given to

reduce swelling and thus distortion of brain structures.

Mapping of the PAG - Extracellular Field Potentials

Stimulation of peripheral nerves

The PLNs were stimulated by a single pulse or a train of 3 pulses of 0.5 msec

width at 1000 EZz, with an intensity 2-5 t h e s the threshold canent required to elicit a cord

dorsum potentid or a pelvic-pudendal reflex In the absence of a reflex or cord dorsum

recording, a maximum intensity of approximately 2-3 mA was used The central cut ends

of the other peripheral nerves (SPUD, SFP, and CCF) were stimulated with single pulses

30 of 0.2 msec wideth at an intensity of 5 times the threshold ciirrent r e q d to eiicit a cord

dorsum potential

Extracellular field potential recordings in the PAG

Single, monopolar tungsten microeIectrodes with 60 pm-tip diameters and

resistances of 9-12 MOhms were used to search the PAG h m A6.0 to P2.5. Distances

between transverse planes ranged b m 05-1 mm and in each track recordings were made

at depths h m 2-8 mm below the surface of the midbrain. In three experiments

mstrocaudal sûarching was restricted to APO-Pl5 to permit more data to be obtained in

searching in the medioIateral plane. Here, distances between tracks ranged nom 0.5 to 1.0

mm. The signals were amplified (gain=loOOX), fitered (bandpass=l Hz-3kH.z). and

displayed on an oscilloscope. Cord dorsum and PAG field p0tentia.k were digitized at

sampling rates ranging from 2-10 kHz (MacLab Scope, AD Instruments Inc.). The

digitized sigaais were averaged over 16 sweeps and the averages stored on a Macintosh

cornputer.

PAG field potential data handling

Maximum field potential locations for each of the nerves were detemiined and

mapped onto standard outlines of the cat midbrain b m a stereotaxic atlas of the cat brain

(Snider and Niemer, 1961). For the PLN stimulation, our hypothesis assumes that there is

a maximum PLN field potentid which could be elicited in the PAG. Therefore, maximum

responses fuaher than lmm from the border of the PAG were disregardeci and the next

largest field potential located in or within 1 mm of the PAG was considered to be the

maximum.

Mapping of the Spinal Cord - Single Units

Stimulation of the PAG

Prior to PAG stimulation, the bladder was distended with 5-1 0 mL of saline. Two

strategis were used to determine the appropriate placement of the PAG stimulating

electrode. ûne strategy involveci eliciting bladder contractions. Sites in the caudal PAG

(AO.IP1.5) were shulated with constant current pulses of 3 mA, at 50 Hz, in search of

an optimum site for evoking PAG-evoked bladder contraction. The other strategy used

PLN-evoked field potential recordings in the PAG to detamine the optimum PAG site for

antidromic studies.

For most experiments, a single stimulating tungsten microelectrode (60 p - t i p

diameter, 9- 12 MOhms) was placeci in the midbrain at a location which when stundated

elicited a rise in bladder pressure (measured by the bladder transducer and chart recorder).

Insulation was removed for a distance of 1130 pn h m the electrode tip to increase the

suface area and reduce the resistance of the electrode. Several arrangements of indifferent

electrodes were tried, but nonnaliy, the indifferent ektrode was placed either on muscle or

cerebellum. In two experiments two stirnulating electrodes were used, separated by a

distance of 1.8 mm h m the indifferent electrode. In this case, one stimulating electmde

penetrated the left PAG while the other penetrated the right, with the indifferent elecaode at

m i m e .

Once a location in the PAG was found that could elicit a large bladder response, the

stïmulating electrode was kept at that PAG location. For the single unit searches in the

spinal cord, the PAG was stimulated with a train of 4-5 0.5 msec pulses of 3 mA inteI1Sity

at 333 Hz. Trains were delivered at an intertrain interval of 2 sec.

32 Single unit recordings in the spinal cord

Using a micloelectrode pulier (Narishige Scientinc Instrument Laboratory, PE-2).

recording glas microelectrodes were drawn fiom 1.2 mm X 0.68 mm (outer and b e r

diameter, respectively), 4" hlamented glass capillaries (A-M Systems Inc.). They were then

med with a solution of 4M NaCL A microelectrode manipulator (Tramertex, her ican

Medicd Systems) mounted on a custorn-built Lundberg arch was used to move the

recording electrode to search the spinal wrd for activated cells.

The spinal cord was seanihed in the caudal to mstral direction h m the SYS3

junction to SUS2 junction in steps of 150pn. At each rostmcaudal plane, the

microeiectrode was moved fmm the midline in lateral steps of 150-200 jim untü the dorsal

mot entry zone was reached The search was stopped in a track when a depth of 2ûûû-2700

pm was reached Extracellular si@ were amplified (gain= 1000X). nItered (band pas=

300 H z - 3 W ) and displayed on an oscilloscope. The signals were also digitized at a

sampling rate of 21 kHz and displayed and stored on a Macintosh cornputer (SpiLe 2.2;

140 1 plus, Cambridge Electronics Design).

Peripheral nerve stimulation for spinal cord unit recordings

Units antidromically or orthodromically activated by PAG stimulation were tested

for peripheral nerve inputs. The PLN was stimulateci with a train of 2-3 0.3 msec wide

pulses at 333 Hz and an intensity of 5 tirnes the threshold current for producing a cord

dorsum potential or a maxiniun of 3 mA The other peripheral nerves were stimulateci with

single 0.2 msec wide pulses at an intensity of 5 times the threshold current for producing a

cord dorsum potential Units responsive to PAG and peripheral nerve inputs were

subjected to a collision test

Criteria used to confirm antidromic activation

(1) Unit discharge at constant latency h m PAG stimulus.

(2) Stimulus-locked following of a bief train of stimuli in the PAG at a rate exceeding 333

Hz (usuaIly lm Hz).

(3) Collision of orthodromie and antidromic action potentials during a critical intemal,

Histology

Recording and stimulation sites in the PAG were marked by passing 5û-100 pi of

cunent through the elecwde for 2-45 mia In the PAG field potenbal experiments. the

animals were perfused transcardially with a solution of NaNe, followed by 4%

paraformaldehyde in phosphate b s e r (0.1 M, pH 7.4). The fixeci brainstem was removed

and post-hxed in 4% parafonnaidehyde and transferred into 20% sucrose for

cyroprotectioa F i micrometer sections were cut with a cryotome and stained with

thionin. The location of the electrolytic lesions were determinecl according to a cat brain

atlas (Snider and Niemer, 196 1).

Spinal cord recording locations were marked by cuthg off and leaving the tip of

the glas deetrode in the spinal cord The segment of the spinal cord containing the

electrode tip was removed and placed in 10% fornalin The cord was freeze sectioned with

a cryotome. F i micrometer sections were stained with 2% thionin and the micmelectrode

tracks were identifed. In these experiments the brainstem was pemised through the carotid

artery with 350 mL of 0.9% saline followed by 350 mL of 10% forniaiin. Brauistem

processing was as desCnbed above.

34

Statistical Test

The Mann-Whitney Rank Sum test was used to compare median values of central

conduction velocity determined in the two types of neurophysiological studies.

RESULTS

Neuroanatomicai Experiments

PAG retrograde tracer injections

The objective of this part of our wodc was to use retmgradely transported tracers to

fïnd neurons in the spinal cord with direct projections to the PAG. Of the three tracers used

(Table 1). only WGA-HRP injections into the PAG d t e d in retmgradely labelled

neurons in the lumbosacral spinal cord at the h v a i time examineci, some of the survivd

t h e s were too long. Figure 2 depicts the achial site of WGA-HRP injection and degree of

spread for the tracer for cases W93 and #8 10, with survival times of 4 and 3 days,

respectively. The main injection site for case #8lO was AI%, while W93 injection was at a

more rostral position of A2.5-A3.0.

Injection at more caudal regions of the PAG resulted in more retrograde1y labelled

cells in the spinal cord (#810). even though twice the volume of WGA-HRP was used in

case #793 (Table 1 and Figure 2). Comparing the two PAG injection experiments, there

were more spinal neurons projecting to caudal PAG since the total number of retrogradel y

labelled spinal neurons was 127 with an injection at AW, versus 75 with an injection at

A2.5-A3.0 (Table 2). Labelhg for #793 was M t e d to segments S2 and S3 with no

retrograde labelling in more rostral regions of the cord. For case #8 10, remgrade labelhg

occurred in segments L7 to S3 (Figure 3A and 3C). By combining the data nom the two

cases, 50% of the labelling occurred in S2, while 23% occumd in SI. 17% in S3. and

104b in L7. The total number of retrogradely labelled neurons in S2 was estimated to range

from 48 to 293.

36 Labelling in the spinal cord was prwiominantly contralaterat In both cases there

w a e cluste15 of retrograde1y labelled nemm at the sacral parasympathetic nucleus and the

central canal at the S2 level (Figure 3A and X). Labelled œlls were found in the lateral

parts of lamina V and W in the S2 and S3 segments. This pattern of labelhg was more

apparent in the S2 segments. In L7 and S1 segments, l e s labelhg was seen in the sacral

parasympathetic nucleus but in S1 there was a cluster of PAG projecting neurons around

the central canal and in lamina W extending ventrolaterally. There were a few labelled

neurons along the lateral collateral pathway of S 1. There was some labelling in the ventrai

hom in the S2 and S3 segments, but especially in S3 (#793) along the lateral band of the

sacral parasympathetic nucleus neuroas.

Figure 4 contains camera lucida drawings of retrogradely labelled neurons of

various shapes and sizes, seen at different locations and in different orientations. One type

of neuron displayed a M o r m (or bipolar) soma with two main dendrites (or one dendrite

and an axon) onented in opposite directions. This type of neuron was most often observed

in lamina X Another type of neuron that was retrogradely labelled had a triangular soma

with two apical and two basal fibers extending from the soma (one of which is an axon).

There were also multipolar, round or polygonal somata with dendrites radiating in all

directions. The diameter of the somas for these labelled neurons ranged from 20-80 p.

The number of dendrites extending h m these retrograde cells varied h m 0-4. The larger

neurons, with multiple dendntic extensions, were most often seen around the central canaL

These dendrites tended to extend horizontally towards the sacral parasympathetic nucleus or

across the d o d commissure. The dendrites of neurons found in the lateral coilateral

pathway or lateral band tended to extend in the direction of the sacral parasympathetic

nucleus in a vertical manner.

Primary Ptrerent terminai distribution in spinal cord.

HRP injected into the bladder wall labelleci afferent t e m h i s at the dorsal mot

entry, lamina 1, Lissauer's tract, along the laterai collateral pathway, and the lateral band in

the S2 segment (Figure 3B). There was no labelling along the medial collaferal pathway

leading to the dorsal commissine ( i a m i ~ X). Only ipsilateral & h g of the bladder

afferent terminals was observed Bladder afferent terminal distribution overlapped with

regions containhg neurons retrogradely labelled h m the PAG dong the lateral coilaterai

pathway and the sacral parasympathetic nucleus

c-Fos induction by bladder distension.

Having attempted to localize sacral spinal neurons projecting to the PAG. an

activity-dependent marker, c-fos protein, was used to detemine whether these neurons

might be related to bladder function. c-Fos. an immediate early gene, may be induced in

neurons by various chemical and mechanical stimuli (Weinberg, 1985; Hunt et al, 1987).

Bladder distension can activate the expression of this gene (Birder and deGroat, 1992).

In the absence of bladder distension (n=l), c-€os was expressed in the spinal cord

in L7-S3 @5gure 5) with liale reaction product discemible in S2 and S3. C-Fos protein

immunoreactivity was detected in the dorsal commissm (lamina X) and dorsolateral s a d

parasympathetic nucleus. Bladder distension (n=5) induced c-fos expression on both sides

of the spinal cord (Figure 5'). Labelled neurons were located along the lateral collateral

pathway, near the dorsal mot entry zone and along the lateral edge of the dorsal hom, and

in lamina V, W and X (Figure 5). The c-fos-positive cells fouad in L7, S 1 and S3 were

more concentrateci around the cenaal canal ( i a m i ~ X). In the experiments in which the

bladder was repeatedly distende4 the majority of the c-fos immunoreactive neurons was

found in S2 (Table 3).

Figure 2: Retrograde Tracer Injection Sites in the PAG. Schematic drawing of the site of

5% WGA-HRP injection and degree of spread for the tracer for cases #8 10 (A) and #793

(B). IIlustrations are actual tracings of the cat midbrain sections. Out of the 3 AP levels

injecteci with the tracer? the main injection site for case #8 10 was AEQ while for #793

injection. it was at A2.ÉA3.0.

Table 2: HRP Cell Counts. Total c d counts for remgradely labelled ce& found in 20 sections with the most labelhg for cases #8 10 and W93.

A

case #810 # Ce&

L7 S1 S2 S3 total#

20 47 48 12 127

15.70% 37.00% 37.80% 9.40% 100%

case #793

L7

O

O

# CelIs

S1 S2 S3 total #

O 52 23 75

O 69.30% 30.70% 100%

Figure 3: Distribution of Spinal Cord Neurons Projecting to the PAG and Bladder AfTerent

Temllnals in the Sacral Cord Retrogradely labelled celis in tbe lumbosacral spinal cord

after injection of 5% WGA-HRP into the PAG in cases #8 10 (A) and #793 (C). WGA-

HRP injected into the bladder waiI ipsilaterally labelled afferent terrninals at the dorsal mot

entry, lamina 1, Lissauer's tract, dong the laterd collateral pathway in the S2 segment (B).

Figure 4: Iliustrations of Spino-PAG Tract Cells. Camera lucida drawings of S2 spinal

cord neurons retrogradely labelled following injection of 5% WGA-HRP into the PAG.

Each neuron is numbered and ttiese numbers correspond to the numberings in the

representative spinal wrd section. The bar lines in the section indicates the position, and

orientation of the axond and deadritic extensions for each neuron.

44 Figure 5: Bladder Distension Increased c-Fos Immunoreactivity in S2.

Fos-immunoreactive cells in representative sections h m L7 (AT B. C), SI @, ET F), S2

(G, H). and S3 (I, J) in experiment #785, with repeated bladder distension over 2 hrs (A,

B. D, E. G, I) and a conttol experiment in which the blad&r was drained for 2 hrs (C, F.

H, J). Panels B and E are rnagnified photos of the boxed areas in paneis A and D. Arrow in

panel E indicates c-fos in DC; c-fos cluster. are indicated in panel G by: srnail arrow. LDH

(also referred to as lateral collateral pathway); medium size amwow, SPN; large amw, DC;

smail arrowhead, MDH; large arrowhead, LVH (also referred to as lateral lamina WI) Sale

bars = 500 p.

Table 3: Sum of c-Fos Immunoreactive CeIls in Each Spinal Cord Segment

Blad&r distension for 2 hrs increased c-fos immunoreactivity in S2-as indicated by the sum

of c-fos positive cells (n=5) in each of the spinal cord segments; control (n=l).

Spinal Cord Segmental Total # c-fos positive cells Total # c-fos positive celis

Levels Control B Iadder Distension

L7 1545 149

S 1 1742 2520

S 2 1332 12115

S 3 905 41 10

46 The -test induction of the fos protein by bladder distension also occurred in S2, with a

srnaller increase occurring in S3 and a decrease in L7 and S 1 (Figure 6). Bladder distension

induced c-fos expression in ail five zones of S2 but the greatest induction occurred in the

DC and the sacral parasympathetic nucleus (Table 4 and Figure 7). In L7. bladder

distension suppressed the expression of the c-fos gene in ai l five zones. A decrease in Fos-

immmoreactivity o c c d in 4 zones in the S 1 segment. An induction of c-fos was

observed in the medial dorsal horn of S l with bladder distension. In S3, c-fos was induced

in the medial dorsal hom. dorsal commissure and sacral parasympathetic nucleus but

suppressed in the lateral d o r d horn and lateral ventral hom (Figure 7).

Figures 3 4 B. and C. and Figure 543, show that the distribution of c e h

wtrograddy labelied f?om the PAG overlaps with the Fos-immunoreactive cells in S2.

These spinal neurons are also located within the vicinity of the HRP 1abeUed afferent

terminais. W e were unable to obtain more definitive evidence that retrogradely Iabelled ceh

were related to bladder fiinctions (Le. were Fos-immunoreactive in bladder distension

experiments) due to technical ciifficul tics

Spinal Cord Segments

Figure 6: Distribution of c-Fos Immunoreactive Cells in the Lumbosacral Cord The bar

graph illustrates the degree of c-fos protein induction in different spinal cord segments. The

greatest induction of c-fos by bladder distension occurred in S2, with a smder increase in

S3, and a decrease in L7 and S 1, The number of c-fos immunoreactive neurons in each

segment was expressed as a percent of the total of c-fos neurons in L7-S3. and this

percentage was averaged for 5 experiments. Bars indicate the standard deviation

Table 4: Sum of c-Fos Immunoreactive Ceils in Different Zones of Each Spinal Cord

Segment The major increase in c-fos imrnunoreactivity. following bladder distension for 2

hrs, occurred in the zones DC and SPN of S2. as iadicated by the sum of c-fos positive

cek (n=5) in each segmental zones.

Spinal Experimental MDH

Levels Group

Conml

bladder

distension

Control

bladder

distension

Control

bladder

distension

Control

bladder

distension

LDH

529

32

467

479

38 1

2043

465

327

SPN

447

43

508

610

478

381 1

237

1660

LVH

8

O

35

1

66

429

4

217

Spinal Cord Regions in each Segments

Figure 7: The Dorsal Commissure in S2 is the Major Site of c-Fos Indi

The bar graph depicts the area of greatest c-fos induction in each spinal cord segment

Bladder distension induced c-fos expression in all five zones of S2 but the greatest

induction occurred in the DC and the SPN, The number of c-fos neurons in a zone was

expressed as a percent of the sum of c-fos neurons in the 5 zones, and then averaged

(n=5). The control groups are L7-CTRL, SI-CTRL, S2-CTRL, S3-CIRL and the

experimental groups are L7, SI, S2, S3. MDH, medial dorsal hom; LD& lateral dorsal

horn; DC, dorsai commissure: SPN, sacml parasympathetic nucleus; L W , lateral ventrai

hom. Bars represent the standard deviation for the bladder distension group.

Neuroph ysiological Experïrnent

PAG extracellular field potentials

Multiphasic field potentials could be elicited in the PAG by stimulation of the PLN

in 13 of 20 experiments. Data from a representative experiment are shown in Figure 8. The

fastest component of the field potential illustrated in Figure 8B was not consistently present

in other field potentials elicited by PLN stimulation. Central Iatencies for the field potentials

(component 2) recordeci in the PAG were measmd as the difference between PAG and the

cord dorsum potentials evoked by different peripheral nerves. The median and range of the

centrai latencies of the fint 4 components of the field potential figure 9) for each

peripheral newe stimulated are presented in Table SA The median latency for contralateral

PLN stimulation was similar to the ipsiiateral PLN stimulation (T'able 6A), but faster than

SPUD and SFP. The median central conduction velocity caldated in the field potential

studies was detemllned to be not significantly dinerent fiom the median conduction

velocity calculated in the spinal unit studies using the MaunWhitney Rank Sum Test

Assuming no signiricant contribution h m synaptic delays, all median conduction

velocities Lie in the range of A6 fibers. Although the maxUnum velocities correspond to

those of large diameter myelinated (A) fibers. We did not &termine whether the different

components were due to pathways with different conduction velocities or due to differently

routed pathways.

The PLNevoked field potentials diminished perceptibly with distance in the

dorsoventral 8B). anteroposterior (Figure 10A) and mediolateral (Figure 10B)

dimensions. The field potential recordings for SPUD and SFP display similar

characteristics to those for PLN stimulation. They were multiphasic (Figure 9) and the s k

Figure 8: PAG Field Potentials Evoked by PLN Stimulation. An example experiment in

which PLN stimdation-evoked potentials recorded (B) d o m a track in PAG at P 1 .O (A).

Each trace averages responses to 16 consecutive single stimuli. Arrows indicate diflferent

components of the multiphasic response. The maximum for the second component was

found in the dorsal PAG (as indicated by 'rnax'. in panel A). CAq, cerebral aqueduc~ SC,

superior co~cdus; FL, fasciculus longitudinalis medialis; LDT, laterodod tegmental

nucleus; W. nucleus raphe magnus.

Figure 9: Multiple Components in PAG Field Potential Recordings Representative PAG

field potential recordings foIlowing PLN, SPUD, and SFP stimulations demonstrating

rnultiphasic repenses. Arrows indicate different components present. The arrows are

numbered and these numbers correspond to the component numbers in table 4 (median CV

estimations).

Table SA: Median Latencies for Different Components of tk MuItiphaSc Eeld Potential

Respooses Penpherd nerve stimulation produces a multiphasc respome (differeflt

components with ciiffirent latencies) in the PAG. Latencies for the different components

correspond to the numbered m w s in figure 9.

L-PL median 7.8 msec

min. 2.3 msec

max. 9.5 msec

SPUD median 1 1 .O msec

min- 7.6 msec

max. 15.0 msec

SFP median 12.0 msec

min. 2.9 rnsec

max- 23.9 msec

Cpt 2

12.6 msec

7.4 msec

20.9 msec

19.5 msec

10.3 msec

33.3 msec

18.3 msec

10 msec

28.2 msec

Cpt 3

18.4 msec

9-6 msec

36.6 msec

22.9 msec

12.9 msec

42.9 msec

25.6 msec

17.3 msec

47.0 msec

Cpt 4

58.6 msec

42.1 msec

7 1 .O msec

80.8 msec

60.0 msec

123.9 msec

54.1 msec

33.1 msec

74.0 msec

Table SB: Median Conduction Veiocities for Different Compooents of the Muitiphasic field

Potentiai Responses. Penphed nerve stimulation produces a rnultiphasic response

(different components with different CVs) in the PAG. C V s for the different components

correspond to the numbered arrows in figure 9.

Cpt 1 Cpt 2 Cpt 3 Cpt 4

L-PL median 45.4 dsec 33.7 m/sec 24.1 misec 8.4 dsec

min. 37.3 mlsec 16.9 m/sec 9.7 m/sec 8.4 m/sec

max. 153.9 m k c 47.8 d s e c 36.9 d s e c 5.0 d s e ç

SPUD median 32.8 mkec 20.5 &sec 20.4 dsec 5.1 misec

min. 23.6 dsec 10.6 m/sec 8.3 dsec 2.9 m/sec

max. 46.6 m k c 34.4 m/sec 27.4 dsec 5.9 d s e c

SFP median 36.9 rn/sec 22.7 mfsec 17.9 dsec 8.3 dsec

min. 14.8 misec 12.6 &sec 7.5 dsec 4.8 m/sec

max. 122.1 m/sec 35.4 rn/sec 20.5 dsec 10.7 d s e c

Table 6A: Median Latacies, for the 2nd component of the field potentid response in the

PAG for ipsilateral and contralateral PLN stimulations.

Median Latency Range (msec)

(msec)

contralateral PLN 10.0

ipsilateral PLN 9.0

SPUD 19.5

SFP 18.3

Table 6B: Median Conduction Velocities, estimated for the 2nd component of the k l d

potential response in the PAG for ipsilateral and contratateral PLN stimulations.

Nerves Median CV (m/sec) Range (misec)

contralateral PLN 38.8

ipsilateral PLN 35.1

Figure 10: Size of PLN-EvoW Field Potentials Varies with PAG Recording Site.

Representative traces demomtrating that the field potential decreases as the PAG recording

electroàe is moved in the anterior-posterior (A) and medio-lateral (B) direction.

58 of the field potentials incffased as the recording electrode appmhed a population of

n e m m in the PAG responsîve to SPUD (Figure 1 1A) and SFP 11B) stimulation.

At the location in which the maximum &Id potential was evoked following the

stimulation of the L-PLN, SFP and SPUD stimulation did not evoke a maximum field

potential ( Figure 12A) (les than 25% of maximum). In this figure, at sites at which

maximum field potentials were recoakd for SPUD and SFP, L-PLN stimulation elicited a

submaxirnal response (50% of maximum) (Figure 12B).

In individual experiments, the PAG was searched over some AP distance. An

example of the results h m one experiment in which w k s were made at 3 AP levels are

illusaated in Figure 13. A maximum response was &tected in the domlateral region of the

PAG (APO) ipsilateral to the PLN stimulation site. Smaller responses were detected on the

contralateral side of the PAG.

Figure 14 illustrates the distribution of maximum PAG field potentials found in 13

experiments following PLN. SPUD, and SFP stimulation. Most of the maximum field

potentials elicited by PLN stimulation, were found at more caudal levels in the PAG

(-P1.0). Although most of these activated neurons are concentrated in the dorsal region.

there were also bladder responsive neurons in the ventrolateral region of the PAG.

Stimulation of SPUD and SFP nerves elicited field potentials within the vïcinity of

those evoked by PLN stimulation in the PAG but the maximal did not occur at the sites of

PLN maxima (Figures 11 and 12). The distribution of SFP and SPUD sensitive neurons in

the PAG ranged from P2.0 to A2.5 for SPUD and P2.O to A3 J for the SFP (Figure 14).

There is a cluster of points for SPUD sensitive neurons in the P0.5 - P1.0 level at an

intemediate region of the PAG. The distribution of newons responsive to each nerve

stimulation are not the same (Figure 14). This is compatible with a 'private' pathway for

bladder input.

A SPUD B SFP

Figure 1 1: Peripheral Nerve Stirnimulation Evoke SimiIar Field Potentiai Respoases.

Representative field potential recordings illustrating characteristics for SPUD- and SFP-

evoked responses. They are multiphasic and the response increased as the recording

electrode approached a population of neurons in the PAG respollsive to SPUD (A) and

SFP (B) stimulation.

L-PL

SFP 1

SPUD 1:

L-PL SFP SPUD

Figure 12: Different Areas of the PAG Are Responsive to Different Peripheral Nerve

Inputs- Representative traces of field potential recordings in caudal PAG Uustrating a site

which elicited a maxirnum for PLN stimulation not responding to SPUD or SFP

stimulation (A). At sites which maximum SPUD- and Sm-evoked field potentials were

de- L-PLN stimulation elicited a submaximal response (B).

Figure 13: Pattern of Field Potential Responses to R-PW Stimulation. Schematic drawing

of the distribution and tracks of R-PLN-evoked field potentials in the PAG (A1.0-APO)

greater than 50% of the maximal recording in experirnent 960730. Both sides of the PAG

were searcheci in 3 AP levels. The maximum field potential (large filleci chle) in the

dorsdateral PAG, is ipsilateral to the stimulation site. Responses 7699% (medium) and

5 1-758 (small) of the maxima surrounds the maxima and diminishes with distance-

Figure 14: Distribution of Maximum Field Potential Recordings in the PAG.

The distribution of field potential maximas differ for each nerve stimulation. The different

symbols distinguishes the different nerves while their location. on the right or left side of

the PAG, indicates contralateral or ipsilateral stimulation, respectively. Dots. PLN;

squares. SPUD; opencircle, Sm.

63 The question of ipsilated versus contraiateral transmission of bladdes impulses

was adâressed in this snidy. There may be a slight ipsilateral dominance in bladder

trammision (Figure 15A). Athough inputs h m SPUD appear to dernonstrate a

contralateral Pansmission (Figure 15B) while SFP inputs seem to demonstrate an ipsilateral

dominance (Figure 1 SC), there were only 3 cases in which both sides of the PAG searched

for SPUD and SFP. Therefore, these resuits are inconclusive.

Spinal Cord Units

The placement of the stimulating elecaode in PAG was detemineci by: (1) the size

of the bladder contraction response to stimulation of the PAG; andor (2) the size of the

field potential recordings in the PAG foliowing stimulation of the PLNs.

PAG stimulation in the region where bladder responses were produced also often

produced an increase in blood pressure of 25 to 75 rnrnHg. However, there were

experiments in which PAG stimulation produced a strong blood pressure response but not

bladder. The site of PAG stimulation producing a pressor response was usually very

distinctive as evidenœd by a gradualiy increasing response as the electrode was moved

d o m a track (Figure 16).

PAG stimulation also produced a rise in intravesical pressure which appeared to be

dependent on bladder volume. although this point was not systematically studied. The

biadder response ranged fkom an increase of 1.0 to 18 cm &O in 15 experiments. Figure

16 illustrates a representative experiment in which PAG stimulation produœd bladder

pressure responses which increased from 3 cm %O at 3.0 mm below the sUTfaçe of the

midbrain to 12 cm %O at 6.0 mm below the surface. It is not known if stimulation of more

v e n d areas would have elicited larger bladder contractions, since ttiese regions were not

searcheci. Bladder activity elicited by PAG stimulation was always small compared to those

elicited by stimulation of the PLNs at 10 Hz (Figure 16).

Figure 15: IpsÜateral versus ContraIateral Transmission. Histograms illustrating the

ipsilateral and contralateral transmissions of bladder information. PLN (n=13) (A) and SFP

(n=3) (C) information are conveyed in an ipsilateral manner while SPUD (n=3) (B) is

contralateral.

-3 -4 -5 -6 rev. -6

Figure 16: Effects of PAG Stimulation. PAG stimulation (A) at 50 Hz produced a large

bladder (B) and blood pressure (C) response, unWce L-PLN stimulation at 10 Hz which

produced a larger bladder but not blood pressure response. Bladder response to PAG

stimulation increased as the recording elecaode was moved in the dorsoventral direction

66 In two experiments, the PAG stimulating sites were detennined by

searching for the W e s t field potential responses in the PAG that codd be produœd with

PLN stimulation This response was con€iied by stimulating at this PAG site and

examining the size of the bladder response. It was found that at PAG sites which showed a

field potential in response to PLN stimulation, PAG stimulation produced blad&r

con tractions.

Spinal neurons antidromically activated from the PAG

In 13 cats, 17 uni& were found in the lumbosacrai spinal cord which were

considered to be antidromicaIly activated h m the PAG (TabIe 7) based on: (1) consistent

latency of response; (2) reponse foilowed a high fquency of PAG stimulation (333-

1000Hz). The distribution of these uni& is iUustrated in figure 17. They were located

conttalateral to the PAG stimulation site. Some appeared to be located in the white matter

(5/17) based on micrometer readings. There is a possibiüty that these five discharges are

due to axonal activation and not soma1 discharges since this was not tested at the time of the

recording. Antidromically activated neurons were primarily located in the dorsal hom with

the exception of two neurons in the v e n d hom and 3 in the intemediate area. Of the 17

neurow antidromically activated from the PAG in this study, 4 (23.5%) received inputs

horn the peripheral nerves we had prepared for stimulation (Figure 17). They were mainly

located in S2 and responded to SPUD only (lamina VIi), SPUD/SFP/CCF m i n a V) and

SFP/CCF (iamina V) (Figure 17). ûnly one (1.9%) neuron. found in lamina W of rosaal

S3. received an input from PLN (Figure 18). It is not hown if t h - neuron would have

been responsive to other periphed nerve stimulations since it was lost before they could be

tested.

In 3 of 4 cases confirmation that the peripheral input reached the it~ltidrornically-

activated neuron was obtained by using a collision test

Table 7: Counts of PAG Antidromically and Orthodromidy Activated Neurons in the

Sacral Cord The majority of the newons aictivated (antidrornicaily or orthodromically) by

the PAG did not respond to the test nerve stimulations.

# antidrornic % of totai # orthodromic % of total

cells #antidromic cells # orthodromie

ce1 1s cells

total # 17

PAG only 13

PAG & PLN 1

al1 nerves 3

no PLN

al1 nerves

(incLPLN)

Figure 17a: Disaibution of Lumbosacral Neurons Activated by PAG Stimulation PAG

stimulation at APO represented by a dot (A) antidromically (B) and synaptically (C)

activated neurons in S2 and S3 @) contralateral to the PAG stimulation sites. Stars indicate

PAG activated neuroas.

Figure 17b: Distribution of Lumbosanal Nemm Activated by PAG Stimulation PAG

stimulation sites (dots) at PL0 (A) antidromicaily activated neurons in S2 (B & C) and S3

(D) contralateral to the PAG stimulation sites. PAG stimulation also synaptically activated

neurons in L743 @-H). Asterisks indicate PAG activated neurons; stafi represent PAG

activated nemm receiving peripheral input.

Figure 18: Sacral Spinal Neuron Which Projects to the PAG Conveys PLN Input. Neuron

in S3 (star in F), was antidrornically activated by PAG stimulation at P1.0 (dot in E) (333

Hz), and it responded with a latency of 41 msec (C). Stimulation of the PAG (50 Hz)

produced bladder (A) and blood (B) pressure responses. L-PLN stimulation activated this

neuron at a latency of 12 msec @). Amws indicate start of stimulation for the PLN or

PAG. Calibration bar in @) refers to panels C and D.

Figure 19 Uustrates a representative experiment An S2 dorsal hom projecting neuron was

identified by antidromic activation at a constant latency of 5 msec h m the con-

caudal PAG (P1.0) (Figure 19F). The neuron had a latency of 5.5 msec (Figure 19D) and

followed a train of 3 stimuli in the PAG at 100 Ek @gure 19C). This neuron also

responded to SFP (Figure 19D), SPUD and CCF stimulation. Figure 19E demonstrates

collision of SFP stimulation evoked and antidromic PAG stimulation evoked discharges at

an interstimulus delay of 8 msec. Collision of SPUP and CCF- stimulation evoked

discharges with the PAG-stimulation evolrwi discharge was also confirmed at an

interstimulus interval of 8 msec. These d t s indicate that the spinal neuron projecting

directly to the PAG rewived afferent input fmm all uiree peripheral nerves. However, it did

not have an input fiom PLN.

The median latency for neurons that were antidromically activated by PAG

stimulation was 11.5 msec (n=17) with a range of 5-60 rnsec. Using a conduction distance

of 361 mm ( h m the cord dorsum electrode to the PAG electrode), the median conduction

velocity was estimateci to be 3 1.4 dsec. Only one conduction distance was measured in

these spinal unit searches but the average conduction distance for the field potential studies

was 355 mm (n=l 1). Using the latency from the projecting neuron which also responded

to L-PLN stimulation (iarnina VII), the conduction velocity was caîcuiated to be 8.8 &sec.

The conduction velocity for the neuron responding to only SPUD (lamina VII) was 12.4

m/sec. The antidmrnicaJly activated neurons receiving convergent peripheral input from

SPUDISFPKCF (lamina V) and SFEVCCF (lamllia VI) had conduction velocities of 72.2

m/sec and 40.1 mkc, respectively.

PAG stimulation also activated spinal neurons synaptically. These neurons were

found more frequently (1W177) than those which were antidromically activated (171177)

(Table 7). The characteristics of synaptic activation were: (a) variable latency for single unit

nriag, (b) burst firing, (c) failure to foilow a train of 4 stimuli at 333-1000 Hz

Figure 19: Spinal Neuron, Receiving SFP Input, Projects to PAG. A neuron in S2 (G) was

antidromically activated by PAG stimulation at P1.0 (F) with a latency of 5 msec @).

Stimulation of the PAG produced bladder (A) and blood (B) pressure responses. The

neuron also responded to SFP nerve stimulation with a latency of 3 msec. The unit

followed PAG stimulation at 100 Hz (C) and responded to both SFP and PAG stimulation

at an interstimulus interval of 10 mec @) but not 8 msec (E). Small arrows indicate SFP

stimulation; medium size arrows indicaie PAG stimulation. Large m w indicates where the

PAG stimulus-evoked discharge was expected to appear. Calibration bars in (E) refer to

panels C, D, and E.

73

An example of such a neuron is presented in Figure 20. In addition to the PAG. this neuron

was activated by stimulation of GPL but not &PL. SPUD and SFP inputs were not

exarnined Only 61160 (3.8%) of these cells received an input from PL, SFP and SPUD

(Table 7). Spinai neurons that were synaptically activated from the PAG were found mostly

in S2. distributeci from lamina V to the ventral horn and ventral white matter (Figure 17),

with a median latency of 14.0 msec (n=120) and a range of 6-34 msec. The conduction

velocity was estimated to be 25.8 mlsec.

In experiments in which orthodromically or aatidromically activateà neurons did not

respond to PLN inputs, there were indications that the PLNs were functioning. Indication

of functional PLNs include PLN responses in other backs. bladder contraction following

PLN stimulation, and the presence of PLN cord dorsum potentials or reflex responses.

Spinal neurons which were antidromically activated h m the PAG but did not respond to

P M . SPUD, SFP or CCF, could be receiving input from other pelvic viscera (bowel. sex

organs) which were not studied.

Convergence of merent input

During the search for spinal neurons antidromidy activated nom the PAG, 27

neurons were encountered that were activated by FLN stimulation but not by PAG

stimulation. M y one of these, found in the ventral hom of S2, was a f b t order

intemeuron receiving PLN input There were twice as many higher order (37) intemewons

as fiirst or&r (16) intemeurons. The criteria used to distïnguish between fmt and higher

order intemeurons was the latency of the response recorded in the spinal cord. If the

latency for the response was shorter than 3 msec, then the neuron was considered to be a

6rst order intemeuron. Some first order intememm for SPUDlSFPfCCF input also acted

a s higher order intemeurons for PLN input (i.e. a neuron responds faster for one input

74 compared to another). The distribution of the higher order and nrst order intemewons were

similar (Qwe 2 1).

Effets of PAG stimulation on spinal newon activity

Although it was not searched for coosistently, interactions between PLN and PAG

stimulations were observed in 5 spinal nemns. These interactions were observed to be

inhibitory (Kgure 2 2 4 and facilitatory (Figure 22.B). Co-stimulation of the PAG and G

PLN was required to elicit a consistent response h m a sacral spinal neuron (Figure 22A

and 22B). A late component of a response elicited by R-PLN stimulation was depresseci

with CO-stimulation of the PAG (Figure 22C and 22D).

Figure 20: PAG Orthodrornically Activateci Spinal Neuron

Neuron in S2 (dot in D) which was synaptidy activated by ipsilateral PAG stimulation

(iarge dot in C). Responses of the sacral spinal neuron to LPAG (A) and GPL (B)

stimulation are illustrated.

Figure 21: Distribution of Spinal Fust and Higher ûrder htemeurons in Sacral Spinal

Cord Panels show distribution in sacrai segment SI-S3 of fmt (x) and higher order (small

dots) interneurons responding to stimulation of left SPUD, SFP, andor CCF. Ceils which

are fmt order intemeuron for SPUD, SFP or CCF and receive PLN input are indicated by

squares. Those which receive higher order inputs including P M , are indicated by open

circles. One ceil was a finit order intemeuron for PLN and responded to ody PLN

stimulation (large dot).

Figure 22: PAG and PLN Interactions. PLN stimulation did not elicit a response (A) in an

S2 neuron (dot in E) but CO-stimulation of PL and PAG stimulation at APû (star in F) did

elicit a response (large arrow in B). In the same experiment, another S2 neuron (dot in G)

responded to PLN stimulation (C), but CO-stimulation with PAG (star) d t e d in inhibition

of a component of PLN response (D). Large m w Ui D indicates where the unit should

have appeared. Smaller m w s indicate the start of the stimulation.

DISCUSSION

The act of mictuntion is compriseci of a storage ( e g ) and an expulsion phase.

These phases require opposite functions nom the bladder and the u r e k The bladder acts

as a reservoir to store urine and also as a pump to expel it during voiciing. During urine

expulsion, the urethra opens, dilates and becornes cornpliant to allow urine flow. It is

closed and contracted during storage. The neural components controlling the complex

events of rnicnirition involve highly complex central and peripheral, afferent (sensory) and

effereat (motor) autonomie pathways which are integrated and coordinated by cephalic

control centers, spinal cord nuclei, and peripheml gangiia

M o u s mo&k of bladder control postulated that bladder information was camied

via the spinal cord to Barrington's nucleus in the dorsolateral pontine tegrnentum. This was

recognized as the center for bladder conml as lesions of this area abolished micturition.

Recently, it has k e n proposed that the periqueductal gray (PAG) region of the brainstem

may be involved in michirition Our hypothesis is that the PAG plays a role in the

mictwition reflex pathway.

The aims in this research project were: (1) to l o c h the bladder responsive celis in

the PAG and spinal cord; (2) to characterize this ascending pathway.

There were two approaches which were taken to locaiize, iden-, and characterize

bladder sensitive neurons projecting from the sacml spinal cord to the PAG. One approach

involved nemanatomical tracing studies while the other entailed neurophysiologicai

studies.

Retrograde eracing techniques were applied to establish a direct projection from the

sacral cord to the PAG. This part of the neuroanatornical study was abandoned upon the

79 publication of an article by Vmderhorst et al <19%) which provided retrograde and

anterograde evidence of direct pmjections fmm the sacral spinal cord to the PAG.

Anterograde tracïng and c-fos induction studies were used to reved the conveyance of

bladder-related information in this ascending pathway.

Electrophysiological techniques were used to test the hypothesis that there are

bladder sensitive cek in the PAG. The PAG was searched for field potentials evoked by

PLN stimulation. To M e r characteize the ascending projeçtion h m the sacral spinal

cord to the PAG, projecting neurons were identified by antidromic activation from the

PAG, and tested for PLN and other identified peripheral nerve inputs (SPUD. SFP, and

CCF) to detennine the specif~city of this pathway.

Neuroanatomical Experiments

The mode1 for the micturition reflex pathway is quickiy changing to include the

periaqueductai gray region Our interest in this pathway however. lies with the neurons

projecting from the spinal cord to the PAG. Neuroanatornical studies by Blok et al (1995)

have shown that thete are very few nemm in the spinal cord with direct projections to

Barrington's nucleus. Retrograde labelhg revealed direct spinal cord projections u, the

PAG (Vanderhorst et al, 1996). In th& shidy. injections into the intemediate area of the

PAG at PO.5, resdted in predominately condateral retrograde labelhg of neurons in

caudal S2. These resuits correspond with ou. retrograde fmdings. Of nine retrograde

experiments. only two using WGA-HRP resulted in labelhg in the sacral cord The other

WGA-HRP retrograde experiments used a longer sufvival time than 4 days and thus the

tracer could have been metaboliml prior to andysis.

80 In the present study, contralateral retrogradely labelleci ceh were rnainly observed

in the SZS3 segments of the spinal cor& These d t s are also compatible with our

bladder tracing data

The objective of our retrograde study was not localkation of an area of the PAG

which receives projection fiom the d The objective was to establish a direct projection

h m the spinal cord to the PAG, therefore the tracer volume injected hto the PAG was not

small or discrete. Now that it has k e n established that there are spinal projections to the

PAG, it would be more usehl to identify the specific region of tbe PAG that is receiving

these projections by making smaller injections. A possible concem with our retrograde

study is the degree of tracer diffusion at the site of injection. It should be noted that

although the most caudal area of the PAG shown in figure 2 was at the level of Pl 5, the

tracer could have ciifhiseci into more caudal areas. These caudal areas may consist of

structures which have ken shown to regdate micturition. For example the tracer codd

have reached Barrington's nucleus, the locus coedus, or parabrachial nucleus. Therefore,

it is possible that the retrogradely labelied n e m m in the spinal cord are projecting to these

sites and not the PAG. But as mentioned earlier, studies have already shown a paucity of

projections h m the lumbosacral cord to the Barrington's nucleus of the cat (Blok et al,

1995). With regards to the locus coeruius, it has been shown by Aston-Jones et al (1986)

that previous reports of dorsal horn projections to the locus coenrlus (Cedarbaum and

Aghajanian, 1978) were inaccurate and were instead found to projet to the Iaterally

adjacent parabrachial nucleus or to the pontine central gray media1 to locus coerulus. There

are contlicting resdts for studies examining the d e of the locus coeruius in micnuition.

Whüe Sasa and Yoshimura (1994) and Yoshimura et al (199ûa,b) have provided evidence

for locus coenrlus involvement in micturition, Satoh et a1 (1978) have show that bilateral

destruction of this area did not result in urinary disorders (Satoh et al, 1978).

81 Studies have shown spinai projections to the parabrachial nucleus (Blomqvist et ai,

1989). This nucleus is known to mceive information €rom the bladder and to project to

Barrington's nucleus (Valentino et ai, 1994). Furthemore, elecaical and chernical

stimulation of the parabrachial nucleus in the rat resulted in bladder contraction (Lumb and

Morrision, 1987). This evidence implies that the parabrachial nucleus may also have a d e

in the micturition pathway. However, its d e may be more reiated to viscerd nociception

since retrogradely labelled spinoparabrachial tract c e k expressed c-fos protein foliowing

formaiin irritation of the bladder @ing et ai, 1994). It shodd aiso be noted that although

Barrington's nucleus does receive projections from the parabrachial nucleus, it receives

more extensive projections h m the PAG (Wentino et al, 1994). niese findings indicate

that although the btainstern bladder control circuitry may consist of the ventrolateral PAG

and Bamington's nucleus, it may also involve other brainstem structures.

Vanderhorst et al (1996) made discrete tracer injections into various divisions of the

PAG, limiting the spread of the tracer into other regions of the brainstem which may be

involved in the micturition reflex pathway. Tbeir injection sites which did not include the

locus coeruius or the parabrachial nucleus, resdted in retrograde labelling in the spinal

cord.

Based on our findings, the spinal cord areas with neurons projecting to the PAG

consist of the lateral band, the sacral parasympathetic nucleus, and regions around the

central canal (including the dorsal commissure). These projections are bilateral but

predominately contralateral. In an antemgrade tracing study where HRP was administered

udaterally to the PLN, labeiied dorsal mot ganglion neurons were found only ipsilaterally

(Nadelhaft and Booth, 1984). Some of the bladder afferent inputs which are conveyed to

one side of the sacral spinal cord have access to a population of neurons projecting to both

sides of the PAG, but predominantly contralateral, Sacral preganglionic nemm are also

labelied ipdaterally h m the P W (Nadelhaft and Booth, 1984) but they have long axon

82 collaterals projecting to the opposite side of the cord (Morgan et al, 1991). nie latter

kding implies that the crosshg of bladder information occiirs at the spinal cord level in the

efferent pathway. However, the question of communication between opposite sides of the

cord and brainstem at the afferent level =mains unclear.

In the present saidy. retrogradely labeUed neurons pmjecting to the PAG were

found in lamina X (dorsal commissure). They were bipolar neurons widi dendrites

extending across the dorsal commissure. This indicates that the crossing of the micairition

pathway could occur at the level of the sacrai cord before the information is conveyed to the

PAG. By contrast, NadeIhaft and Vera (1995) ushg pseudorabies virus tracing, found

cells with dendrites extending into the dorsal commksure, but not crossing to the other side

of the spinal cord. Nevertheles, there is evidence for afferent fibers crossing in the sacral

spinal cord. Although Ueyama et al (1984) found that tenninals of SPUD afferents could

be found ipsilaterauy in lamina 1 of the sacral segment and graciüus nucleus, bilateral

labehg of terminais could &O be visrtali;rf!d in lamina lII and IV and in the dorsal

commissural grey matter. Matsushita and Tanami (1983) have also reported afferents

cmssing the midline in dorsal and central commissures of the sacral cord in the cat

In our anterograde study, afhent fiber terminai labelhg occurred ipsilateral to the

injection site in the bladder wd. This corresponds to other antemgrade studies in which

HRP was applied to PLNs (Nadelhafl and Booth, 1984; deGroat et al, 1981) and in

retrograde studies (Downie et al, 1984). Studies using pseudorabies virus also showcd

hbehg in ipsilateral intemediolaterd area before it is observed in other areas of the corci,

such as the dorsal gray commissure (NadeIliaft and Vera, 1995). With time pseudorabies

Wus travelled to the brainstem and eventually labelled the contralateral intermediolateral

regions of the spinal cord.

Retmgradely labeiled celis were observed in the ventral hom, along the lateral edge

of caudal S2 and S3 spinal cord sections. Inis area in the ventral horn corresponds to the

83 lateral band region of the sacral parasympathetic nucleus (de Groat, 1981 ; Morgan et al,

1991,1993). A similar clustering of neurons were c-fos immunoreactive in our bladder

distension study. Retrogradely IabeIled tells in this area were also observed at the S2 and

S3 level in the study by Vanderhorst et al (19%). They mentioned that these snall clusters

were fonned at distinct rostrocaudal intexvals of 220-300 um.

Although the ventdateral lamina W region does not appear to receive bladcier

afferent tenninaIS in our study, the neurons in this region were synaptically activated by

bladder distension (figure 5G). Furthermore, anatomical studies in which sacrai

preganglionic neurons were labelled by retrograde HRP traciag methods revealed that the

sacral parasympathetic nucleus CO& of three divisions: a dorsal band of neurons located

in lamina V and VI, a lateral band of nemm located in lamina W, and an interband region

which lies between the dorsal and lateral band regions (deGroat et ai. 198 1; Morgan et al.,

1979; Nadelhafi et ai., 1980,1986; Mawe et al, 1986). Electrophysiological and axonal

tracing studies demonstrateci that neurons in the dorsal band innemate the large intestine

while those in the lateral band innenate the bladder (deGmat and Ryall, 1969; Morgan et

al, 198 1). This implies that in our study, neurons in the ventdateral region of lamina W

which projects to the PAG may be involved in bladder-s-c activites. But in our

anterograde tracing study, bladder afferent tenninals were not labelled in this region

(figure2C). In contrast Nadehafi et al (1980) revealed antemgrade HRP labelled axons

extending ventrally into lateral lamina W. However, the absence of aEerent terminal

labelling in this region in our studies may be attributed to s m d tracer volume used to

achieve labelling in the more distal fields such as the lateral lamina W.

The interband region of the sacral parasympathetic nucleus consists of intemeurons

and spinal tract neurons projecting to at least L2 and Tl3 (deGroat and Ryall, 1969;

Morgan et al, 1981). These internewons are well-positioned to receive viscerd aî3erent

84 cohterals (figole 23) and in tum may be involveci in intersegmental and supraspinal

The specifïcity of bladder aerent terminal labelhg in anterograde studies would

depend on where dong the PLN the tracer was applied since the PLN separates distally into

distinct branches which can be traced to different effector organs (Langley and Anderson,

1895,1896). Since the tracer was injected into the bladder wall in our study, the tenniaals

that were labelled could be attributed to bladder afferents terminating in S2, near the dorsal

mot entry. dong the lateral coilateral pathway towards the sacral parasympathetic nucleus

and more medially. nie distribution of the labelled bladder afZerent tenninals corresponds

with the location of spinal tract neufons in the second sacrai segment of the spinal cord that

were labelled remgradely foilowing the unilateral application of HRP to cut axons in the

dorsdateral fimiculus at the fmt lumbar level (deGroat et al, 1981).

The pattern of afferent terminal labelling in the present snidy corresponds closely

with other anterograde studies (deGroat et ai, 1981) with the exception that in our study,

the medial coiiaterai pathway extendhg towards the dorsal commissure was not ~ ~ ~ ~ ~ e d .

This was unexpected since it has been dernonstrated that the projections to the medial

collateral pathway and dorsal commissure are rehtively weak for colon afferents not

bladder (deGroat, 1986).

Our anterograde tracing study also did not resdt in labelling of preganglionic

neurons and their processes in contrast to other studies in which HRP was applied to the

cur PLN (Morgan et al, 1981; Nadelhaft and Booth, 1984). It is possible that a longer

survival time is required to achieve bladder afferent labeiiing in the medial collateral

pathway and lateral band 1~egions. There may be a difference in transport times for different

fibers. Another expianation for these ciifferences in results is that an insufficient volume of

the tracer was injected to £dl the ce11 bodies and dendrites of preganglionic neurons, or the

fibers projecting to the medial collateral pathway and lateral band regions. In the case of

85 non-labeiled preganglionic neurons, it is also possible that the tracer was not picked up by

the efFerent fibers with tracer injection into the bladder wall.

Our studies show that the areas which receive bladder aEerent tenninals overhp

with the regions sending projections to the PAG. Therefore, bladder af fe~nts reaching the

sacral cord via the PLNs. tenninate near the dorsal root entry, dong the Iaieral coliateral

path and in the sacral parasympathetic nucleus and in the vicinity of neurons projecting to

the PAG.

c-Fos protein induction by bladder distension

Although the above hd ings provide M e r support for the involvement of the

PAG in the mictuntion refiex pathway in tenns of neuroanatomical connections, it does not

provide funct iod support for our hypothesis. Tracing studies do not provide information

about the specificity of the ascending pathway for bladder transmission.

The functional signifcance of the S e r m connectivity was studied using the

presence of c-fos protein as a macker for neuronal activity. The expression of the protein

product of this immediate eariy gene was inducible in urethane anestheshed rats by bladder

distension (Birder and deGroat, 1992). In the present study, we found that c-fos protein

expression was inducible in chlordose anesthesized cats by bladder distension. The S2

spinal cord segment contained the major@ of the n e m n s that were immunoreactive for c-

fos protein. The localization of c-fos protein in cells in S2 reflects the larger number of

urinary tract afferents projecting to this segment, as demonstrated by our antemgrade

tracing studies.

It should be noted that negative results with c-fos technique are mcult to interpret.

Increased c-fos expression implies that murons have received synaptic input. However, the

86 failure to detect c-fos expression does not necessarily mean that neurons have not been

synaptically activated. It is possible that some n e m m do not express c-fos uuder any

conditions, or they may produce leveis of c-fos protein below the threshold for àetection

with immunocyochemical techniques. There are even certain types of synaptic activation

which rnay in- c-fos expression. whereas other types rnay nor For example, Birder

and deGroat (1992) reported the failure of sphincter motoneurons to express c-fos in

response to a constant infusion of saline into the bladder. Synaptic activation of the

sphincter motoneurons was demonstrated by extenial u r e W sphincter contraction during

voiding. In accordance with the fïndings of Birder and m a t , sphincter motoneurons. in

our c-fos induction study, did not display imrnunoreactivity for the c-fos protein. Other

reports have also indicated that c-fos levels are below threshold for detection in

motoneurons (Jenkins and Hunt, 1991).

Following bladder distension. c-fos protein immunoreactivity was detected in five

major areas of the S2-S3 spinal segments: medial dorsal horn, lateral dorsal hom, dorsal

commissure, sacral parasympathetic nucleus, and lateral ventral hom zones which (with the

exception of lateral ventral hom) correlates with findings in the rat (Birder and ffiroat,

1992). These areas reœive PUD and PLN afferents innemathg the urethra (Thor et al,

1989) and bladder (Nadelhaft and Booth, 1984), respectively. Birder and deGroat (1992)

reporteci h m their selective denervation experiments that pudendal nerve afferents

conveying somatic information activated ceh prirnarily in the dorsal CO-ure and

medial dorsal horn zones. wheceas PLN afferents carrying information from the viscera

activated cells in the dorsal commissure and sacral parasympathetic nucleus regions.

Therefote they suggested that the dorsal commissure region may be important for

viscerosomatic interaction since it exhibited the highest induction of c-fos expression by

PLN and SPUD stimulation. The medial dorsal hom and sacral parasympathetic nucleus

regions exhibited greater specifïcity and rnay be important for processing somatic and

87 viscerd inputs, respectively. They also reported that the dorsal commissure region was

important for p m g nociceptive information. Therefore, in our study, bladder

distellsion r e s u l ~ g in an increased expression of c-fos in the dorsal commissure region of

the S2 and S3 segments could be attribut& to greater viscerosomatic interactions or painfid

stimulation of the bladder* Our hding of an increase in c-fos positive œlls in media1 dorsal

hom in the S1 and S2 segments indicates greater PUD proceshg by the neurons in this

area with bladder distension A greater viscerai proceshg occurred in the sacral

parasympathetic nucleus region of S2 with bladder distension, a s indicated by the increase

in neuronal activity (increase in c-Fos protein expression) in this area.

Although, there was an uicrease in c-fos expression in the medial dorsal horn,

lateral dorsal horn, sacral parasympathetic nucleus and dorsal commissure regions of S2

following bladder distension, the regions of &reatest induction was the dorsal commissure

followed by the sacral parasympathetic nudeus. This suggests that bladder distensions or

reflex bladder contractions preferentially activate ce& in the dorsal commissure and sacral

parasympathetic nucleus. These findings are in agreement with data h m Birder and

deGroat (1992).

Data from our studies demonstrate that the location of the c-fos positive neurons

overlaps the sacral cord region where WGA-HRP retrograde1y labelled neurons projecting

to the PAG were found This Unplies that these synaptically activated c-fos positive

neurons may be involved in an ascending projection systern to the PAG. This postulate is

supported by other preliminary reports (Birder et al, 1990) demonstrating that a percentage

of the c-fos positive ceh after bladder imitation were spinal tract neurons pmjecting to the

hypothalamus or to the region of the pontine micturition center and others were

pregangiionic neurons sending axons to the pelvic ganglia (deGroat et al, 1992). Ding et al

(1994) were able to induce c-fos-iike protein in neuronal ce11 bodies within the sacral

parasympathetic nucleus by chemicaiiy irritating the bladder* They found that more than

88 5W6 of these fos immunoreactive neurons were retrogradely iabeUed with fluoregold

injected into the parabrachial nucleus. This indicates that some non-preganglionic nemns

within the sacral parasympathetic nucleus may trammit noxious information h m the

bladder to the parabrachial nucleus.

The neuroanatomical hdings, provide presunptive evidence that the connections

h n the bladder to the sacral cord and h m the sacral cord to the PAG rnay be conveying

bladder afferent information to the PAG. Neurophysiological studies were undertaken to

provide M e r support for the functional significance of these connections and to

characterize this ascending pathway in terms of the specificity of the information it

conveys.

NeurophysioIogicaI Experiments

PAG extracellular field potentials

We were able to evoke field potentials in the PAG by stimulating the PLN. This

response suggests that there may be neurons in the PAG reœiving bladder information.

The distribution of maximum field potentials (figure 14) indicates that the region which is

most responsive to PLN stimulation is around P0.5 - P1.O. This is consistent with

neuroanatornical data showing that the PAG at the P1.0 level is the major target of

Iumbosacral afZerents (Blomqvist and Craig, 1991; Vanderhorst et al, 1996; Yezierski,

1988; Blok et al, 1995).

Our neurophysiological resuits revealed maximum field potentials for PLN

stimulation in two regions of the PAG, the dorsolaterai and ventrolateral regions. Within

the PAG, spinal projections terminate in lateral PAG at PO5 (Vanderhorst et al 1996) in

cats. Liu (1983) found that tracer injections into the ventdateral PAG of the rat resdted in

the most neuronal labelling at the lumbosaaal region, as compared to dorsolateral PAG

89 injection. Furthemore, Blomqvist and Craig (1991) found that injection înto the lumbar

cord resuited in terminal labehg in lateral PAG at P1.0 but labelling in two areas in caudal

PAG (P1.5-P2.0) in the cat: dorsolateral and ventrolateral PAG.

HEW trachg shidy in cats revealed projections to Barrington's nucleus fmm lateml

regions of the PAG (Blok et al, 1995). Cameron et al (1995), however, demonstrated

greater Phaseolus vulgaris-leucoagglutinin (PHA-L) labelling in Barrington's center of the

rat following tracer injection into dorsoiateral PAG than ventrolateral PAG.

Based on these controversial hdïngs, it is evident that the circuitry within the PAG

is very complex and unclear with respect to the micturition pathway. The different species

used in different laboratones may add to the complexity of the circuitry involved in the

mictwïtion pathway. Furthemore. not all of the projections have to be involved in the

mic turition pathway.

The neuroaatomical studies mentioned above do not provide fiinctional evidence

for the axonal projections h m the sacral cord to the PAG and from the different regions of

the PAG to Barrington's nucleus. Neurophysiological studies in rat have demonstrated an

optimum site in the dorsal PAG for recording short latency PLNevoked field potentials

(Noto et al. 1991). The same study also revealed an optimum stimulation site in the ventral

PAG to elicit PLN discharges. This suggests rhat there are two regions in the PAG

involved in transmitting bladder information. which corresponds with our field potential

data (figure 14). The role of the nemns located in the dorsolateral PAG io the micturition

pathway is unclear. But it is known that the ventrolateral PAG is important in supporthg

reflex rnictuition since preliminary studies (S. Matsuura, G.V. Allen, J. W. Downie

unpublished) show that cobalt blockade of synaptic transmission at sites in the ventrolateral

PAG interrupts micturition in the urethane anesthetized rat

In a study on spinal neurons projecting to unloiown supraspinal targets, ascending

axons responsive to PLN stimulation also responded to pudendal and hypogastric nerve

91 However, the PAG covers a fairly large area, so it is possible that lesioned areas did not

cover the location of the newons involved in micturition

In rhis study, stimulation of more caudal ~egions of the PAG not ody elicited

bladder contraction. but also antidrornically activated 17 sacral spinal cord nemns. This

may seem to be a low yield but one explanation for the low yield in antidromically activated

neurons is that we were not stimulating in the right area of the PAG rezeiving bladder

transmission from the s d cord. The PAG sites which when stimulated elicit a large

bladder response may not correspond to the site to which spinal projections tenninate. Even

the location of the maximum PW-evoked field potentials in the PAG may not correspond

to the termination site of the ascending pathway in the PAG if the projection teminates onto

a mail nucleus or if the termiaals of the pmjecting n e m m spread diffusely in the PAG.

Since retrograde studies have s h o w sacral neurons pmjecting to lateral PAG in cats

(Vanderhorst et al, 1996), stimulation at this site may result in more identiocation of PAG

antidrornicaliy activated neurons conveying PLN input

It is possible that the activity of the projecting neurons are suppressed by the

anesthesia used during our search for single in the sacral spinal cord. However, on-

going studies in decerebrate cats (J.W. Downie and HJ. Du, unpublished) have not

produced a higher yield in antidromicaliy activated neurons respoasive to PLN stunulation,

Although a yield of 17 antidrornically activated newns in the sacral cord seems

low, it corresponds with our fiidings in retrograde tracing experiments and tracing and

neurophysiological results from other laboratories. Vanderhorst et al (1996) only found an

absolute count of 115 retrogradely labelleci neurons in S2 fmm an injection of WGA-HRP

into the ventdateral PAG (P1.0). In our retrograde study, we estimated a minumum of 48

and a maximum of 293 labelleci ce& in S2. Bearing in mind that only a srnail area of the

PAG was stimuiated in each experiment in our spinal cord unit studies, the yield of 17

antidrornically activated neurons sounds reasonable. FiIrthemore, Yezierski and Schwarz

92 (1986) only found 13 antidrornicaIly activated neurons in L7-S 1 foilowing stimulation of

13 sites within the caudal PAG using an array of 2- 4 stimulating electrodes.

'Ik median conduction velocity for the pmjecting neurons found in this study (3 1.4

mfsec) was similar to the conduction velocity estimated for spinomesencephalic rract œlls in

the lumbosacral cord antidromically activated h m the PAG (Pl 5 ) in lamina 1, V-VIII

(34.9 f 2 1.1 dsec) (Yezierski and Schwartz, 1986). Yezierski and Schwartz (1986)

mentioned that variations in estimated conduction velocities may be amibuted to cells

projecting to different midbrain levels; cells in different hinctional classes; ceils in different

spinai hmhae. Spinomesencephalic projecting cells originating in the superficial spinal

Iaminae (1 and II) have been reported to have slow conduction velocities (mean: 14.1 f 5.7)

while cek in lamina III-N and VII-VIII conduct faster (mean: 56.3 f 20.8 mlsec). The

range they reported for cells found throughout lamina 1-MII that were activated by

stimulation of the PAG at AKI was 8.8-102 mkc. In our study, the only antidromically

activated œll that received PLN input had a conduction velocity of 8.8 dsec, which would

f a within the large range reported by Yezierski and Schwarz (1986), but this neuron was

Iocated in lamina VlL

The estimated median conduction velocity for the ascending axons to the PAG

conveying bladder information in our spinal cord antidromic unit study (3 1.4 m/sec) was

not signincantiy different fiom the one estimated in the field potential recordings (33.7

m/sec). The latency for the second component of the multiphasic field potential response

was used to calculate these conduction velocities. This latency was used because it was the

fastest component which was consistently present

According to our centrai conduction veIocity estimations. the most common fibers

used by these projectïng neurons to convey bladder information to the PAG are Adelta

fibers. McMahon and M o d n (1982a) estirnated that the conduction velocity for fibers

93 conveying PL input between U and the brainstem to be about 30 dsec, which

corresponded with the conduction velocities detedeci for other autonomie spino-bulbar

pathways (Coote Bi Dowmnan. 1966.20-30 dsec for car& and rend nerves). Their

calcdations supports the median conduction velocity estirnated in die present study.

DeGroat (1975) reported a conduction velocity of 1û-11 dsec for the ascending fibers to

Barrington's nucleus (using a latency of 3 0 msec from PLN e v o M field potentials in

the rostral pontine areas (Bmhgton's nucleus) and a conduction distance of 400 mm). The

latency of PLN-evoked potentials recorded in Barrington's nucleus @eGn,at, 1975; Lalley

et al. 1972) is longer than the latency recorded in the PAG in our shidy. A finding

compatible with the afferent infornation king relayed through the PAG before it miches

Barrington's nucleus.

We sought to determine whether the population of sacral spinal ceUs projecting to

the PAG contained a subgroup that responded exclusively to PLN input It was reasoned

that such a population would be most k l y to represent the afferent limb of the micturition

reflex mode1 proposed by Blok and Holstege (1994). However, it was recopized that an

earlier study failed to find ascending nemm with purely PLN input (McMahon and

Morrison, 1982a). This led to a hypothesis in which the specificity in the micturition reflex

was detennined by PL afferent gating of the spinal cord output rather than by bansmission

of bladder-specinc afferent idormation to the brainstem (McMahon and Monison. 1982a).

One dficulty with the latter study is that the supraspinal target of the asçending neurons

was not defined.

In the present study, information from three peripheral nerves converged onto a

sacral spinal neuron located in the dorsal hom and traveled in the same axon to a

ventrolateral region of the PAG (P 1 .O) (figure 19). Although it is not known how eady

dong the transmission path the convergence of input occurs. the central latency of the

mponse to the peripheral nerve stimulation indicates that chis projecting neuron was not a

94 first order internewon for any of the three nerve inputs. Different peripheral neme inputs

can travel and terminate ont0 specific neurons in the sacrai cord where they may interact

with other intemettrons in the cord before the information is conveyed to the PAG.

Neurons appear to act as fïrst order interneucon for one input but serve as a higher order

internemon for another type of input (figue 22). Of the 4 antidromidy activated sacral

neurons, only one was a k s t order intemeuron (for SFP input). This neuron &O served as

a higher order internewon for SPUD and CCF mmmision.

Although our data demonstrate a convergence of different peripheral nerve inputs

ont0 spinal-PAG projecting ceUs, the inputs that converged onto these projecting nemm

did not include the PLN. nierefore these d t s do not negate the possibility of a pnvate

pathway for bladder input to the PAG. It does, however, provide evidence for a non-

specific pathway for SPüD, SFP and CCF input to the PAG. The fact that there was an

antidromically activated neuron that responded only to SPUD input suggests that there may

be a sWc and non-smc pathway for the aansmission of SPUD inputs to the PAG.

These data would fit with the data h m the field potential study in which maximum FPS

were found for SPUD in a wide range of AP levels (A2.5-P2.O). and h r e was a cluster of

maximas in the lateral (intemediate) region of the PAG at P0.5-P1.0.

The pudendal nerve conveys afferent information from other structures besides the

urethra. It conveys information £kom somatic structures such as the skin of penis, clitoris

and perineum (Vanderhorst et al, 1996). striated muscles of the pelvic floor (Sato et al,

197 8) and the urerhral and anal mucosa (Kwihara et al, 1980). Input fmm visceral

structures such as the vagina, and part of the uterine cervix are a h conveyed by these

nerves (Vanderhorst et al. 1996). It is possible that different types of input conveyed in the

PUDN separate at the levei of the spinal cord and travel in different paths to enninate at

diffexent regions of the PAG responsible for diffe~ent functions. while other PUD inputs

converge with other peripherai nerve inputs ont0 spinal projecting neurons.

95 The PAG has already been shown to be involved in a variety of functions such as

lordosis (Sakuma and Pfaff, 1979). cardiovascular regdation (Lovick, 1993). vocalization

(Zhang et al, 1994) and pain modulation (Bandlet et al, 1991; Carrive and Bander, 1991).

Vanderhorst et al (1996) observed that in cats, neurons remgradely filied by KRP injection

to the lateral PAG were located in regions that overlapped with pelvic and pudendal afferent

terminais in the sacral cord. Also in cats, Blok and Holstege (1994) demonstrateci that the

lateral part of the PAG containecf neufons projecting to Barrington's nucleus. 'Ibis is the

region that, in our work, contains a clustering of maximum field potentials for SPUD

stimulation (figure 14). Therefore, neurons in the lateral PAG which receive projections

h m the sacral cord (in an area of PUD afferent tenninations) could convey PUD

information to Bamington's nucIew, more specifically the L-region, where neurons

projecting to Onuf 's nucleus are located (Holstege et al, 1986). Puciendal aEerent

information related to rnicturition may be conveyed in a private pathway to the lateral region

of the PAG. The other inputs carried by the pudendal nerve rnay terminate in other regions

of the PAG related to lordosis, or pain modulation. This is supported by our fmding that

only 4 of the l? ceus we found projecting directly to the PAG received inputs fiom our test

nerves which implies that the major part of this pathway is not directly related a lower

urinary tract function.

The fmding that only 1 out of 4 nemns which was antidrornicdy activated from

the PAG was found to receive a PLN input suggests that there are very few direct

projections to the PAG conveying bladder-specific information. Even so, this is also

evidence for ci-t projections to the PAG h m the sacral cord conveying PLN input. It

was unfortunate that this antidromicaily activated neuron which received PLN inputs

(figure 18F) was lost before other peripheral nerve inputs could be tested to answer the

question of selectivity in the ascending pathway for michnition.

96 Although the yield of antidromically activated sacrai spinal cord neufons was low.

there were numerous neurons in which were synapticaily activated nom the PAG. These

nemns which were orthodromicaily activated wese located in the ventral horn and white

matter of the sacrai spinai cord, contralateral to the site of stimulation in the lateral and

ventrolateral PAG. Even though this could represent a direct PAG to spinal cord pathway

(Mouton and Holstege, 1994). previous work suggest that PAG stimulation could also

cause activation of other descending pathways fkom, for example, raphe nuclei (Chandler et

al. 1994) or ventral medulla (WiIlis, 1988) (generally inhibitory) or even from Barrington's

nucleus (Wrely to be excitatory).

Yezierski and Schwan (1986) found that PAG stimulation inhibited not only

background activity or responses evoked by low or high intensity cutaneous stimuli , but

also responses to the stimulation of deep smicnires such as joint andlor muscle. Because

the PAG regions receiving sacral spinal cord input can exert inhibitory or facilitatory effecu

on s d neurons receiving PLN inputs (figure 22). this projection may constitute an

important Serent limb of a negative or positive feedback loop involved in the control of

spinal neurons involveci in michintion.

The regions of the PAG receiving input fiam the sacral cord have been shown to be

involved in the descending control of spinal neurons (Liebeskind et al, 1W3; Oliveras et al,

1976). actions of morphine (Yaksh et al, 1976). sexual (Hansen and Gummesson, 1982)

and locomotor behaviours (Skulrety. 1963). This area is also h o w n to influence or elicit

motional responses such as fear and anxiey, defensive behaviours and vocalization

(Behebani, 1995). Spinal-PAG projecting cells provide aEerent inputs to an area important

in the motivational e t i v e dimensions of pain. The perception of pain is closely

associated with bladder dysfunction. In the absence of micturition, nIling of the bladder

produces a feeling of urgency and severe discornfort, leading to pain. Therefore. pain input

97 (due to bladder overdistension) could be mediated by afferent fifibers in the PLN. along with

other inputs such as bladder fùllness. and conscious desire to void

Conclusion

To conclude and speculate on the significance of our fïndings, inputs related to

bladder fimction enter the sacral spinal cord at S2. to terminate around the dorsal mot entry.

Lissauer's tract, and along the lateral coilateral pathway. The information is then conveyed

to spinal interneurons located mainiy in the sacral parasympathetic nucleus. Although the

micturition pathway has been shown to be polysynaptic. the &gree of synapsing that

occurs in the cord (or in the brainstem) is not known. What is known is that eventually.

bladder-related information is conveyed by spinal projecting neurons (lamina V-VU) to the

ventdateral PAG (P1.0). This conclusion is supported by our fudings of retrugradely

labelleci neurons from WGA-KRP injections into the PAG within the vicinity of c-fos

induced expression by bladder distension. PLN stimulation activating neumns in the

dorsolateral and ventrolateral PAG and PAG stimulation resulting in an increase Ui bladder

pressure imply a role for the PAG in bladder functions. The latency for these evoked

responses in the PAG was shorter than those reported in Barrington's nucleus which is

consistent with bladder inputs king conveyed to the PAG before they are transmitted to

Banington's nucleus. These fhdings support our hypothesis that the PAG is involved in

the basic micturition reflex. But the question of selectivîty in input transmitted in the

ascending limb of the michintion pathway remains imsolved.

Significance

The proceshg of bladder afferent activity for micturition at the level of the spinal cord and

brainstem is not weU understood. However. this is important in the developement of our

understanding of the organization of this physiological process. This pmject should

98 provide a stmt in the unravehg of the various roles of ascending bIad&r affixent

infoxmation and addressing the question of convergence and redundancy in these

pathways. The devance of this infornation cm be seen in sensory dysfunction and spinal

cord injury where blad&r control is disturbed at the se11sory level. An understanding of the

bladder afferent pathway may aid in the development of pharmacologial interventions to

manage bladder dysfunction.

Appendix A

DAB (brown) immunohistochemistry protocol for Fos protein and *biotin

dextran detection.

1. Place h e d spinal cord specimens in sumse bufXer (20% in O. 1 phospate buffer)

ovemight

2. Section using k z i n g mirotorne and collect sections in 0.05 M phosphate buffesahe

(PBS).

3. Wash 3 times in PB S (1 minute per wash).

4. Make 2% rabbit senun (dilute 200 pL aliquot of rabbit senun into 10 mL of 1% Triton-

X in PBS).

5. Make blocking senun (dilute 200 pL aliquot of rabbit senun into 4.5 mL of 2 4 rabbit

se- and add 100 mg bovine serum albumin).

6. Pellet sections and incubate for 90 minutes in 1 mL of blocking serum.

7. Wash 3 t h e s in PBS.

8. Pellet sections and add 1 mL of 2% rabbit senun plus primary antibody 1: 1000.

Incubate sections overnight at room temperature on shaker.

9. Wash 3 times in PBS and peliet

10. Incubate for 1-2 hours in 1 mL of BioRas 1: 1000 (Biotuiylated Rabbit Anti-Sheep to

2% rabbit senun),

11. Make up ABC solution 30 minutes pnor to use (see notes below).

12. Wash sections 3 times and pellet

13. Incubate for 1-2 hours in 1 mL of ABCelite 1 : l W (Avidin-Biotin-peroxidase

Corn piex).

14. Wash sections 3 times in PBS and pellet

15. Weigh out a srnail amount of DAB (diaminobenzidine). Wear gloves.

16, Dissolve the DAB in a smaU amount of distilled water.

17. Add appropriate volume of 0.1 M phosphate buffer such that the DAB is at a

concentration of O 5 mg/mL.

18. Make up fie& hydrogen peroxide (dilute 1 mL of 30% hydrogen peroxide into 100 mL

of distilled water).

19. Rem sections in DAB until the sections start to turn brown (approximately 20 minutes)

by adding 5 mL of 0.5 mg/mL of DAB solution and 150 of diluted hydrogen

peroxide to the sections.

20. Pour the DAB solution into the waste DAB bottle and wash the sections 3 tirnes in

PBS.

2 1. Store sections in the fridge.

* for biotin dextran, the proceshg of sections start at step #Il.

Appendix B

Nickel enhanced DAB (black) immunohistocbemistry Protocol.

Repeat steps 1-16 in Appendbc A for standard DAB (brown) immunohistochemistry

except al l stages must be done in TBS not PBS or PB.

Make up 0.6% ammonium-nickel sulfate solution (0.6 g (NH4)2Ni(Sû4)2 in 100 mL

of TBS. pH 8.0).

Add 0.6% ammonium-nickel solution to DAB such that DABM is at a concentration of

0.16 mg/mL.

Make up nesh düute hyârogen peroxide (dilute 1 mL of 3û% hydrogen peroxide into

100 mL of distilled water).

React sections in DABm solution untü they start to tum gray (approximately 3

minutes) by adduig 5 mL of the 0.16 mg/mL D M i solution and 80 pL of the

hydrogen peroxide to the sections.

Pour the DABINi solution into the waste DAB bottle and wash sections 3 times in TBS.

Store sections in the fndge.

Appendu C

Histochemi*stry for the deteetion of WGA-HRP or HRP.

Make Solution A: dissolve 200 mg sodium nitrofemcyaaide into 185 mL of distilled

water and then add 10 mL of acetate buf5er (pH 3.3). StV for approximateIy 5 minutes.

Make Solution B: dissolve 15 mg 33 '5 5'-tetramethyl bemidine (TMB) into 7.5 mL

of absolute ethanol by gentiy heating (37-40 OC).

Mùr solutions A and B (Solution C) for approxirnately 15-20 seconds and add to

sections.

Incubate for 20 minutes in the da*

Repare fresh 0.3% hydrogen peroxide and add 2 mL of 0.3% hydmgen peroxide per

100 mL of Solution C to the sections.

Incubate for 20 minutes in the da&

Wash sections 3 times with post reaction solution (dilute 50 mL of acetate bdfer, pH

3.3. in 950 mL distilled water) and store sections in this solution in the fndge.

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CHARACTERIZATION OF THE ASCENDING MICTURITION …1986). the organizing center for the coordination of bladder function The basic micturition reflex pathway was thought to involve the