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INTRINSIC AND BXTRINSIC NEURAL PATHWAYS TO THE

LARGE INTBSTINE OF THE CAT

BY

Johnson W. McRorie, Jr.

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Neuroscience Program and Department of Physiology
Michigan State University
1992

ABSTRACT

INTRINSIC AND EXTRINSIC NEURAL PATHWAYS TO THE

LARGE INTESTINE OF THE CAT

BY

Johnson W. McRorie, Jr.

Extrinsic Afferent Pathways. The distribution of visceral
afferent fibers from sacral dorsal root ganglia to the
pelvic nerve, pudendal nerve, large intestine, urinary
bladder and urethra was studied in the cat. Also, the
relation of these fibers to neurons in parasympathetic
colonic ganglia, pelvic plexus ganglia, urinary bladder
ganglia and the colon-rectal myenteric plexus was deter-
mined. Wheatgerm agglutinin-horseradish peroxidase (WGA-
HRP) and unconjugated wheat germ agglutinin (WGA) were
injected into sacral dorsal root ganglia (81-83) bilateral-
ly. Antibodies against neurofilament were used to label
neuronal somas and processes within the myenteric plexus,
pelvic plexus, urinary bladder and parasympathetic colonic
ganglia. Anterogradely transported WGA-HRP and WGA were
detected in peripheral afferent projections in branches of
the pelvic and pudendal nerves.

Axial orad and aborad sacral visceral afferent projec-

tions to the colon and rectum-anal canal involved ascending
and descending afferent fibers, respectively, within colo-
nic and rectal fiber bundles, branch points of fiber bun-
dles and interganglionic fiber tracts within the myenteric
plexus. Dense arborization patterns of afferent fibers, some
with varicosities, were detected in colon and rectal myen-
teric plexus ganglia. Afferent fibers were detected in
approximately 9% of proximal, 16% of mid and 20% of distal
colon myenteric plexus ganglia. Afferent fibers were traced
from the myenteric plexus to the circular muscle layer of
the colon. They were distributed around the circumference of
the colon, parallel to the long axis of circular muscle
fibers. Diffuse fibers, small bundles and dense arborization
patterns of afferent fibers were detected on circular
muscle fibers. No sacral visceral afferent fibers were
detected in the longitudinal muscle layer. In submucosa,
sacral visceral afferent fibers were sparse, appearing as
interrupted short fragments within interganglionic fiber
tracts. No afferent fibers were detected in submucosal
plexus ganglia.

For urinary bladder, afferent fibers were detected as
sparse fragments in serosal bundles and extrinsic muscle
layers of all bladder regions. Afferent fibers were visual-
ized in the submucosa of the neck region only. No afferent
fibers were detected in mucosa.

When sacral dorsal root ganglia were injected bilater-

ally, afferent fibers were detected in approximately 64 % of

proximal, 68 % of mid and 77 % of distal urethral sections.
They were detected in serosal bundles, longitudinal and
circular smooth muscle layers, submucosa, mucosa and striat-
ed muscle of distal urethra and external urethral sphincter.

When sacral dorsal root ganglia were injected ipsilat-
erally and the pelvic nerve was sectioned ipsilaterally,
pudendal nerve afferent fibers were detected predominantly
in distal and mid urethra and external urethral sphincter.
When sacral dorsal root ganglia were injected ipsilaterally
and the pudendal nerve was sectioned ipsilaterally, pelvic
nerve afferent fibers were detected in proximal and mid
urethra and the submucosal region of distal urethra. Fibers
were not detected on striated muscle fibers of distal ureth-
ra or external urethral sphincter.

Sacral afferent fibers were detected in 18 of 18 para-
sympathetic colonic ganglia, 60 of 61 pelvic plexus ganglia
and 37 of 42 urinary bladder ganglia. They were detected in
fiber bundles on the border and center of the ganglia. 100
percent of colonic and pelvic plexus ganglia and 97 percent
of urinary bladder ganglia had fibers in proximity to gan-

glion cell bodies.

In summary, these data show that sacral visceral affer-
ent fibers project axially both orad and aborad over rela-
tively long distances through colonic and rectal fiber
bundles, respectively, where they provide innervation to
some colonic and rectal effector structures. Sacral viscer-

al afferent fibers also project through pelvic and pudendal

nerves to innervate the urinary bladder, urethra and exter-
nal urethral sphincter. Afferent fibers occurr in proximity
to neurons in some myenteric plexus ganglia and nearly all
sacral parasympathetic colonic ganglia, pelvic plexus gan-

glia and urinary bladder ganglia.

Intrinsic Neural Pathways. The morphology and projections
of myenteric plexus neurons through colonic fiber bundles in
cat colon were determined using in-situ retrograde transport
of HRP and fast blue. Myenteric neurons were found to
project from at least 5 to 59 mm orad (mean: 42 mm) or
aborad (mean: 54 mm) through colonic fiber bundles. Approx-
imately 73% of labeled cells were in ganglia within 2.8 mm
of colonic fiber bundles in the axis of circular muscle
fibers: none was beyond 7.7 mm. There were 2 soma morpholo-
gies. One type (Dogiel type I) had a mean soma diameter of
40.5 um and had a rough somal surface. There were few if
any short, broad dendrites, but its one long process extend—
ed to a branch point of an adjacent colonic fiber bundle.
The other type (Dogiel type III) had a mean soma diameter of
26.4 pm, a smooth somal surface and few if any fine den-
drites. It also projected a single long axon to colonic
fiber bundles. There were twice as many Dogiel type III
neurons.

In summary, these data show that myenteric neurons in
the cat colon project both orad and aborad over relatively

long distances through colonic fiber bundles where they form

another intrinsic neuronal pathway for the myenteric plexus.

Extrinsic Parasympathetic and Sympathetic afferent Pathways.
The distribution of neurons in parasympathetic prevertebral
and sympathetic prevertebral and paravertebral ganglia that
project processes to at least mid-colon through colonic
fiber bundles was determined using retrograde transport of
fast blue.

For parasympathetic prevertebral ganglia, a range of 44
to 477 neurons were labeled per animal (mean i SE, 233.1 i
49.0). This anatomical data supports previous electophysio-
logical studies (Krier and Hartman, 1984). Together the data
suggest that neurons in parasympathetic colonic ganglia
provide synaptic input to myenteric and/or submucosal
plexus neurons and/or directly innervate colonic effector
structures.

For sympathetic prevertebral ganglia, the mean number
of neurons (: SE) labeled per animal were: inferior mesen-
teric ganglion (IMG), 2,755 i 660; superior mesenteric
ganglion (SMG), 356 i 131; and coeliac ganglion, 1,415 i
874. For lumbar paravertebral chain ganglia, the mean number
of neurons per experiment was 780 i 314. These data show
that a subpopulation of neurons in sympathatic prevetebral
and paravertebral ganglia project long postganglionic pro-
cesses through colonic fiber bundles to at least the mid-
colon region. The functional significance of this anatomi-

cal arrangement is unknown. It is likely that these neurons

are noradrenergic and innervate blood vessels, myenteric
plexus ganglia and intestinal smooth muscle (Furness and

Costa, 1974).

ACKNOWLEDGEMENTS

I would like to thank Dr. Jacob Krier for his guidance,
support and the opportunity to work in his laboratory. I
would also like to thank the members of my graduate commit—
tee: Dr. Thomas Adams, Dr. James Galligan, Dr. Seth Hootman,
Dr. Ralph Fax and Dr. Cheryl Sisk for their guidance and
help throughout my graduate program. I would like to thank
the neuroscience program for its support of my research and
travel.

I would like to thank Jane Walsh for her expert ad-
vice, technical assistance, hard work and friendship.

Finally I would like to thank my wife, Susan, who kept
life in proper perspective throughout my graduate program
and continues to be an endless source of inspiration and

love.

viii

TABLE OF CONTENTS

Page
INTRODUCTION ........................................... 1
HISTORY ................................................ 3
Sacral Afferent Pathways .......................... 3
Postganglionic Efferent Pathways ................. 14
Intrinsic Neural Pathways ........................ 19
Colonic and Rectal Fiber Bundles ................. 25
Specific Aims .................................... 29
METHODS.......... ........... ....... ................ ... 30
CHAPTER 1: Sacral and Lumbar Afferent Pathways ........ 41

A. Density and Distribution of Sacral Dorsal
Root Ganglia Neurons that Project Peripheral
Processes to Colonic Fiber Bundles

B. Distribution of Sacral Visceral Afferent Fibers in
Parasympathetic Colonic Ganglia, Pelvic Plexus
Ganglia, Colonic Fiber Bundles, Myenteric Plexus
and Extrinsic Muscle layers of Large Intestine

C. Distribution of Sacral Visceral Afferent Fibers to
Urinary Bladder Ganglia, Urinary Bladder, Urethra
and External Urethral Sphincter

CHAPTER 2: Intrinsic Neural Pathways .................. 116

CHAPTER 3: Prevertebral and Paravertebral Pathways to Colo-

nic Fibers Bundles.. ....................... .141
SUMMARY ........... ..... ................................ 159
LITERATURE CITED ....................................... 161

ix

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Distribution of Neurons in Sacral Dorsal Root
Ganglia

Profiles of Ganglia Containing Afferent Fibers in
the Myenteric Plexus

Distribution of Sacral Afferent Fibers to Urinary
Bladder

Distribution of Sacral Afferent Fibers in Cat
Urethra

Distribution of Sacral Afferent Fibers in Tissue
Layers of Cat Urethra

Distribution of Neurons in Parasympathetic Colonic
Ganglia

Distribution of Neurons in Sympathetic Preverte-
bral Ganglia

Distribution of Neurons in Sympathetic Lumbar
Paravertebral Ganglia

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

8:

LIST OF FIGURES

Drawing of sacral spinal cord with sacral dorsal
root ganglia, pelvic nerve and colon

Drawing of colon wall with attached pelvic nerves
and colonic fiber bundles - arrows at pelvic brim
and IMA

Drawing of colon wall with attached pelvic nerves
and colonic fiber bundles - arrows at IMA

Drawing of sacral spinal cord with sacral dorsal
root ganglia, urinary bladder and urethra

Fluorescent photomicrograph of sacral dorsal root
ganglion section retrogradely labeled with fast
blue

Distribution of neurons in sacral dorsal root
ganglia that project peripheral processes to
colonic fiber bundles

Brightfield photomicrograph of sacral spinal cord
section with anterogradely labeled afferent
fibers

Fluorescent photomicrographs of sacral afferent
fibers in pelvic nerve and colonic ganglia

9: Fluorescent photomicrographs of sacral afferent

10:

12:

13:

14:

fibers in pelvic plexus ganglia

Drawing and photomicrographs of colonic fiber
bundle with anterogradely labeled sacral afferent
fibers

Distribution of sacral afferent fibers in colonic
fiber bundles

Fluorescent photomicrographs of rectal fiber
bundles

Camera lucida drawings of sacral afferent fibers
in colonic fiber bundle with attached myenteric

plexus ganglia

Camera lucida drawing of sacral afferent fibers

in myenteric interganglionic fiber tract

xi

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

15:

16:

17:

18:

19:

20:

21:

22:

23:

24:

25:

26:

27:

28:

Camera lucida drawing of varicose sacral afferent
fibers myenteric plexus ganglion

Camera lucida drawing of sacral afferent fibers
in myenteric plexus ganglia of colon and rectum

Fluorescent photomicrographs of sacral afferent
fibers and adjacent myenteric plexus and colonic
ganglion neurons

Camera lucida drawing of sacral afferent fibers
on circular muscle

Fluorescent photomicrographs of sacral afferent
fibers in pelvic nerve, pudendal nerve, bladder
ganglia and pelvic plexus ganglia

Fluorescent photomicrographs of sacral afferent
fibers on urinary bladder smooth muscle

Camera
sacral

lucida drawing and photomicrographs of
afferent fibers in urethra

Camera lucida drawings and photomicrographs of
sacral afferent fibers on urethra and external
urethral sphincter striated muscle fibers

Fluorescent photomicrographs of sacral afferent
fibers in urethra mucosa and submucosa

Camera lucida drawing and photomicrographs of
myenteric neurons that project processes to
colonic fiber bundles

Distribution of myenteric neurons in distal
colon in relation to axial distance from colonic
fiber bundles

Distribution of neurons in mid-distal colon and
mid-proximal colon in relation to axial distance
from colonic fiber bundles

Distribution of myenteric neurons lateral to
labeled colonic fiber bundles

Bright-field photomicrograph of Dogiel type I
neurons

xii

Figure

Figure

Figure

Figure

29:

30:

31:

32:

Bright-field photomicrograph of Dogiel type III
neurons

Drawing of colon, prevertebral ganglia and para-
vertebral ganglia

Bright-field photomicrograph of colonic ganglia
neurons that project processes to colonic fiber
bundles

Fluorescent photomicrograph of fast blue labeled

neurons in sympathetic prevertebral and paraver-
tebral ganglia

xiii

INTRODUCTION

The mammalian large intestine, which represents the
terminal portion of the gastrointestinal tract, is comprised
of the caecum, proximal, mid and distal colon, rectum and
anal canal. It is responsible for the storage, mixing,
transport and evacuation of intestinal contents. The motili-
ty of the large intestine is under the influence of both the
electrical properties intrinsic to smooth muscle cells and
the nervous system. The electrical properties of the smooth
muscle are thought to set up a basic pattern of contractile
activity which is then modulated by the autonomic nervous
system.

The autonomic nervous system is classically divided
into three major subdivisions: sympathetic, parasympathetic
and intrinsic (Langley, 1921). The lumbar sympathetic divi-
sion that innervates pelvic vicera is comprised of pregan-
glionic and postganglionic neurons. The preganglionic neu-
rons are cholinergic and their soma are located in the
intermediolateral cell columns of the lumbar spinal cord.
The postganglionic neurons are adrenrgic and their soma are
located in lumbar sympathetic chain ganglia (paravertebral)
and inferior mesenteric, superior mesenteric and coeliac
ganglia (prevertebral). The major nerve trunks for these
sympathetic postganglionic fibers include inferior splanch-
nic nerves, lumbar colonic nerves and hypogastric nerves

(Fig. 30).

The sacral parasympathetic division that innervates
pelvic viscera also includes preganglionic and postganglio—
nic neurons. The preganglionic neurons are cholinergic and
their soma are located in the intermediolateral columns of
the sacral spinal cord. Sacral preganglionic fibers project
through the pelvic nerves to innervate postganglionic neu-
rons in prevertebral and possibly intrinsic ganglia. Prever-
tebral postganglionic neurons are located in ganglia on the
serosal surface of the pelvic viscera (colonic ganglia and
urinary bladder ganglia). The postganglionic pathways in-
clude colonic fiber bundles (Fig. 1) and fiber bundles on
the serosal surface of the urinary bladder and urethra.
Afferent fibers originate from sensory neurons located in
dorsal root ganglia in both lumbar and sacral spinal regions
(Figs. 1 and 4). The intrinsic division of the autonomic
nervous system is located within the walls of the gastroin-
testinal tract and includes the myenteric (Aurebach's)
plexus and the submucosal (Meissner's) plexus.

The overall objective of this dissertation is to study
the intrinsic and extrinsic neural pathways to the large
intestine of the cat. One major focus of this dissertation
will be to determine if both intrinsic and extrinsic neurons
utilize colonic fiber bundles as a peripheral nerve trunk to
innervate effector structures in the colon. A second major
focus of this dissertation will be to examine the peripheral
distribution of sacral afferent fibers to the large intes-

tine, prevertebral ganglia, urinary bladder and urethra.

HISTORY

EXTRINSIC NEURAL PATHWAYS

1. SACRAL APPERENT PATHWAYS.

A. ANATOMY: DISTRIBUTION OF NEURONS IN SACRAL DORSAL ROOT
GANGLIA THAT PROJECT VISCERAL APPERENT FIBERS TO PELVIC
NERVE. Primary afferent neurons in sacral dorsal root
ganglia (DRG) are known to project to the pelvic nerves of
the cat, monkey and rat (Morgan et al., 1981: Nadelhaft et
al., 1983; Nadelhaft and Booth, 1984). In retrograde studies
of cat and monkey, labeling the ipsilateral pelvic nerve
with horseradish peroxidase (HRP) demonstrated sacral dorsal
root ganglion neurons numbering 3,676 and 2,992, respec-
tively (Morgan et al., 1981; Nadelhaft et al., 1983). The
greatest percentage of retrogradely labeled DRG neurons was
located in $2 (80%). In similar retrograde studies of rat
pelvic nerve, 95% of labeled cells were found in L6 and SI
(lumbosacral) DRGs (Nadelhaft and Booth, 1984). A mean of
1500 neurons was found, most having a small soma (17 x 25
um) and central processes that entered Lissauer's tract
(Nadelhaft and Booth, 1984). The visceral afferent compon-
ent of dorsal root ganglia neurons is relatively small
(approximately 2%) when compared to the population of spinal
afferents that innervate skin and deep somatic structures

(Janig and Koltzenburg, 1990).

ANATOMY: DISTRIBUTION OF NEURONS IN SACRAL DORSAL ROOT
GANGLIA THAT PROJECT VISCERAL APPERENT FIBERS TO LARGE
INTBSTINE. Sacral visceral afferent fibers are known to
project to the distal colon of rat and cat (Keast and de
Groat, 1992; Kawatani et al., 1985). In retrograde tracing
studies of rat and cat, dye (Fast Blue, True Blue or Fluoro
Gold) injections into distal colon labeled neurons in sacral
dorsal root ganglia (Keast and de Groat, 1992; Kawatani et
al., 1985). The predominant distribution of labeled neurons
was 51 for rat and $2 for cat. The actual number of retro-
gradely labeled neurons was not reported in these studies.

Degeneration techniques have also been used to study
the distribution of sacral afferent fibers to cat large
intestine (Schofield, 1962). Degeneration of extrinsic
fibers was accomplished by sectioning spinal nerves central
or peripheral to dorsal root ganglia. Afferent fibers were
detected as numerous degenerating fragments in myenteric
plexus ganglia.

There is a paucity of information on the number and
distribution of neurons in sacral dorsal root ganglia that
project peripheral processes to the large intestine. The
peripheral distribution of sacral visceral afferent fibers
to the large intestine has also not been studied with modern
tracing techniques. This dissertation will study the distri-
bution of neurons in sacral dorsal root ganglia that project
processes to at least mid colon using retrograde tracing

techniques. The distribution of sacral afferent fibers to

the large intestine will also be studied using anterograde
tracing techniques.

ANATOMY: DISTRIBUTION OF NEURONS IN SACRAL DORSAL ROOT
GANGLIA THAT PROJECT VISCERAL AFFERENT FIBERS TO URINARY
BLADDER AND URBTHRA. Retrograde axonal tracing techniques
have been used to determine the distribution of primary
visceral afferent neurons in sacral dorsal root ganglia that
project peripheral processes to urinary bladder (Downie et
al., 1984: Applebaum et al., 1980) and urethra (Downie et
al., 1984). One study (Downie et al., 1984) identified
small populations of neurons in sacral DRG, predominantly in
$2. The mean values of retrogradely labeled afferent neu-
rons were 81 to urethra, 159 to detrussor muscle of the
urinary bladder and 178 to bladder base. The second study
injected all regions of urinary bladder and identified a
similar small population (mean = 365 neurons) distributed
to all 3 sacral dorsal root ganglia (Applebaum et al.,
1980). When considered against the estimated 7300 neurons in
sacral dorsal root ganglion that project peripheral process-
es to cat pelvic nerves (Morgan et al., 1981), afferent
innervation of the bladder and urethra represents less than
5%.

Degeneration techniques have also been used to study
the distribution of sacral axon terminals in the cat urinary
bladder (Uemura et al., 1973: Uemura et al., 1975). Sacral
dorsal root ganglia were surgically ablated and degenerating

afferent terminals were detected in the urinary bladder by

electron microscopy. These studies showed that sacral affer-
ent fibers were distributed equally to all bladder regions
(Uemura et al., 1973). Degenerating afferent terminals
represented less than 5% of total axon terminals associated
with smooth muscle fibers and submucosa in cat urinary
bladder (Uemura et al., 1973; Uemura et al., 1975).

Projection of sacral afferent fibers across the midline
of urinary bladder and urethra was demonstrated in degenera-
tion and retrograde tracing studies. Degeneration studies of
sacral afferent terminals in cat urinary bladder showed
approximately one third of sacral afferent fibers cross the
bladder midline to innervate the contralateral side (Uemura
et al., 1973: Uemura et al., 1975). Retrograde tracing
studies, in which HRP, bisbenzamide, nuclear yellow or fast
blue were injected into ipsilateral urinary bladder and
urethra, also demonstrated that sacral dorsal root ganglion
neurons project afferent fibers across the midline in cat
urethra and rat and cat urinary bladder (Downie et al.,
1984; Applebaum et al., 1980). This redundant bilateral
innervation by sacral afferent fibers may represent a safety
feature in bladder function.

Previous studies of sacral visceral afferent pathways
have relied on retrograde tracing and degeneration tech-
niques. The former provides cell numbers, morphology and
distribution while the latter provides the distribution of
degenerating fiber fragments. The anterograde tracing tech-

niques used in this study allowed me to visualize sacral

visceral afferent fibers with relative continuity over
centimeter distances in proximity to neurons and colonic

effector structures.

B. IMMUNOHISTOCHEMISTRY OF PRIMARY AFFERENT NEURONS AND
FIBERS. Sacral dorsal root ganglion neurons in mammals
are immunoreactive for substance-P (SP), calcitonin gene-
related peptide (CGRP), somatostatin (SOM), galanin, opioid
peptides, neurokinin A, peptide histidine isoleucine amide,
vasoactive intestinal polypeptide (VIP), choleycystokinin
(CCK), angiotensin II and bombesin (Buck et al., 1982: de
Groat, 1987: de Groat et al, 1983; Hokfelt et al., 1975:
Keast and de Groat, 1992: Klein et al., 1990). These pep-
tides are distributed in afferent fibers of pelvic viscera,
visceral afferent neurons in sacral dorsal root ganglia
(lumbosacral in rats) and at sites of afferent termination
in spinal cord.

In double-labeling experiments, retrograde tracers were
injected into the colon and urinary bladder of rats and cats
(Keast and de Groat, 1992). The retrogradely labeled dorsal
root ganglia were then double-labeled for one of five pep—
tides (SP, CGRP, VIP, enkephalin (ENK) or SOM). Neurons
innervating pelvic viscera and immunoreactive for peptides
had the following relative distribution: CGRP>SP>VIP>ENK>SOM
(Keast and de Groat, 1992). The three most numerous peptides
in sacral (lumbosacral in rat) dorsal root ganglia are

summarized below.

For sacral (lumbosacral in rat) dorsal root ganglion
neurons that project peripheral processes to the colon,
CGRP-immunoreactive neurons had the greatest percentage of
co-localization (Keast and de Groat, 1992). 70% of rat
lumbosacral and 45% of cat sacral dorsal root ganglia neu-
rons innervating the colon were immunoreactive for CGRP. SP-
immunoreactivity was detected in 38% of rat and 33% of cat
colon afferent neurons. VIP-immunoreactivity was detected in
only 1% of rat and 18% of cat colon afferent neurons.

For rat lumbosacral dorsal root ganglion neurons that
project to urinary bladder, 52% were immunoreactive for
CGRP, 29% for SP and 11% for VIP (Keast and de Groat, 1992).
C. PHYSIOLOGY OF SACRAL AFFERENT FIBERS: LARGE INTESTINE.
Sensory information from the colon and rectum (sense of
fullness, urge to defecate, pain impulses, chemical and
mechanoreceptive input) is conveyed to the sacral spinal
cord via afferent fibers in the pelvic nerve (de Groat and
Krier, 1978; de Groat et al., 1981: Haupt et al., 1983:
Janig and Koltzenburg, 1990: Morgan et al., 1981). When a
mechanical stimulus was applied to cat anal mucosa or the
colon was passively distended, afferent discharges were
detected in sacral (SI-$2) dorsal root fibers (Bahns et al.,
1987: Jani and Koltzenburg, 1991). A total of 59 82 afferent
units were identified electrically in cat pelvic nerve
(Janig and Koltzenburg, 1991). Of these, 61% responded only
to passive distension of the distal colon, while 39% re-

sponded only to mechanical stimulation of the anal mucosa.

This suggests that pelvic afferent units consist of distinct
populations that respond to specific stimuli.

The colonic afferents were thin myelinated and non-
myelinated fibers with a median conduction velocity of 3.2
m/s (Janig and Koltzenburg, 1991). They were classified as
myelinated phasic afferents, responding only transiently to
colonic distension (median conduction velocity 8.0 m/s), and
non-myelinated tonic afferents, discharging throughout the
distension (median conduction velocity 1.7 m/s). For tonic
units, increases in colonic distension resulted in increased
discharge frequencies. In-situ recordings of visceral affer-
ent fibers in cat inferior splanchnic nerves demonstrate
that afferent fibers respond in a graded fashion to colon
distension (Blumberg et al., 1983). These studies suggest
that the colon is innervated by mechanoreceptive afferents
that encode the degree of distension by increases in dis-
charge frequency.

A relatively new class of non-myelinated afferent
fibers has been described for colon. Electrophysiological
studies demonstrate that approximately 95% (202/213) of
non-myelinated afferent units projecting from sacral dorsal
root ganglia (82) to pelvic nerve do not respond to mecha-
nincal stimulation of cat colon or anal canal (Janig and
Koltzenburg, 1991). The authors suggest that these silent C-
fibers may be mechanoreceptors that are active only during

inflamation.

REFLEXES OF SACRAL AFFERENT FIBERS TO COLON: SPINAL REFLEX.

Afferent fibers in sacral dorsal roots play a role in colo-
nic reflexes. In in-situ studies of cat mid-distal colon
(de Groat and Krier, 1978), measurements of colonic motility
were performed simultaneously with electrophysiological
recordings of colonic fiber bundles. Distension of the
colon or rectum, or electrical stimulation of pelvic nerve
afferent fibers resulted in increased efferent firing in
colonic fiber bundles and sustained propulsive contractions
associated with defecation. The responses were blocked by
transection of the sacral dorsal roots or the pelvic nerves,
indicating a spinal pathway reflex. The sacral reflexes were
mediated by non-myelinated afferent and preganglionic effer-
ent fibers (de Groat and Krier, 1978; de Groat et al.,

1981).

REFLEXES OF SACRAL AFFERENT FIBERS TO COLON: AXONAL REFLEX.
Afferent fibers are known to relay sensory information to
the central nervous system and play a role in reflexes. It
has also been suggested that afferent collaterals in the
periphery may release neurotransmitter substances on enteric
neurons to modulate local circuits (Delbro and Lissander,
1980; Delbro et al., 1981: de Groat et al., 1987). Stimula—
tion of sympathetic (lumbar splanchnic nerves) and parasym-
pathetic (vagus nerve) nerve trunks produced contractions of
cat colon and stomach, respectively, that were not blocked
by nicotinic (hexamethonium) or adrenergic (guanethidine)

antagonists (Delbro et al., 1983; Fandriks and Delbro, 1985;

10

Delbro and Lisander, 1980; Fandriks et al., 1985; Delbro et.
al., 1981). These contractions were greatly reduced by
atropine and substance-P antagonists. This suggests that
the pathway for this contraction involves cholinergic-mus-
carinic and SP receptors. These contractions may be mediated
by antidromic activation of substance-P containing afferent
fibers which in turn activate cholinergic postganglionic

motor neurons (Fandriks et al., 1985).

D. PHYSIOLOGY OF SACRAL AFFERENT FIBERS: URINARY BLADDER AND
URBTHRA. Afferent fibers emanating from sacral dorsal root
ganglia mediate sacral parasympathetic reflexes to urinary
bladder and urethra (micturition)(de Groat and Booth, 1984;
de Groat and Kawatani, 1989). In contrast to colon reflexes
which are mediated predominantly by non-myelinated C-fibers
(de Groat and Krier, 1978; de Groat et al., 1981), electro-
physiological studies in cat have shown that urinary bladder
reflexes involving the sacral spinal cord are mediated by
both myelinated and nonmyelinated afferent fibers (de Groat
et al., 1981; Habler et al., 1990).

In cat urinary bladder, electrophysiological studies
have shown that myelinated afferent units have low
thresholds and firing frequencies that rise with increasing
intraluminal pressure during distension and isovolumetric
contractions (Iggo, 1955: Floyd et al., 1976: Bahns et al.,
1987). Iggo was able to demonstrate that the same afferent
unit was stimulated by both passive distention and active

contraction of the cat urinary bladder (Iggo, 1955). This

11

suggests that bladder mechanorective afferents encode the
degree of intraluminal pressure by increases in discharge

frequency.

SACRAL AFFERENT FIBERS TO URINARY BLADDER AND URETHRA:
BENSATION. Stimulation of urinary bladder afferents in
humans by gradual bladder distension elicits sensations of
fullness, urge to micturate and eventually pain (Nathan,
1956). Nathan was able to distinguish several sensations
associated with micturition and identify their source by
studying patients that had suprapubic cystostomies and
urethral ligation at the bladder neck. The "desire to mictu-
rate " was attributed to urinary bladder distention and
contractions. By connecting the suprapubic catheter to a
manometer and instilling water at ever increasing pressures,
the urge to micturate became stronger. The sensation that
"micturition is imminent" was attributed to the urethra by
mechanically stimulating the urethra in the same patients.
Differences in sensation may be due in part to the type
of afferent receptors in the urinary bladder. Light and
electron microscopic studies have demonstrated a relative
absence of specialized nerve endings in cat urinary bladder,
suggesting that afferent receptors are free nerve endings
(Fletcher et al., 1970; Uemura et al., 1974; Uemura et al.,
1975: Uemura et al., 1973: Fletcher and Bradley, 1970;
Fletcher and Bradley, 1978). These free nerve endings may

act as chemoreceptors (Habler et al., 1990), thermoreceptors

12

(Fall et al., 1990: Nathan, 1952) and mechanoreceptors
sensitive to passive distension, active contraction, mucosal
deformation and bladder position (Habler et al., 1990:
Fletcher and Bradley, 1978; Iggo, 1955).

A relatively new classification of visceral afferent is
a high threshold mechano-chemoreceptor (Habler et al.,
1990). In electrophysiological studies of cat urinary blad-
der, recordings of afferent fibers in sacral dorsal roots
demonstrated a population of C-fibers that were not activat-
ed by innocuous or noxious increases in intravesicular
pressure in normal bladder. When the bladder was inflamed by
injection of mustard or turpentine oil, the previously
silent C-fibers responded to both the irritant and increases
in intravesicular pressure. This suggests that these silent
C-fibers may be involved in the sensation of pain associated

with mucosal inflamation of the urinary bladder.

SACRAL AFFERENT FIBERS TO URETHRA: MECHANO-RECEPTORS. The
differences in bladder and urethra sensation may also be due
to distinct populations of afferent units that respond to
specific stimuli (mechanoreceptors). Urethral afferent units
responded to mechanical stimulation of the urethral mucosa,
but did not respond to distension or isovolumetric contrac-
tion of cat urinary bladder (Bahns et al., 1987). Light
microscopic studies (haematoxylin and eosin) in cat urethra
have identified specialized nerve terminals and free nerve

endings (Garry and Garven, 1957). The nerve terminals were

13

identified as pacinian corpuscles and "cucumber" or "sau-
sage" shaped terminals. Pacinian corpuscles were identified
in mucosa and external muscle layers of the urethra, pre-
dominantly in distal urethra and external urethral sphinc-
ter. Rapidly adapting specialized nerve terminals may

be responsible for the detection of mucosal mechanical
stimulation associated with urinary flow in urethra (Fletch-

er and Bradley, 1978: Garry and Garven, 1957: Nathan, 1956).

2. POSTGANGLIONIC EFFERENT PATHWAYS

A. PARASYMPATHETIC COLONIC GANGLIA. Sacral parasympathetic
preganglionic fibers provide excitatory input to the large
intestine that is thought to regulate colonic motility and
defecation (de Groat and Krier, 1978). Electrical stimula-
tion of the pelvic nerve elicits contractions of the colon
and action potentials in colonic fiber bundles in cats (de
Groat and Krier, 1976). Both effects are markedly reduced by
ganglionic blocking agents, indicating the presence of
synaptic relays that may involve neurons in parasympathetic
colonic ganglia and enteric ganglia.

Neurons in parasympathetic colonic ganglia may project
processes to colonic effector structures through colonic
fiber bundles. When colonic fiber bundles in cat distal
colon are electrically stimulated, antidromic potentials are
recorded in 62% of neurons tested in colonic ganglia (Krier

and Hartman, 1984). This indicates that postganglionic

14

fibers of colonic ganglion neurons project processes through
colonic fiber bundles to at least distal colon regions. The
distribution of colonic ganglia neurons that project pro-
cesses to mid and proximal colon via colonic fibers bundles
is unknown. This dissertation will determine this distribu-

tion using retrograde tracing techniques.

B. SYMPATHETIC PREVERTEBRAL AND PARAVERTEBRAL POSTGANGLIONIC
PATHWAYS. The lumbar sympathetic nervous system is com-
prised of cholinergic preganglionic fibers arising from
lumbar spinal cord and noradrenergic postganglionic fibers
arising from prevertebral and paravertebral ganglia (Baum-
garten, 1982; Furness and Costa, 1974: Szurszweski and
Krier, 1984). The preganglionic fibers synapse with postgan-
glionic neurons, which in turn provide adrenergic innerva-
tion to enteric ganglia and colonic effector structures
(Gabella, 1979: Jacobowitz, 1965; Manber and Gershon, 1979;
Norberg, 1964; Norberg and Hamberger, 1964; Szurszewski and
Krier, 1984).

In early studies of the large intestine, Langley and
Anderson determined that electrical stimulation of lumbar
spinal nerves in rabbits, cats and dogs decreased the motil-
ity of the large intestine and increased internal anal
sphincter tone (Langley and Anderson, 1895). Electrical
stimulation of sympathetic lumbar colonic nerves decreased
colonic tone and depressed spontaneous and neurally evoked

contractions of colon external muscle layers (Langeley and

15

Anderson, 1895). In these studies electrical stimulation
was obtained by placing electrodes directly on isolated
whole nerves and supplying a "weak tetanizing induction
shock" that could be "distinctly felt" when placed on the
investigaters tongue. Motility changes were qualitatively
described after direct observation of the viscera.

The number, distribution and electrophysiology of
neurons in cat prevertebral and paravertebral ganglia that
project processes to hypogastric and lumbar colonic nerve
trunks have been studied and are summarized below.

LUMBAR SYMPATHETIC CHAIN GANGLIA. Neurons in lumbar sympa-
thetic chain ganglia project processes to cat inferior
mesenteric ganglion (IMG) through inferior splanchnic nerves
(Baron et al., 1985 II). When ipsilateral cat inferior
splanchnic nerves were labeled with HRP, approximately 1569
neurons in lumbar sympathetic chain ganglia were labeled.
These neurons were distributed predominantly to Lz-L5 gan-
glia. Similar retrograde tracing studies of ipsilateral
hypogastric nerve demonstrated that approximately 350 neu-
rons in lumbar sympathetic chain ganglia had fibers that
continued through the IMG to the hypogastric nerve in cat
(Baron et al., 1985 I).

Postganglionic fibers from lumbar sympathetic chain
ganglia are also found in the pelvic nerve. When ipsilateral
cat pelvic nerve was labeled with HRP or true blue, approx-
imately 120 cells were labeled in lumbar sympathetic chain

ganglia (Kuo et al., 1984). Electrophysiological evidence

16

showed that stimulation of the sympathetic chain from L3 to
L6 and occasionally as high as L2 elicited firing in the
ipsilateral pelvic nerve (Kuo et al., 1984). They proposed
that these sympathetic postganglionic fibers passed through
the sacral paravertebral ganglia to the pelvic nerve. The
distribution of lumbar sympathetic chain ganglia neurons
that project processes to mid and proximal colon through
colonic fiber bundles is unknown.

Intracellular recording and injection techniques were
used to describe the morphological and electrophysiological
characteristics of neurons in cat lumbar paravertebral
ganglia (Percy et al., 1988). Two distinct morphologies,
spherical (mean soma area; 730 pmz) and fusiform (mean soma
area 540 umz), were detected in a 2:1 ratio, respectively.
The two morphologies could not be distinguished electrophy-
siologically. Intracellular recordings of lumbar sympathetic
chain neurons indicated that 86% had myelinated fibers and
14% had non-myelinated fibers projecting to lumbar splanch-
nic nerves or to the lumbar sympathetic chain (Hartman and

Krier 1984).

INFERIOR MBSENTBRIC GANGLION (IMG). The IMG projects pro-
cesses through two postganglionic trunks, the lumbar colonic
nerves and the hypogastric nerves (Baron et al., 1985a and
b). When cat lumbar colonic nerves were retrogradely la-
beled with HRP, approximately 24,000 neurons were identified
in the IMG (Baron et al., 1985 III). In a similar study, cat

hypogastric nerves were retrogradely labeled with HRP and

17

32,300 neurons were detected in the IMG (Baron et al., 1985
I). The subpopulation of IMG neurons that enter the colon
via colonic fiber bundles to innervate colonic effector

structures is unknown.

SUPERIOR MESENTERIC GANGLION (SMG) AND COELIAC GANGLION.
The number and distribution of neurons in SMG and coeliac
ganglia that provide innervation to the colon is relatively
unexplored. When lumbar colonic nerves were labeled with
HRP, cell bodies in SMG were detected in only 2 of 5 cats
(six cells and seven cells)(Baron et al., 1985 III). The
distribution of neurons in the coeliac ganglion was not
reported. The distribution of neurons in these prevertebral
ganglia that project processes through colonic fiber bundles
to at least mid colon is not known.

The SMG and coeliac ganglia receive excitatory input
from the colon. Electrophysiological studies in guinea pig
demonstrated that both coeliac and SMG neurons received
excitatory input from the colon that was increased by colon
distension and abolished by cutting the intermesenteric
nerves (Kreulen and Szurszewski, 1979). This suggests the
possibility of a peripheral reflex loop between these pre-
vertebral ganglia and enteric ganglia of the colon (Kruelen
and Szurszweski, 1979). The postganglionic fibers may pro-
ject to the colon via hypogastric nerves and colonic fiber
bundles.

The number and distribution of neurons in sympathetic

prevertebral and paravertebral ganglia that project process-

18

es to at least mid colon will be determined using retrograde
tracing techniques.

3. INTRINSIC NEURAL PATHWAYS: MYENTERIC PLEXUS. Intrinsic
nerves of the large intestine are located within intercon-
nected ganglia of the myenteric and submucosal plexus (Costa
and Furness, 1976: Gabella, 1979: Gershon, 1981). The myen-
teric plexus, located in the connective tissue layer between
the longitudinal and circular smooth muscle layers, consists
of large ganglia interconnected by numerous interganglionic
fiber tracts. The myenteric plexus has been described mor-
phologically, immunocytochemically, and electrophysiologi-
cally in an attempt to correlate these characteristics with
function (Dogiel, 1899; Christensen et al., 1984: Christen-
sen, 1988; Costa et al., 1982; de Groat, 1987: Gunn, 1959;

Gunn, 1968).

MORPHOLOGY. Dogiel was the first to describe three cellular
morphologies for myenteric neurons (Dogiel, 1899) and his
work remains the standard for enteric neuron morphology.
Using methylene blue to stain the myenteric plexus of large
and small intestine in man, guinea—pig, dog, cat, rabbit and
rat, he identified three distinct morphologies, Dogiel type
I, II and III (Dogiel, 1899). Dogiel type I cells have a
rough somal surface, short, broad dendrites and a long axon.
Dogiel type II cells have a smooth somal surface, numerous
fine processes, and no discernible long axon. Dogiel type
III cells have a smooth somal surface, several fine den-

drites and one long axon. Dogiel speculated that type I

19

cells were motor neurons and that type II cells were sensory
neurons. The three morphological types of myenteric neurons
described by Dogiel have been verified in the cat ileum
(Gunn, 1959). Using silver impregnation and methylene blue
techniques, Gunn was able to distinguish Dogiel types I, II
and III as well as a fourth smaller cell type (mean soma
diameter approximately 15-20 umz) not previously described.
The fourth cell type was weakly stained with the silver
impregnation technique, had few discernible processes and
was found predominantly in the interganglionic fiber tracts.
Gunn speculated that these small cells were also neurons.
The distribution of ganglia in the myenteric plexus
has been described in numerous mammals (Christensen et al.,
1983; Christensen et al., 1984; Santer and Baker, 1988).

2

The distribution of myenteric neurons/cm has been deter-

mined in the small and large intestine of young and aging
rats using the NADH-tetrazolium reductase method (Santer and
Baker, 1988). In young rats (6 months) a mean colonic value

2 was determined while aging rats (24

2

of 14,214 neurons/cm
months) had a mean colonic value of 5,128 neurons/cm
(colon). This represents a 64% decrease in the number of
neurons/cm2 in the colon of aging rats.

Silver impregnation techniques were used to determine
the density and distribution of myenteric plexus ganglia in
the distal colon of eight mammals (Christensen et al.,

2

1984). The range of mean ganglia/cm in distal colon was 40

ganglia/cm2 (dog) to 794 ganglia/cm2 (rat). The cat had a

20

2

mean value of 42 ganglia/cm and the opossum had 45 (America

opossum) to 264 (Australian opossum) ganglia/cmz. This is in
contrast to an earlier study in which, using the same tech-
niques, Christensen reported only 18 ganglia/cm2 in the

distal colon of the opossum (specific species not identi-

fied) (Christensen et al., 1983).

IMMUNOCYTOCHEMISTRY. In recent years, a plethora of neuro-
peptides have been identified immunocytochemically in neu-
rons and/or fibers of the myenteric plexus that may play a
role in neurotransmisssion: substance P (SP), somatostatin
(SOM), calcitonin gene-related peptide (CGRP), cholecystoki-
nin (CCK), dynorphin (DYN), enkephalin (ENK), galanin (GAL),
gastrin releasing peptide (GRP), neuropeptide Y (NPY),
neurotensin, peptide HI, neurokinin A and vasoactive intes-
tinal polypeptide (VIP) (Bornstein et al., 1984; Schultzberg
et al., 1980; Costa et al., 1980; Costa and Furness, 1983;
Ekblad, 1987; Furness et al., 1983; Furness et al., 1985:
Furness and Costa, 1979: Furness and Costa, 1982; Fujimori
et al., 1989; Jessen et al., 1980; Llewellyn-Smith, 1987:
Pearse and Polak, 1975). Immunoreactivity for SP, VIP, CGRP,
CCK, NPY, ENK, and SOM was observed in cell bodies as well
as fibers of the myenteric plexus of rat and guinea pig
small intestine (Bornstein et al., 1984: Costa et al., 1980:
Ekblad, 1987: Schultzberg et al., 1980: Furness et al.,
1985). Immunoreactivity to neurotensin was observed only in

fibers (Shultzberg et al., 1980). In most instances, the

21

myenteric plexus had a greater density of peptidergic neu-
rons than did the submucosal plexus (Shultzberg et al.,
1980). In contrast, VIP containing neurons were found in
greater number in the submucosa (Shultzberg et al., 1980,
Furness et al., 1981).

The role of many of these neuropeptides in gastroin-
testinal function remains unknown, although in recent years
some progress has been made in determining some physiologi-
cal functions of several peptides. Ascending contraction
(contraction orad to a bolus) and descending relaxation
(relaxation aborad to a bolus) was demonstrated in isolat-
ed segments of guinea pig colon (Costa and Furness, 1976).
Using ig-vitro preparations of guinea-pig distal colon and
rectum, Costa and Furness studied enteric reflexes by apply-
ing localized distension and recording subsequent changes in
circular muscle activity. Distension of the colon produced a
contraction on the orad side and relaxation on the aborad
side of the stimulus. These effects were abolished by inter-
ruption of the myenteric plexus but not affected by removal
of the mucosa or submucosa. The ascending excitatory path-
ways were partly blocked by hyoscine (cholinergic-muscarinic
antagonist) and methysergide (5-hydroxytryptamine (5-HT)
antagonist) and by making the preparation tachyphylactic to
S-HT. The ascending excitatory pathways apparently involve
cholinergic neurons as well as S-HT-like neurons. The de-
scending inhibitory pathway did not respond to cholinergic

or adrenergic antagonists and is presumed to be nonadrener-

22

gic and noncholinergic in nature. Neuropeptides may play a

role in these nonadrenergic, noncholinergic effects.

Vasoactive intestinal polypeptide (VIP). One peptide sug—
gested to play a role in descending relaxation is VIP
(Farhrenkrug et al., 1978: Furness and Costa, 1978). Myen-
teric neurons labeled with antibodies to VIP project vari-
cose processes less than 1mm to underlying circular muscle
in both orad and aborad directions, aborad to other myenter-
ic neurons for 2-10mm, and aborad to submucosal ganglia up
to 15mm (Furness et al., 1985).

VIP has been suggested to play a role in intestinal
vasodilation (Costa et al., 1980: Fahrenkrug et al., 1978),
intestinal wall relaxation (Costa et al., 1980; Costa and
Furness, 1983: Furness et al., 1981, Grider et al., 1985a:
Grider et al., 1985b: Grider and Makhlouf, 1986) and in-
creased transepithelial transport of water and electrolytes
(Costa and Furness, 1983). VIP neurons were 'S' type (fast
EPSPs) and had two morphologies, Dogiel type I (Katayama,

1986) and Dogiel type III (Furness et al., 1981).

Calcitonin gene-related peptide (CGRP). CGRP is reported to
be a potent vasodilator in rabbit skin (Brain at al., 1985)
and rat mesentery (Han et al., 1990a: Han et al., 1990b)
and inhibits smooth muscle contractor responses in mouse
vas deferens (Al-Kazwini et al., 1986). In in-situ studies,

human or rat CGRP mixed with 133Xe was injected into rabbit

23

skin to measure 133Xe clearance (Brain et al., 1985). Their
results show that femtomole doses of CGRP induced microvasc-
ular dilation and increased blood flow, suggesting that
local CGRP release may be involved in control of blood flow.
Rat and Human CGRP were also used to study their ef-
fects on contractor responses of mouse vas deferens (Al-
Kazwini et al., 1986). Both rat and human CGRP were effec-
tive at inhibiting contractions of mouse vas deferens evoked
by either electrical stimuli or acetylcholine. CGRP did not
alter the uptake of [3H]-noradrenaline or the
fractional release of [3H]-noradrenaline from preloaded vas
deferens, suggesting CGRP does not function by interference
with adrenergic mechanisms. These observations suggest that
CGRP exerts its effects through post-junctional CGRP recep-

tors.

Substance-P (8P). SP has been shown to be excitatory to
myenteric neurons (Katayama and North, 1978). When SP is
iontophoretically applied directly onto myenteric neurons of
the guinea-pig (region of gastrointestinal tract not identi-
fied), SP causes a depolarization that is unaffected by
hexamethonium (cholinergic-nicotinic antagonist), atropine
(cholinergic-muscarinic antagonist), naloxone (opiate antag-
onist) or enkephalin (Katayama and North, 1978).

SP also has a contractile effect on guinea-pig ileal
longitudinal smooth muscle (Matthijs et al., 1990), cat

stomach and large intestine (Delbro et al., 1983; Fandriks

24

et al., 1985: Fandriks and Delbro, 1985) and canine small
intestine (Neya et al., 1989). In-vitro experiments on
fura-Z loaded guinea pig ileal longitudinal smooth muscle
demonstrate that intracellular calcium concentrations and
contractile force increase in a dose dependent manner when

exposed to SP (Matthijs et al., 1990).

4. COLONIC AND RECTAL FIBER BUNDLES.

In addition to neurons, ganglia and interganglionic
fiber tracts in the myenteric plexus, portions of the gas-
trointestinal tract also contain large fiber bundles. The
distribution of these fiber bundles has been described in
human lower esophagus, stomach, small intestine, mid and
distal colon and rectum using siver impregnation techniques
(Kumar and Phillips, 1989).

The morphology and distribution of fiber bundles has
also been described in numerous experimental mammals using
silver impregnation techniques (Christensen and Rick, 1985:
Christensen and Rick, 1987: Christensen, et al., 1983:
Christensen et al, 1984). Fiber bundles in the large intes-
tine originate within the pelvic plexus and pass orad to the
colon (Fig. 2) and aborad to the rectum and anal canal. For
the cat colon, six to eight colonic fiber bundles ascend
orad beneath the serosal surface of the distal colon and
between the external muscle layers. They project consider-
able distances to the mid and proximal regions of the colon.

Silver impregnation techniques also identified rectal fiber

25

bundles in the cat (Figure 4 of Christensen et al., 1984),
but they were not mentioned in the text of the paper.

Colonic fiber bundles in cat have been shown to contain
sacral parasympathetic preganglionic fibers (de Groat and
Krier, 1978), parasympathetic postganglionic fibers (Krier
and Hartman, 1984; de Groat and Krier, 1978) and sympathet-
ic postganglionic fibers (de Groat and Krier, 1979). Colonic
fiber bundles also contain afferent projections from neurons
in sacral dorsal root ganglia in cat and dog (de Groat and
Krier, 1978; Fukai and Fukada, 1984).

Sacral parasympathetic preganglionic fibers provide
excitatory input to the large intestine that is thought to
regulate colonic motility and defecation (de Groat and
Krier, 1978). Electrical stimulation of the pelvic nerve
elicits contractions of the colon and action potentials in
colonic fiber bundles in cats (de Groat and Krier, 1976).
Both effects are markedly reduced by ganglionic blocking
agents, indicating the presence of synaptic relays that may
involve neurons in parasympathetic colonic ganglia and
enteric ganglia.

Colonic ganglia were identified histologically on the
surface of cat distal colon (Fig. 2)(de Groat and Krier,
1976). In a later study, electrical stimulation of cat
colonic fiber bundles produced antidromic potentials in
approximately 62% of the colonic ganglion neurons tested.
This suggests that some neurons in parasympathetic colonic

ganglia project postganglionic fibers orad in colonic fiber

26

bundles to at least distal colon (Krier and Hartman, 1984).

Several reflexes have been shown to be dependent on the
integrity of colonic fiber bundles (Fukai and Fukada, 1984).
In in-situ studies of dog colon in which the colon wall was
ligated at mid-colon but colonic fiber bundles were intact,
mechanical stimulation of the anus and distension of the
rectum and proximal colon elicited contractions in colon and
rectum and colonic fiber bundle discharges. Ligation of
colonic fiber bundles at mid-colon did not diminish colonic
or rectal contractions at the site of stimulation, but did
abolish contractions and fiber bundle discharges beyond the
ligations. This demonstrates that ano-colonic, recto-colonic
and cola-colonic reflexes are dependent on the integrity of
colonic fiber bundles. These experiments, however, left the
pelvic nerves intact, so it is unknown if these reflexes
involving colonic fiber bundles are central in origin,
peripheral or both.

Peristalsis may be mediated by enteric neurons (Costa
and Furness, 1976). In an in-vitro study, ascending contrac-
tion (contractions orad to a bolus) and descending relaxa-
tion (relaxation aborad to a bolus) were present in isolated
segments of guinea pig colon in response to a mechanical
stimulus (bolus). These peristaltic waves were abolished by
interruption of the myenteric plexus (Costa and Furness,
1976). Myenteric neurons were postulated to be responsible
for the peristaltic wave that propelled a bolus through an

isolated guinea pig colon. Colonic fiber bundles were not

27

mentioned in this study, but have been demonstrated in
guinea pig distal colon by silver impregnation techniques
(Christensen et al., 1984).

Colonic fiber bundles have branch points interconnec-
ting them with the myenteric plexus as they ascend the colon
and are known to give off myelinated and non-myelinated
processes over their entire length (Christensen and Rick,
1987). It is not known, however, if myenteric neurons uti-
lize colonic fiber bundles as another intrinsic neural
pathway, possibly having a role in these reflexes. This
dissertation will utilize retrograde tracing techniques to
determine if myenteric neurons project processes orad and/or

aborad through colonic fiber bundles.

28

Specific Aims

The specific aims of this dissertation are:

1. Sacral Afferent Pathways.

a. Determine the distribution, number and diameter of
neurons in sacral dorsal root ganglia (S1 - S3) that project
peripheral processes to colonic fiber bundles.

b. Determine the distribution of sacral visceral affer-
ent fibers to mucosa, submucosa, myenteric plexus, external
muscle layers and serosa of large intestine.

c. Determine the distribution of sacral visceral affer-
ent fibers to mucosa, submucosa, external muscle layers and
serosa of urinary bladder.

d. Determine the distribution of sacral visceral affer-
ent fibers to mucosa, submucosa, external muscle layers and
serosa of urethra and external urethral sphincter.

e. Determine the distribution of sacral visceral affer-

ent fibers to prevertebral ganglia.

2. Postganglionic Efferent Pathways to the Large Intestine.
a. Determine the number and distribution of neurons in
parasympathetic colonic ganglia that project processes to
colonic fiber bundles.
b. Determine the number and distribution of neurons in
sympathetic prevertebral and paravertebral ganglia that

project processes to colonic fiber bundles.

29

3. Intrinsic Neural Pathways of the Colon.
a. Determine the number, morphology and distribution of
myenteric neurons which project processes to colonic fiber

bundles.

METHODS

Anesthesia

Male or female cats were used in all experiments. In
experiments involving neuroanatomical tracing techniques,
cats were initially anesthetized with intraperitoneal
injections of pentobarbital sodium, 34mg/kg. A peripheral
intravenous (i.v.) line was established (0.9% NaCl) and
anesthesia was maintained with dilute pentobarbital sodium
(1:5 0.9% NaCl) as needed. Body temperature was maintained
during surgery with a K-thermia pad (38.6°C) and respira-
tions were spontaneous. After appropriate survival periods
the animals were reanesthetized with pentobarbital sodium
(50mg/kg; i.p.). Tissues were removed and the animals were

euthanized.

Sterile Surgical and Labeling Techniques
Experiments involved animal recovery and survival
periods. Surgical procedures were performed in a surgical

suite under aseptic conditions. After anesthesia, the surgi-

30

cal sites were shaven and scrubbed with a betadine prepara-
tion. All surgical instruments and drapes were autoclaved

prior to procedures.

Anterograde Tracing

A mid-line dorsal surgical incision was made expos-
ing the lumbar and sacral vertebral segments ( L6 — S3).
Laminectomies were performed on the sacral vertebral seg-
ments (81 - S3) and the sacral dorsal root ganglia ($1 -

S3) were identified bilaterally (Fig. 1).
sunaqmntuu

...... a)

9 ”” .1 ' I.
lgl‘E‘h»~«.!‘§!!!!!"'

Dorsal Root Ganglia

33

}
I
\

‘WWM'--v ”mum

\ .
| . \
I

x I | \ I
\
‘ I W '
Aborld \\"' Orad
.‘ ¥ ‘ '
, ‘c‘.l<lll \ l ' I .
- - ‘ L--l~~LL-t-~ Law-mi "'1""'r“.‘*'4‘L“—:~"—¥“ ' +—"

Colonto Ganglton
Figure 1. Drawing of cat sacral spinal cord, sacral dorsal

Polvlo larva Coloolo Floor Bundles

 

 

 

 

root ganglia (81'33)' pelvic nerve, colonic ganglion and
colon with colonic fiber bundles. WGA-HRP was pressure
injected into sacral dorsal root ganglia (51'33) bilaterally
or ipsilaterally. Long arrow indicates axis of longitudinal
muscle fibers. Short arrow indicates long axis of circular

muscle fibers.

31

A 2% solution (distilled deionized water) of wheatgerm
agglutinin (WGA)(Vector), either unconjugated orconjugated
to horseradish peroxidase (WGA-HRP, Vector) was pressure
injected into the ganglia via a glass micropipette (tip
diameter range, 18-25 pm). The micropipettes were sealed
to a 1 pl syringe with heated sealing wax. Pressure injec-
tions of volumes ranging from 0.5 to 1 pl were made bilater-
ally (n = 9) or ipsilaterally (n = 3) at two or three sites
for each dorsal root ganglion ("n" refers to the number of
cats unless otherwise specified). In the 3 ipsilateral
injection experiments, the ipsilateral sacral ventral roots
($1 - S3) were sectioned to rule out incidental labeling of
efferent fibers. After injection, the muscle layers and skin
were closed. Following time periods ranging from 72 to 120

hours, animals were reanesthetized and perfused.

Retrograde Tracing

A midline abdominal incision was made exposing the
large intestine and allowing identification of the bilateral
pelvic nerves and their neural connections with parasympa-
thetic colonic ganglia and colonic fiber bundles (DeGroat
and Krier, 1976: DeGroat and Krier, 1978)(Fig. 2). Five
millimeter segments of 2 to 6 colonic fiber bundles were
dissected from the serosal surface of the mid colon at the
level of the inferior mesenteric artery or at the distal
colon one cm orad to the pelvic brim. The distal colon is

defined as the region extending 1.5 cm below the pelvic brim

32

/Pelv1c Nerve Colonic Fiber Bundles

 

Aborad

 

 

 

Orad

 

 

 

 

 

\ ‘ 'I '.t -L-l---i'l----?--~--:-r' "3,. ‘.‘"l“"{'+'P".'1["+-‘(1':"r“1"f”}j"r';"i1‘ ‘H

——-—\ Colonic Ganglion L—L.

 

Figure 2. Drawing of one side of cat distal and mid colon
with attached pelvic nerves, colonic ganglia and colonic
fiber bundles. Filled bars at orad end indicate transection
site of colonic fiber bundles at level of inferior mesenter-
ic artery (IMA). Asterisks indicate transection sites of
colonic fiber bundles 1cm orad to pelvic brim. HRP or Fast
Blue was placed on central cut end (side continuous with
pelvic nerve) or peripheral cut end (side continuous with
colon) of transected colonic fiber bundles. Long arrow
indicates axis of longitudinal muscle fibers and colonic
fiber bundles. Short arrow indicates axis of circular muscle

fibers. Bar = 10mm.

33

to 1.0 cm below the inferior mesenteric artery. The proximal
colon is defined as the region extending 2.0 cm below the
ileocecal sphincter to 4.0 cm above the inferior mesenteric
artery. The mid colon is defined as the region between the
proximal and distal colon. Central or peripheral cut ends of
colonic fiber bundles were placed into small chambers
containing 10 ul of 15% horseradish peroxidase (HRP) solu-
tion ( Sigma Type VI) or 8% Fast Blue (Sigma). The open
end of the chamber was sealed with Vaseline. After a 90
minute incubation period, the colonic fiber bundles were
removed from the chambers and the abdomen was closed. After

36 to 96 hours, animals were reanesthetized and perfused.

Perfusion

Animals were anesthetized (pentobarbital sodium, 50
mg/kg,i.p.) and given 1000 units of heparin intravenously.
The thorax was opened and the animal was transcardially
perfused with ice-cold filtered 0.9% sodium chloride/0.05%
sodium nitroprusside, followed by ice-cold filtered 4%
paraformaldehyde (Immunocytochemical and Fast Blue experi-
ments) or 1% paraformaldehyde/1% glutaraldehyde (HRP experi-
ments) phosphate-buffered fixative and then ice-cold fil-

tered 0.05 M phosphate buffer.

Dissection and Tissue Preparation

After fixation, all tissue to be sectioned was first

34

cryoprotected by sequential equilibration in 10%, 20% and
30% sucrose buffer. Cryoprotected tissue was then sectioned
at 30 to 40 pm on a freezing microtome and processed either
free floating or mounted on gel coated slides. Tissue to be
processed as whole mounts was dissected free and stored

free-floating in 0.1 M phosphate buffered saline (PBS).

Large Intestine. The large intestine was cut open along its
mesenteric border and pinned flat with the mucosal border
up. The mucosa/submucosa was first removed as a single
sheet, followed by removal of the circular muscle layer.

From the mucosa/submucosa layer, two to three 1 cm2

pieces
from each colonic region (proximal, mid and distal) were
removed and cryoprotected before sectioning at 40 um. Each
square centimeter of mucosa/submucosa (approximately 2 mm
thick) produced 25 to 50 (40 pm) sections. Using a dissect-
ing microscope with polarized optics, successive circular
muscle strips (lengths, 2 cm to 5 cm in long axis of the
circular muscle fibers: widths, 1 mm to 5 mm) were dissect-
ed free from the underlying connective tissue layer. The
connective tissue layer containing the myenteric plexus of
the cat colon proved to be too thick in distal and mid colon
regions for conventional processing techniques. For distal
and mid colon, the longitudinal muscle layer was separated
as a continuous sheet from the adherent connective tissue

layer containing the myenteric plexus and colonic fiber

bundles. Sheets of myenteric plexus were cut into rectangu-

35

lar sections (2 cm to 3 cm lengths in long axis of the
longitudinal muscle, 4 cm to 5 cm in long axis of circular
muscle fibers). For proximal colon the connective tissue
layer containing the myenteric plexus was much thinner and
could not be seperated as a sheet. Whole mount sections of
myenteric plexus adherent to the longitudinal muscle layer
were prepared for proximal colon. For the longitudinal
muscle layer, successive segments (lengths, 2 cm to 5 cm
in long axis of the longitudinal muscle fibers ; widths, 2
cm to 5 cm) were prepared. The myenteric plexus, circular
muscle strips and longitudinal muscle were processed free

floating and whole mounted.

Urinary Bladder and Urethra. The urinary bladder and urethra
were removed and separated at the bladder neck-proximal
urethra. The urinary bladder was first hemisected into right
and left halves, then divided into dome (apex), ventral
wall, dorsal wall, trigone and neck regions. One to two 1

cm2

sections of bladder wall from each of 8 bladder regions
and the entire bladder neck were sliced in horzontal section
on a freezing microtome at 40 um. Each sqaure centimeter of
urinary bladder wall (approximately 1 to 2 mm thick depend-
ing on bladder distension) produced 20 to 45 (40 um) sec-
tions. In 2 animals, sections of bladder wall (2 mm x 10 mm)
were also sliced in cross section at 40 pm. The urethra was

divided into equal thirds by length. For unilateral injec-

tion experiments the urethra was hemisected into right and

36

left halves. For 4 animals, a 2 mm length of urethra from
each region was cut in cross section at 40 pm. The remainder
of the 6-10 mm long sections of urethra were cut in horizon-
tal sections at 40 pm. The external urethral sphincter was
dissected from the distal urethra and cut in horizontal or
cross section at 40 pm. Some slides were counterstained with

neutral red to identify tissue layers.

Histochemical Processing

Peroxidase. HRP labeled neurons and fibers were visualized
by incubating tissue with tetramethyl benzidine dihydrochlo-
ride (TMB) for 12 to 18 h at 4°C (Mesulam, 1978) followed by
3,3'-diaminobenzidine (DAB) for 5 min at room temperature
(22-25°C) (Lemann, 1985; McRorie et al., 1991). The tissue
was rinsed in PBS and dehydrated in serial dilutions of
ethanol followed by xylene. All tissue was then
coverslipped in Permount and allowed to dry for 24 hours.
Immunohistochemistry. Antibodies to the cytoskeletal pro-
tein neurofilament were used to label neuronal somas and
processes within the myenteric plexus, pelvic plexus ganglia
and colonic ganglia (Sternberger and Sternberger, 1983).

The tissue was incubated overnight in neurofilament antibody
(mouse anti-neurofilament, Sternberger-Meyer, SMIBZ 1:250-
500 and SMIB3 1:500 PBS/2-10% fetal calf serum/1% triton X-
100 non-ionic detergent), rinsed three times with PBS/1%
triton X-100 and incubated in a secondary anti-mouse anti-

body conjugated to fluoroscein isothiocyanate (FITC) or

37

tetramethylrhodamine isothiocyanate (TRITC) (Cappell, 1:100
PBS/1% triton X-100) for 2 hours at room temperature. This
was followed by rinsing the tissue three times in PBS/1%
triton X-100 and twice in PBS. The sheets of myenteric
plexus and the pelvic plexus and colonic ganglia sections
were mounted/coverslipped with a PBS/glycerol/p-Phenylene-
diamine dihydrochloride buffer to retard the fading of
fluorescence (Johnson et al., 1981). This technique was also
combined with the anterograde neuroanatomical tracer wheat
germ agglutinin (WGA). The tissue was incubated overnight in
a moist chamber at 4°C with goat primary anti-sera to WGA
(1:200, Vector) and mouse monoclonal antibodies to neuro-
filament (SMI32 and SMI33, 1:500, Sternber-
ger-Meyer)(PBS/0.5-2.0 % Triton X-100/5 % fetal calf serum).
The tissue was then rinsed in PBS/0.5-2.0 % Triton X-100 and
incubated with biotinylated rabbit anti-goat (Vector). The
tissue was next rinsed with PBS/0.5-2.0 % Triton X-100 and
incubated with TRITC-conjugated avidin and FITC-conjugated
rabbit anti-mouse secondary antibody (1:100: PBS/0.5%
triton X-100) for 2 hours at room temperature (22-25°C). The
tissue was then rinsed in PBS and mounted in
PBS/glycerol/p-Phenylenediamine dihydrochloride buffer to
retard the fading of fluorescence (Johnson et al. '81)..
TRITC labeled primary afferent fibers and FITC labeled
myenteric neurons and processes were visualized by fluores-

cence microscopy.

38

Data Analysis

Microscopy. HRP labeled neurons and fibers were viewed under
bright field and fluorescent microscopy using magnifica-
tions between 100 and 1000 X. The thickness of the connec-
tive tissue layer containing the myenteric plexus frequently
precluded the use of bright field optics due to increased
incidence of stray light. It was determined that fluorescent
optics in the 420-490 nm range (cube H2, Leitz) offered not
only less optical interference, but also assisted in visual-
izing glutaraldehyde induced fluorescence. Glutaraldehyde
fixation causes all tissue to fluoresce. The intensity of
this fluorescence is optimal in the 420-490 nm range (green
or FITC). In the connective tissue layer containing the
myenteric plexus, the colonic fiber bundles, ganglia and
interganglionic fiber tracts have a greater density than
surrounding connective tissue layers, causing them to fluo-
resce bright green against a pale green/dark background.
This allowed for photomicrographs and camera lucida drawings
of both the black HRP labeled neurons and fibers as well as
the fluorescent ganglia and fiber tracts surrounding them.
This same phenomenon was observed in other sliced and whole-
mounted tissues fixed with glutaraldehyde. Attempts to
counterstain HRP-labeled tissue frequently resulted in loss
of HRP reaction product. The glutaraldehyde induced back-
ground fluorescence was used to visualize soma and tissue
layers that were not readily discernible under bright field

optics in non-counterstained tissues. Fluorescently labeled

39

neurons and fibers were visualized by fluorescent optics
(Leitz) using magnifications between 100 and 1000X. Perma-
nent records were made by both 35 mm photomicroscopy and

camera-lucida drawings (Leitz).

Cell Counts. When neuronal soma were counted in sectioned
tissue (freezing microtome), the raw numbers were corrected
for double counting by the method of Abercombie (Abercombie,
1946). The formula is as follows: corrected number = raw
number X (slice thickness/mean soma diameter + slice thick-
ness). The mean soma diameter was computed as being one
half of the sum of the long and short axis. Abercombie's
formula yields a number that accounts for soma size and
slice thickness in correcting for potential double counting

in sliced tissue.

40

CHAPTER 1

DISTRIBUTION OF PRIMARY SACRAL AFFERENT FIBERS
TO LARGE INTESTINE, URINARY BLADDER, URETHRA,EXTERNAL
URETHRAL SPHINCTER, AND PREVERTEBRAL GANGLIA
Visceral afferent fibers emanating from sensory neu-
rons in sacral dorsal root ganglia project to pelvic vis-
cera via the pelvic nerve (Langley and Anderson, 1895; 1896;
de Groat and Krier, 1978). Sacral afferent fibers transmit
mechanoreceptive and nociceptive stimuli from the colon
(Floyd and Lawrenson, 1979; Floyd et al., 1976; Haupt et
al., 1983; Bahns et al. 1985: Janig and Koltzenberg, 1990).
Some of the afferent fibers in pelvic nerve and colonic
fiber bundles mediate spinal reflexes during defecation (de
Groat and Krier, 1978).

Sacral afferent fibers also transmit mechanoreceptive
and noiceceptive stimuli from the urinary bladder (de Groat
et al., 1981: Habler et al., 1990: Iggo, 1955: Floyd et al.,
1976; Bahns et al., 1987) and urethra (Bahns et al., 1987).
Afferent fibers emanating from sacral dorsal root ganglia
mediate sacral parasympathetic reflexes to urinary bladder
and urethra (micturition)(de Groat and Booth, 1984: de Groat
and Kawatani, 1989).

The central distribution of sacral visceral affer-
ents from the pelvic nerve to sacral dorsal root ganglia,
dorsal horn spinal gray matter and to the sacral parasympa-

thetic nucleus is well known for several species (Nadelhaft

41

et al., 1980: Nadelhaft et al., 1983; Nadelhaft and Booth,
1984: Mawe et al., 1986: Morgan et al., 1981; Vera and
Nadelhaft, 1990). The subpopulation of sacral afferent
neurons that provide innervation to mid and proximal colon
through colonic fiber bundles is unknown.

The distribution of sacral afferent fibers in guinea-
pig and cat colon external muscle layers and myenteric
plexus ganglia has been described using degeneration tech-
niques (Schofield, 1962). Degeneration techniques have also
been used to describe the distribution of sacral afferent
fibers to the cat urinary bladder (Uemura et al., 1973). The
distribution of sacral afferent fibers to the large intes-
tine, urinary bladder, urethra and prevertebral ganglia has
not been studied using anterograde tracing techniques.

This chapter identifies the origin of neurons in sacral
dorsal root ganglia that project their peripheral processes
to mid colon using techniques of retrograde axonal trans-
port. It also identifies the distribution of sacral visceral
afferent fibers in the pelvic and pudendal nerves, large
intestine, urinary bladder, urethra, external urethral
sphincter and prevertebral ganglia using techniques of
anterograde axonal transport of WGA-HRP and unconjugated

WGA.
MATERIALS AND METHODS
Retrograde Tracing

Experiments were performed on 7 anesthetized cats. A

42

midline abdominal incision was made 5 to 6 colonic fiber
bundles were labeled with 4% fast blue at the level of the
inferior mesenteric artery (Fig. 3). The abdomen was closed
and after 90 to 100 hours, animals were reanesthetized and
perfused.

Sacral dorsal root ganglia ( Sl - S3), lumbar dorsal
root ganglia (L7) and coccygeal dorsal root ganglia (Cgl)
were removed bilaterally and sectioned in the horizontal
axis at 40 um. Fast blue-containing neurons were visualized
by fluorescence microscopy at 100 to 400x. In each horizon-
tal section, counts of neurons were corrected for double

counting (Abercombie, 1946).

Anterograde Tracing

Experiments were performed on 12 anesthetized cats. A
surgical incision was made exposing the lumbar and sacral
vertebral segments (LS-S3). A 2% solution (distilled de-
ionized water) of wheat germ agglutinin (WGA, Vector),
either unconjugated or conjugated to horseradish peroxidase
(WGA-HRP, Sigma) was pressure injected into sacral dorsal
root ganglia (81-83) either bilaterally (n = 9) or unilater-
ally (n = 3) via a glass micropipette (tip diameter 18-25
pm) (Fig. 1). In all three unilateral experiments the ipsi-
lateral sacral ventral roots (81-53) were chronically sec-
tioned (96-120 hours) to eliminate labeling of preganglionic
efferent fibers. In 2 of the unilateral experiments, either
the ipsilateral pelvic or pudendal nerve was sectioned (96-

120 hours) to determine the relative contributions of the

43

Colonic Fiber Bundles

   
 

Pelvic Colonic
Nerves Ganzlia

Aborad

Pelvic L———) Inf h—‘

Mesenteric
Artery

Brim

Figure 3. Drawing of cat colon with attached pelvic nerves,
colonic ganglia and colonic fiber bundles. Fast blue was
placed on central cut end (end continuous with pelvic plexus
and pelvic nerve) of transected colonic fiber bundles at
level of inferior mesenteric artery (arrows). Long arrow
indicates long axis of longitudinal muscle fibers and colo"
nic fiber bundles. Short arrow indicates long axis of circus

lar muscle fibers. Horizontal bar is equal to 10 mm.

44

pudendal and pelvic nerves, respectively. After injection,
the muscle layers and skin were closed. Following time
periods ranging between 72 to 120 hours, animals were rea-

nesthetized and perfused.

Large Intestine. The mucosa/submucosa layer was cut into one
square centimeter segments and 3 to 4 segments were examined
from each intestinal region. For circular muscle layer,
successive circular muscle strips (lengths, 2 cm to 5 cm in
long axis of the circular muscle fibers; widths, 1 mm to 5
mm) were dissected 1.0 cm orad from the pelvic brim to 6 cm
orad to the inferior mesenteric artery (Fig. 3). For the
longitudinal muscle layer, successive segments (lengths,
2 cm to 5 cm in long axis of the longitudinal muscle fib-
ers: widths, 2 cm to 5 cm) were prepared. Sheets of myen-
teric plexus were cut into rectangular sections (2 to 3 cm
lengths in long axis of the longitudinal muscle, 4 to 5 cm
in long axis of circular muscle fibers). For proximal
colon, whole mounts sections of myenteric plexus and colonic
fiber bundles, adherent to the longitudinal muscle layer
were prepared. The myenteric plexus, circular and longitu-
dinal muscle strips were processed free floating and whole

mounted.

Urethra. The long axis of the urethra (3.5 to 5.5 cm),
extending from the bladder neck to the external urethral

sphincter (Fig. 4), was divided into equal thirds by length

45

and defined as proximal (continuous with bladder neck),
distal (continuous with the external meatus) and mid (region
between proximal and distal). For each region of urethra
(proximal, mid and distal), a 2mm length was cut in cross-
section and an 6-9 mm length was cut in horizontal section.
The external urethral sphincter was removed and sliced in
horizontal section.

Urinary Bladder. The urinary bladder was first hemi-sected
then surgically divided into neck, trigone, dorsal wall,
ventral wall and dome (apex). The bladder neck was cut in
horizontal sections. For the remaining bladder, one or two 1

2

cm sections of bladder wall (with mucosa attached) were

removed from each region and sliced in the horizontal plane.

Prevertebral Ganglia. The colonic ganglia, urinary bladder
ganglia and pelvic plexus ganglia were sliced in the hori-
zontal plane. Tissue was processed for TMB/DAB (WGA-HRP) or
immunocytochemistry (unconjugated WGA) and visualized by
fluorescence microscopy using magnifications between 100 and

400 X.

46

Sacral Spinal Cord

 

Donna mun;omutu.

      

Pelvic larva

Anna.

Inumn' mun

 

 

thou"! Vuuwai

Figure 4. Drawing of cat sacral spinal cord, sacral dorsal root
ganglia, pelvic and pudendal nerves, urinary bladder and urethra.
Wheat-germ agglutinin conjugated to horseradish peroxidase was
pressure injected into sacral dorsal root ganglia (81-83) and
transported anterogradely via pelvic and pudendal nerves to

urinary bladder, urethra, external urethral sphincter and prever-

tebral ganglia.

47

RESULTS
Retrograde Labeling of Neurons in Sacral and Lumbar Dorsal
Root Ganglia

The majority of colonic fiber bundles emanating from
both sides of the pelvic plexus were cut at the mid—colon
region (level of the inferior mesenteric artery) (Fig. 3).
When central cut ends of colonic fiber bundles were placed
in a fast blue solution, labeled cell bodies were found in
sacral dorsal root ganglia (Figure 5). The somas had a
blank nucleus, no processes and were filled with fluores-
cent blue granules (mean soma diameter i SE; 29.1 i 0.6 pm,
n = 423).
Sacral Dorsal Root Ganglia. The distribution of neurons in
sacral dorsal root ganglia which project to at least mid
colon of four cats is shown in Table 1. Neurons were dis-
tributed bilaterally in sacral dorsal root ganglia (81'
S3)(Fig..6) with a peak concentration in 82 dorsal root
ganglia. The range of neurons per animal was 660 to 1193
(mean i SE = 946 i 114). The mean percentage of neurons (:
SE) in bilateral sacral dorsal root ganglia was the follow-
ing: 81; 2.5 i 1.3 : 82, 77.3 i 9.8: S3, 20.1 i 10.3. No
neurons were detected in the first coccygeal dorsal root

ganglia.

48

 

Figure 5. Fluorescent photomicrographs of neurons in sacral
dorsal root ganglion (DRG). Colonic fiber bundles were
retrogradely labeled at the level of the IMA with fast blue.
A: Low magnification view of neurons in sacral ($2) DRG
section. B,C: Higher magnification views of DRG neurons.
Somas were partially filled with a blue granular reaction

product. Horizontal bar equals 80 pm in A; 40 pm in B and C.

49

Table 1
Distribution of Sacral Dorsal Root Ganglia Neurons

That Project to Colonic Fiber Bundles

 

LEFT s1 RIGHT s1 LEFT s2 RIGHT $2 LEFT s3 RIGHT s3

1) 3 3 688 145 31 13
2) 12 1 327 273 325 255
3) 24 17 264 320 17 18
4) 15 8 405 391 57 170

 

(n - 4, corrected for double counting by method of Abercombe)

Anterograde Labeling of Soma in Sacral Dorsal Root Ganglia
Sacral dorsal root ganglia (31'83) were pressure
injected bilaterally with wheatgerm agglutinin conjugated to
horseradish peroxidase (WGA-HRP). Dorsal root ganglia were

nearly completely filled with dense reaction product, ob-
scuring individual soma and their processes. In peripheral
regions of the ganglia labeled somas were identified.
Anterograde Labeling of Sacral Spinal Cord

Sacral afferent fibers were anterogradely labeled
bilaterally in the three sacral segments (Si—s3) of the
spinal cord. The greatest density of reaction product
occurred in Lissauer's tract (LT), lateral collateral

pathway (LCP), medial collateral pathway (MCP), and the

50

Figure 6 Distribution of neurons in sacral dorsal
root ganglia that project peripheral afferent processes
to colonic fiber bundles to at least mid colon re-
gion. Colonic fiber bundles were retrogradely labeled
with fast blue. Ordinate; percentage of total neurons
labeled. Abscissa: lumbar (L7), sacral (81-83) and
coccygeal (Cgl) dorsal root ganglia. A-D: each panel
represents data obtained from one animal. Open bars
represent left ganglia, hatched bars represent right
ganglia. Numbers above horizontal bars indicate total

number of cells.

51

Figure 6

 

 

 

 

 

 

A ”1 “F! B ”l
”4 ‘0‘
JO:
”1
”1
go. 145
:01
.3!
o 3 3 n2 0

Percentage of total cells labeled

10‘

.l

 

 

 

 

 

Segmental Level

52

 

 

dorsal gray commissure (DCM). The dorsal horn (Laminae I -
VI) also contained a diffuse reaction product giving the
granules a random appearance. When ipsilateral sacral
dorsal root ganglia (31'83) were injected with WGA-HRP,
afferent fibers and reaction product were identified only in
the ipsilateral LT, LCP, MCP, and dorsal horn (Fig. 7).

For the majority of experiments (7 of 9), no somas
were detected bilaterally in the intermediolateral regions
or ventral horns of the spinal cord. In two experiments
some neuronal soma were identified in the ventral horn of
the spinal cord.

Sacral Afferent fibers: Peripheral Destinations

PREVERTEBRAL GANGLIA

Colonic Ganglia. Nonvaricose sacral afferent fibers were de-
tected bilaterally in pelvic nerve (Fig. 8A) and sacral parasym-
pathetic colonic ganglia (Fig. 8 B-I)(18 of 18 ganglia, n= 9).
When sacral dorsal root ganglia (SI-S3) were pressure injected
ipsilaterally, afferent fibers were detected only ipsilaterally
(n = 2). No afferent fibers were detected in colonic ganglia
after the pelvic nerve was sectioned (n = 1). For the majority
of ganglia, fibers were detected in bundles along the borders and
center of the ganglia (Fig. 8 B,C). Small bundles and dispersed
fibers branched from large fiber bundles in proximity to neuronal

somas in 100% of colonic ganglia (Figs. 8 D-I: 17G,H).

53

Figure 7. Brightfield photomicrograph of cross
section of sacral spinal cord (S2 spinal cord segment).
Ipsilateral sacral dorsal root ganglia (51 - S3) were
pressure injected with wheat germ agglutinin conjugated
to horseradish peroxidase. Ipsilateral sacral spinal
cord (51 - S3) contained sacral afferent fibers in
Lissauers' tract (LT), lateral collateral pathway (LCP)
and medial collateral pathway (MCP). Diffuse reaction
product was also observed in the dorsal gray commissure
(DCM). No labeled somas were detected. Horizontal bar

is equal to 500 pm.

54

Figure 7

 

55

Figure 8. Photomicrographs and camera lucida drawing
of sacral afferent fibers in pelvic nerve (A) and
sacral parasympathetic colonic ganglia ( B-I). A:
horizontal section (40 pm) of pelvic nerve with affer-
ent fibers. Horizontal section of colonic ganglion (40
um) showing afferent fibers in fiber tracts at border
(B) and center (C) of ganglion. D-I: afferent fibers
in proximity to soma of ganglion cells. Drawings in
E, G and I correspond, respectively to photomicrographs

D, F and H. Horizontal bars in A-I are equal to 50 um.

56

Figure 8

 

57

Pelvic plexus ganglia. In 6 animals, 61 ganglia were removed
bilaterally from the pelvic plexus. Nonvaricose sacral
afferent fibers were detected in 60 of 61 ganglia. A similar
bilateral distribution of sacral afferent fibers was detect-
ed in experiments where sacral dorsal root ganglia (51'53)

were pressure injected ipsilaterally.

Similar to colonic ganglia, afferent fibers were de-
tected within fiber bundles along the border of ganglia and
in fiber bundles which course through ganglia (Fig. 19A).
100% (60/60) of pelvic plexus ganglia had sacral afferent
fibers in proximity to neuronal somas (Figs. 19 B-G: 23E-F).
No sacral afferent fibers were detected in pelvic plexus

ganglia after the pelvic nerve was sectioned (n = 1).

Large Intestine

Colonic Fiber Bundles. When sacral dorsal root ganglia (Sl-
S3) were pressure injected bilaterally with wheatgerm agglu-
tinin conjugated to horseradish peroxidase (WGA-HRP) or
unconjugated wheatgerm agglutinin (WGA), afferent fibers
were detected bilaterally within pelvic nerves (Figs. 8A:
19A), colonic branches of the pelvic nerve, colonic fiber
bundles and branch points of colonic fiber bundles (Figs.
10; 13A,B: 16A). When sacral dorsal root ganglia (81-83)
were pressure injected ipsilaterally sacral afferent fibers

were detected only ipsilaterally. No sacral afferent fibers

58

Figure 9. Photomicrographs and camera lucida drawings
of sacral afferent fibers in pelvic plexus ganglia.
Sacral dorsal root ganglia (81-53) were injected with
wheat germ agglutinin conjugated to horseradish peroxi-
dase (A-E) or unconjugated wheat germ agglutinin (F-G).
A: horizontal section of pelvic plexus ganglion (40 pm)
showing afferent fibers in fiber tracts at border
(white arrows) and center (black arrows) of ganglion.
B-E: afferent fibers in proximity to soma of ganglion
cells. Drawings in D and E correspond, respectively
to photomicrographs B and C. F: fluorescent photomi-
crographs of neurofilament labeled somas and processes.
G: same frame showing afferent fibers (arrows) through
center of ganglion in proximity to ganglion cells.

Horizontal bars in A-G are equal to 30 um.

59

 

Figure 9

 

60

were detected in colonic fiber bundles after the pelvic
nerve was sectioned (n = 1).

Afferent fibers were detected continuously or discon-
tinuously in 4 to 8 colonic fiber bundles emanating from
bilateral pelvic plexuses (n = 5). Their density varied
within colonic fiber bundles. In different regions of the
colon sacral afferent fibers either completely (Fig. 10A,B)
or partially filled (Fig. 13A) the colonic fiber bundle.

Afferent fibers in colonic fiber bundles could be
traced orad from the distal colon at the level of the pelvic
brim to the mid and proximal colon at maximal orad axial
distances ranging from 2.6 to 11.3 cm. The maximal axial
orad distances where sacral afferent fibers were detected

within colonic fiber bundles are shown in Figure 11.

Rectal Fiber Bundles. The distribution of sacral afferent
fibers to the rectum and anal canal was also studied (n =
3). The rectum/anal canal region of the cat contains four
to six axially orientated fiber bundles which can be traced
one cm orad to the external anal sphincter (Langley and
Anderson, 1895: Christensen et al., 1984). There are also
branches of rectal fiber bundles that enter the myenteric
plexus.

Afferent fibers were visualized intermittently in
rectal fiber bundles and their branch points (Fig. 12A-C).
Some were visualized in rectal fiber bundles one cm orad to

the striated musculature of the external anal sphincter.

61

Figure 10. Camera lucida drawing and photomicrographs
of sacral afferent fibers in a branched colonic fiber
bundle. Wheat germ agglutinin conjugated to horseradish
peroxidase was pressure injected into sacral dorsal
root ganglia (SI—S3) bilaterally. A: camera lucida
drawings of afferent fibers in branched colonic fiber
bundle. B; bright-field photomicrograph of afferent
fibers in panel A. C: fluorescent photomicrograph of
afferent fibers leaving colonic fiber bundle to enter
plane of myenteric plexus. Horizontal bar in A is
equal to 2 mm. Horizontal bars in B and C are equal

to 50 um.

62

 

Figure 10

 

63

Figure 11. Distribution of sacral afferent fibers in
colonic fiber bundles. Abscissa: experiment number.
Ordinate: maximal orad distances sacral afferent fibers
were visualized in colonic fiber bundles. Orad distanc-
es are expressed in millimeters from the pelvic brim.
Vertical bars represent individual colonic fiber

bundles.

64

 

 

' 3W Ill/H W! Hill! Imfl i i

EEEEEEEEEEEEEEEE

 

Figure 12. A-C: Fluorescent photomicrographs of sacral
afferent fibers in fiber bundles located in myenteric
plexus of rectum. A; afferent fibers in fiber bundle
1.5 cm orad of external anal sphincter. B: afferent
fibers in fiber bundle 1 cm aborad from pelvic brim. C:
afferent fibers in branching fiber bundle 2 cm aborad
from pelvic brim. D: Fluorescent photomicrograph of
sacral afferent fiber in interganglionic fiber tract of
distal colon submucosal plexus. Horizontal bar in D

.equal to 50 pm, and refers also to A-C.

66

 

Figure 12

 

67

The maximal axial aborad distances where afferent fibers
were detected ranged from 3 to 4 cm. No afferent fibers were
detected in rectal fiber bundles after the pelvic nerve was
sectioned (n = 1).

Colonic-Rectal Myenteric Plexus. After leaving branch points
of colonic or rectal fiber bundles, sacral afferent fibers
projected bilaterally to interganglionic fiber tracts and
predominantly to myenteric ganglia that were adjacent to
colonic fiber bundles. When sacral dorsal root ganglia
(51-53) were pressure injected ipsilaterally, afferent
fibers were detected only ipsilaterally in colonic fiber
bundles and in myenteric ganglia that were adjacent to
colonic fiber bundles. No afferent fibers were detected in
myenteric plexus ganglia after the pelvic nerve was sec-
tioned (n = 1).

In the colon, afferent fibers projected either lateral
or orad to a fiber bundle or its branch point (Figs. 13A,B;
15A). No afferent fibers were detected projecting aborad
to interganglionic fiber tracts or to myenteric ganglia.

For the rectum-anal canal, afferent fibers projected either

lateral or aborad to a fiber bundle or its branch point. No
afferent fibers were detected projecting orad to intergan-

glionic fiber tracts or to myenteric ganglia.

In colon myenteric plexus, some afferent fibers re-
mained as small bundles within interganglionic fiber tracts
and coursed in an axial orad direction through 1 to 2 gan-

glia (Fig. 13B) or numerous ganglia (Fig. 14). Others left

68

the afferent bundles and ended as fibers within the ganglia
(Figs. 13B: 14: 15: 16).

Both varicose and nonvaricose sacral afferent fibers
were detected as isolated fibers and arborizations within
myenteric ganglia of the distal, mid and proximal colon
(Figs.13B: 14: 15: 16A-E) or rectum (Fig. 16F,G). Varicose
fibers were visualized in 12 percent of myenteric ganglia
(38 of 313, n = 3) that contained afferent fibers. The
remainder of ganglia contained nonvaricose fibers (275 of
313). The patterns of arborization included some nonvari-
cose fibers with ‘basket—like' arrangements (Fig. 16 D,E).
Other arborization patterns showed varicose afferent fibers
(Fig. lSB-D).

Figure 17 A—F shows sacral afferent fibers and neurons
in myenteric plexus ganglia. Some neurons were in proximity
to branching afferent fibers (Fig. 17A,B). In other gan-
glia, branching afferent fibers (Fig 17D, arrows) followed
the contour of myenteric neurons (Fig. 17C, arrow)._ Other
sacral afferent fibers (Fig. 17F, arrow) were in proximity
to myenteric neurons located at the border of the ganglion

(Fig. 17E).

69

Figure 13. A; Camera lucida drawings of sacral affer-
ent fibers in a bifurcating colonic fiber bundle with
adjacent myenteric plexus ganglia. B; higher magnifi-
cation of segment of colonic fiber bundle with connec-
tion to myenteric plexus ganglia. Afferent fibers were
identified intermittently through colonic fiber
bundle up to a maximal orad axial distance of 6.5 cm
from distal colon. Note, afferent fibers enter
branch point of colonic fiber bundle to enter one
myenteric ganglion. Afferent fibers course through
interganglionic fiber tract to enter and exit another
myenteric ganglion. C: afferent fiber coursed through
myenteric ganglion and interganglionic fiber tract to
circular smooth muscle. Horizontal bar in A is equal
to 5 mm. Horizontal bars in B and C are equal to 400

um and 200 pm, respectively.

70

Figure 13

 

 

 

 

 

71

Figure 14. A: camera lucida drawing of orad projec-
tions of sacral afferent fibers in interganglionic
fiber tracts and ganglia of mid colon myenteric plexus.
Afferent fiber bundles projected orad continuously for
1.5 cm through interganglionic fiber tracts and
ganglia. Asterisks indicate the location of myenteric
ganglia. The most orad projection of afferent fibers
left plane of myenteric plexus to branch onto circu-
lar smooth muscle (arrow). B; higher magnification of
afferent fibers within one myenteric plexus ganglion
indicated in panel A. Note, afferent fibers enter
ganglion as one bundle, branch into an arborization of
fibers and exit as two bundles. C,D: higher magnifica-
tion views of afferent fibers in myenteric plexus gan-
glia and interganglionic fiber tracts indicated in
panel A. Horizontal bar equals 1mm for panel A, 200 pm

for panels B-D.

72

Figure 14

                  

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73

Figure 15. Camera lucida drawings (A, C) and fluores-
cent photomicrograph (B) of sacral afferent fibers in a
myenteric plexus ganglion. A: myenteric plexus gan-
glion with afferent fibers. B: fluorescent photomicro-
graph of afferent fibers in panel A. C; Higher magnifi-
cation of afferent fibers in A and B. Note, multiple
intermittent increases in fiber diameter, suggestive of
varicosities. Horizontal bar equals 200 pm for A, 50

pm for B and 30 pm for C.

74

 

Figure 15

 

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it

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75

Figure 16. Camera lucida drawings of sacral afferent
fibers in ganglia of myenteric plexus in distal colon
(A,B,C), mid colon (D,E) and rectum (F,G). A: afferent
fibers project in an orad axial direction in colonic
fiber bundle and exit colonic fiber bundle at branch
point oriented nearly perpendicular to colonic fiber
bundle to enter myenteric ganglia. B, D and F: location
of afferent fibers within other myenteric plexus gan-
glia. C, E and G; higher magnification of afferent
fibers in B,D and F, respectively. Arborizations of
afferent fibers were unique to each ganglion. Horizon-
tal bars in B and D are equal to 200 um. Horizontal
bars in A, C, E and F are equal to 100 um. G; horizon-

tal bar is equal to 50 um.

76

Figure 16

 

 

77

Figure 17. Fluorescent photomicrographs of sacral
afferent fibers in proximity to neurons in myenteric
(A-F) and colonic ganglia (G-H). Sacral dorsal root
ganglia (S1 -S3) were pressure injected with unconju-
gated WGA. Neuronal somas were visualized by indirect
immunofluorescence of neurofilament (A, C, E and G).
Corresponding photographs of same frame (B, D, F and H,
respectively) show afferent fibers. G-H: afferent
fibers (H, open arrows) course through colonic ganglion
in proximity to neuronal somas (G). Note, large affer-
ent fiber ending (H, closed arrow) in proximity to

neuronal soma (G). Bar equals 50 um and refers to A-H.

78

 

Figure 17

 

79

Table 2 summarizes the distribution of neurons and
ganglia in the myenteric plexus of the colon (n = 6). It
also shows the distribution of ganglia that contained sacral
afferent fiber bundles, arborizations and fibers (n = 3).
Data are expressed as the mean (1 SE) ganglia per square
centimeter and mean number of neurons (1 SE) per ganglia for

each colonic region.

Table 2. Profiles of Neurons and Ganglia in Myenteric Plexus
of Cat Colon

 

 

Parameter Distal Mid Proximal
Colon Colon Colon
Neurons/ganglion 66.7 1 9 1 71.6 1 4.6 81.1 1 13.2
Ganglia/cm2 13.1 1 2 3 11.4 1 1.3 10.3 1 0.7
*Ganglia/cmz 2.5 1 0 7 1.8 1 0.2 0.9 1 0.5

 

Values are mean 15E
* Ganglia containing sacral afferent fibers

The mean percentage of myenteric ganglia containing
sacral afferent fibers decreased from distal colon to
proximal colon. The mean percentage of ganglia (1 SE) was
the following: proximal colon, 8.6 1 5.1; mid-colon, 15.5 1

1.9: distal colon, 20.2 1 4.5.

30

Colon Circular Muscle. Sacral afferent fibers were traced
from the myenteric plexus to the circular muscle layer (Fig.
13C; 14A). They were also detected on circular muscle
strips obtained from the entire circumference of proximal,
mid and distal colon regions (n = 9). A similar distribu-
tion of afferent fibers occurred in experiments where
sacral dorsal root ganglia (51-83) were pressure injected
ipsilaterally.

Afferent fibers were routinely visualized as inter-
rupted small fiber bundles and nonvaricose fibers. They
were orientated parallel to the long axis of the circular
muscle fibers over the entire length (range 2 to 5 cm) of
individual circular muscle strips (Fig. 18 D,E). Some of
the fibers had a club-like appearance (Fig. 18E).

Dense arborizations of afferent fibers were also de-
tected on circular muscle (Fig. 18 A-C). Some fibers were
perpendicular or oblique to the long axis of circular muscle
fibers (Fig. 15 A). Others coursed parallel to the long
axis of circular muscle fibers ( Fig. 15A,B). No afferent
fibers were detected on smooth muscle fibers of the extrin-

sic longitudinal muscle layer.

81

Figure 18. Camera lucida drawing and photomicrographs

of sacral afferent fibers on circular muscle of mid
colon (A), distal colon ( B-D) and proximal colon (E).
A-C: arborization patterns of afferent fibers on circu-
lar muscle. Double arrow indicates long axis of circu-
lar muscle fibers. Note, arrangement of fibers orient-
ed perpendicular, oblique and parallel to long axis of
circular muscle fibers. D; afferent fiber parallel to
long axis of circular muscle fibers. E: afferent fiber
with club-like appearance. Horizontal bar in A is
equal to 100 um. Horizontal bars in B-E are equal to

50 pm.

82

 

Figure

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18

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83

Submucosal Plexus. Few sacral afferent fibers were detected
in colon submucosal plexus. Non-varicose fibers were iden-
tified exclusively within interganglionic fiber tracts (n =

5)(Fig. 120). No afferent fibers were detected in mucosa.

URINARY BLADDER GANGLIA, URINARY BLADDER AND URETHRA

Pelvic Nerve, Pudendal Nerve and Urinary bladder ganglia.
When sacral dorsal root ganglia (S1 -S3) were injected
bilaterally with wheatgerm agglutinin conjugated to horse-
radish peroxidase (WGA-HRP), non-varicose sacral afferent
fibers were detected bilaterally within pelvic and pudendal
nerves (Fig. 19A-B) and in 37 of 42 urinary bladder ganglia
(n = 4). They were in bundles along the borders of ganglia
(Fig. 19C), through central regions (Fig. l9C-F) and in
proximity to neuronal soma (97% (36/37)) (Fig. 19C-F). No
fibers were detected in ganglia after chronic ipsilateral
section of pelvic nerve (n = 1). This indicates that affer-
ent fibers pass through pelvic nerve and pelvic plexus to

enter urinary bladder ganglia.

84

Figure 19. Fluorescent photomicrographs and camera
lucida drawings of sacral afferent fibers (arrows) in
40 um horizontal sections of pelvic nerve (A), pudendal
nerve (B), urinary bladder ganglion (C-D) and pelvic
ganglion (E-F). Note afferent fibers in proximity to
neuron in bladder (C: arrows) and pelvic plexus gan-
glion (E; arrows). Horizontal bar equals 50 pm in A, B,

D and F and 60 pm in C and E.

85

 

 

Figure 19

 

86

Urinary Bladder. There was a paucity of sacral afferent
fibers detected in urinary bladder. Table 3 shows their
regional distribution in tissues of urinary bladder. There
was an abscence of fibers in mucosa and submucosa of bladder
wall and a paucity of fibers in submucosa of bladder neck
(Fig. 20A). Afferent fibers occured in extrinsic muscle
layers of all bladder regions (Fig. 20A-E). No fibers were
detected in urinary bladder after chronic ipsilateral sec-
tion of pelvic nerve (n = 1).

TABLE 3

RELATIVE DISTRIBUTION OF SACRAL AFFERENT FIBERS
IKIIERINAJNTINUKDDERL

 

 

Urinary Serosa Extrinsic Submucosa Mucosa
Bladder Muscle

Apex + + - -

(n-5*)

Dorsal + + - -

Wall (n86)

Ventral + + - —

Wall (ne6)

Trigone + ++ - -

(0‘6)

Neck + ++ + -

(0'5)

("n" indicates number of animals with afferent fibers, "+"
indicates afferent fibers, "+1" indicates relative increase in
number of afferent fibers, "-" indicates no afferent fibers

detected, "*" n I 2 of 5 for serosa: 3 of 5 for extrinsic muscle
layers)

87

Figure 20. A; Fluorescent photomicrograph of sacral
afferent fibers on urinary bladder smooth muscle
(curved arrows) and submucosa (straight arrows) of
bladder neck. Afferent fibers on smooth muscle of
bladder neck (B), dorsal wall (C), trigone (D) and
ventral wall (E). Horizontal bar in E equals 50 um and

also refers to A-D.

88

 

 

Figure 20

 

89

Urethra. Sacral afferent fibers were detected in all re-
gions and layers (Fig. 21A) of the urethra. Their distribu-
tion is shown in table 4. The mean percentage of histologi-

cal sections containing afferent fibers was 69.8%.

Table 4

DISTRIBUTION OF SACRAL AFFERENT FIBERS IN CAT URETHRA

 

 

PROXIMAL MID DISTAL
Total number of 106 144 122
sections
Horizontal 74 114 80
sections
Cross sections 32 30 42
Number of sections 79 93 101
with afferent fibers
Percentage of 64.1 1 16.1 68.4 1 6.2 76.8 1 5.4

total (mean 1 SE)

 

(Horizontal and cross sections a 40 um, n - 4)

Afferent fiber distribution in urethral tissue is
shown in table 5. For urethral serosa, afferent fibers

occured in bundles (n = 4)(Fig. 218). In longitudinal and

90

circular smooth muscle layers, afferent fibers occurred as
bundles and fibers oriented parallel to the long axis smooth
muscle fibers (Fig. 21 C-E). In striated muscle of distal
urethra they were oriented parallel, oblique and perpendicu-
lar to the long axis of muscle fibers (n = 5)(Fig. 22A-D).
In external urethral sphincter, they were parallel to the
long axis of striated muscle fibers (n = 4)(Fig. 23E-F).
Sacral afferent fibers were relatively more numerous in
urethral submucosa. They occurred as fibers (Fig. 23D),
fiber bundles (Fig.23C and E) and arborizations (Fig. 23F)(n
= 5). Some fiber bundles entered the mucosa (Fig. 23A). In
mucosa, afferent fibers occurred as short fragments along

the entire length of the urethra (Fig. 23A-C)(n = 5).

91

'Table 5

DISTRIBUTION

OF SACRAL AFFERENT FIBERS IN TISSUE LAYERS

OF CAT URETHRA

 

 

TISSUE PROXIMAL MID DISTAL
Serosa 46.4 1 9.7 36.9 1 13.4 22.5 1 0.2
(32) (32) (23)
Longitudinal 51.1 1 17 3 45.9 1 7.1 19.8 1 2.4
muscle (33) (42) (18)
Circular 61.4 1 15 4 59.9 1 5.7 15.9 1 4.8
muscle (38) (58) (20)
Submucosa 84.4 1 5 3 88.0 1 7.0 85.7 1 5.6
(64) (87) (93)
Mucosa 41.7 1 21.0 48.1 1 4.6 50.5 1 6.1
(24) (47) (56)
Striated ** ** 46.8 1 8.9
muscle (43)

(n = 4, mean percentage 1 S.E. of labeled sections,
(#) number of sections labeled, ** absence of striated muscle

fibers)

92

Figure 21. Camera lucida drawings and photomicrographs
of sacral afferent fibers in horizontal sections (40
um) of proximal (D,E), mid (A,B) and distal (C) ureth-
ra. A; camera lucida drawing of mid urethra. Serosa
(S), serosal bundle (SB), longitudinal smooth muscle
(LM), circular smooth muscle (CM), submucosa (SM),
mucosa (M) and lumen (L). B: fluorescent photomicro-
graph of fibers (arrows) in serosal fiber bundle. C:
fluorescent photomicrograph of fibers (arrows) parallel
to long axis of longitudinal smooth muscle fibers D:
camera lucida drawing of fibers (arrows) parallel to
long axis of circular smooth muscle fibers. E: bright-
field photomicrograph of panel D. Horizontal bar equals
400 pm in A, 75 pm in B and E, 50 pm in C and 220 pm in

D.

93

 

Figure 21

 

94

Figure 22. Camera lucida drawings (A,C) and photomicro-
graphs (B, D, E, F) of sacral afferent fibers on
striated muscle fibers of distal urethra (A-D) and
external urethral sphincter (E-F). A, C: camera lucida
drawings of afferent fibers (arrows) oriented parallel,
perpendicular and oblique to long axis of striated
muscle fibers. B, D: brightfield photomicrographs
corresponding to panels A and C, respectively. E,F:
fluorescent photomicrographs of afferent fibers (ar-
rows) oriented parallel to long axis of striated muscle
fibers. Panels A-D and F obtained from preparations
with pelvic and pudendal nerves intact. Panel E ob-
tained from preparation where ipsilateral pelvic nerve
was chronically sectioned. Horizontal bar equals 60 pm

in A and C and 75 pm in B, D, E and F.

95

 

 

Figure 22

 

96

IPSILATERAL INJECTIONS

The concept that sacral afferent fibers cross the
midline in the periphery was tested. Sacral dorsal root
ganglia (81 -83) were injected unilaterally with WGA-HRP (n
= 3). Afferent fibers were detected in ipsilateral pelvic
(Fig. 19A) and pudendal (Fig. 198) nerves but were absent in
contralateral pelvic and pudendal nerves. They occurred
bilaterally in urinary bladder (n = 2), urethra (n = 3) and
external urethral sphincter (n = 2). They were detected in
11 of 17 pelvic plexus ganglia (64.7%)(8 of 8 ipsilateral, 3
of 9 contralateral)(n = 2) and in 17 of 19 urinary bladder
ganglia (89.5%)(10 of 10 ipsilateral, 7 of 9 contralateral)
(n = 2).

The distribution of sacral afferent fibers that pass
through pelvic and pudendal nerves was determined. Sacral
dorsal root ganglia (51 -S3) were injected ipsilaterally
with WGA-HRP and either the ipsilateral pelvic or pudendal

nerve was chronically sectioned.

Pelvic Nerve afferents. When sacral dorsal root ganglia
(S1 -S3) were pressure injected unilaterally with WGA-HRP

and the ipsilateral pudendal nerve was chronically

97

Figure 23. Fluorescent photomicrographs of sacral
afferent fibers in proximal (D), mid (A,C) and distal
(B, E and F) urethra mucosa (M) and submucosa (SM). A,
B: Afferent fibers (arrows) in horzontal section (40
pm) cross section (40 pm), respectively. C: fibers in
submucosa (straight arrows) branch within mucosa
(curved arrows). D-F: fibers (arrows) in submucosa. For
panels A, C, D and E both pelvic and pudendal nerves
are intact. Panel F, afferent fibers in urethra submu-
cosa. Ipsilateral pudendal nerve was chronically sec-
tioned. Panel B, mucosal afferent fibers. Ipsilateral
pelvic nerve was chronically sectioned. Horizontal bar

in F equals 50 um and also refers to A-E.

98

 

 

Figure 23

 

99

sectioned, afferent fibers were detected in ipsilateral
pelvic nerve but were absent in ipsilateral pudendal nerve
and the contralateral pelvic and pudendal nerves (n = 1).
They occurred bilaterally in urinary bladder, proximal and
mid urethra and in submucosa of distal urethra (approximate-
ly 5 mm rostral to EUS)(Fig. 23F). Afferent fibers were
detected in 3 of 7 pelvic plexus ganglia (3/3 ipsilateral
and 0/4 contralateral) and 7 of 8 urinary bladder ganglia
(4/4 ipsilateral and 3/4 contralateral). These observations
show that sacral afferent fibers pass through ipsilateral
pelvic nerve to bilaterally innervate urinary bladder,

urethra and urinary bladder ganglia.

Pudendal Nerve Afferents. When the ipsilateral pelvic
nerve was chronically sectioned, afferent fibers were de-
tected in ipsilateral pudendal nerve (Fig. 198), but were
absent in ipsilateral pelvic and contralateral pudendal and
pelvic nerves (n = 1). Fibers occurred bilaterally in EUS
(Fig. 22E) and distal urethra (serosa, striated and smooth
muscle layers, submucosa and mucosa) (Fig. 238) and in
submucosa of mid and proximal urethra. Afferent fibers were

not detected in urinary bladder, pelvic plexus ganglia or

100

bladder ganglia. These results demonstrate that sacral
afferent fibers pass through ipsilateral pudendal nerve to

innervate effector structures in urethra and EUS.

DISCUSSION

This study shows that some neurons in sacral dorsal
root ganglia project afferent fibers to pelvic and pudendal
nerves, large intestine, urinary bladder, urethra and
external urethral sphincter. Afferent fibers also occur in
parasympathetic colonic ganglia, urinary bladder ganglia and
pelvic plexus ganglia.

Some neurons in bilateral sacral dorsal root ganglia
project their peripheral sensory processes to at least mid
colon through pelvic nerves and colonic fiber bundles. Axial
orad and aborad sacral afferent fiber projections to effec-
tor structures in the large intestine occur over relatively
long distances through colonic and rectal fiber bundles,
respectively. In the colon, maximal axial orad distances
from the pelvic brim ranged between 2.6 and 11.3 cm. For
rectum, maximal axial aborad distances from the pelvic brim

ranged between 3.0 and 4.0 cm. Neurons in ipsilateral

101

sacral dorsal root ganglia projected their peripheral sen-
sory processes only to the ipsilateral pelvic nerve, para—
sympathetic colonic ganglia and colonic and rectal fiber
bundles.

Sacral afferent fibers projected orad through colonic
fiber bundles to enter a minority of myenteric plexus gan-
glia of the colon through branch points of colonic fiber
bundles and interganglionic fiber tracts. The majority of
afferent fibers were located in myenteric ganglia and inter-
ganglionic fiber tracts that were adjacent to and obliquely
orad to branch points of colonic fiber bundles. Some fibers
could be traced continuously from branch points of colonic
fiber bundles to one adjacent myenteric ganglion. Others
could be identified continuously in two or three myenteric
ganglia and in their interganglionic fiber tracts. In rare
instances, afferent fibers formed a continuous axial orad
projection in interganglionic fiber tracts and to
numerous adjacent myenteric ganglia. These results indicate
that sacral afferent fibers emanating from neurons in sacral
dorsal root ganglia project orad through colonic fiber
bundles and through myenteric ganglia and interganglionic

fiber tracts.

102

The data show that sacral afferent fibers were detected
in approximately 15% of myenteric ganglia but in nearly 100%
of colonic, pelvic plexus and bladder ganglia. The data
also show sacral afferent fibers present in myenteric gan-
glia that are adjacent to colonic fiber bundles and connect-
ed to them by their branch points. It appears unlikely that
the differences in afferent labeling we report in colonic-
pelvic plexus and myenteric ganglia are related to the
anterograde tracers WGA-HRP and unconjugated WGA. For rat
stomach and duodenum, vagal efferents were detected in a
minority of myenteric ganglia when Eggseolus vulgari§ leu-
coagglutin was used as the anterograde tracer (Kirschgessner
and Gershon, 1989), but a majority of ganglia appeared to
contain both vagal afferent and efferent fibers when Dil was
used as the anterograde tracer (Berthould et al., 1990).

Sacral afferent fibers which coursed through the myen-
teric plexus were traced to colonic circular muscle. Within
circular muscle, there were two morphological arrangements.
One had several intramuscular afferent fiber bundles and
dispersed fibers that ran parallel to the long axis of the

smooth muscle fibers. Afferent fibers were observed on both

103

mucosal and myenteric borders of circular muscle and were
present around the entire circumference of the colon. The
other occurred as a dense arborization pattern with afferent
fiber bundles and dispersed fibers either parallel, oblique
or perpendicular to the long axis of circular muscle fibers.
It is likely that the circumferentially oriented affer-
ent fibers identified anatomically in the present study
within circular muscle correspond to afferents activated by
passive distension of cat distal colon (Janig and Koltzen-
burg, 1991). In their electrophysiological study, a total of
59 afferent units were defined in sacral dorsal roots (S2)
that projected to pelvic nerve. Of these, 61% responded only
to passive distension of the distal colon, while 39% re-
sponded only to mechanical stimulation of the anal mucosa.
Colonic afferents, responding only to passive disten-
sion, were identified as thin myelinated and non-myelinated
fibers with a median conduction velocity of 3.2 m/s (Janig
and Koltzenburg, 1991). Myelinated afferent fibers responded
only transiently to colonic distension (median conduction
velocity 8.0 m/s), and were classified as phasic. Unmyeli-
nated afferent fibers discharged throughout the distension

(median conduction velocity 1.7 m/s), and were classified as

104

tonic. For tonic units, increases in colonic distension
resulted in increased discharge frequencies.

Other afferent fibers identified anatomically within
circular muscle may correspond to another population of non-
myelinated sacral afferent units. Approximately 95%
(202/213) of non-myelinated sacral (S2) afferent units
projecting to the pelvic nerve did not respond to mechanical
stimulation of the colon or anal canal. These silent C-
fibers may only be active when sensitized by inflamation
(Janig and Koltzenburg, 1991: Habler et al., 1990).

In circular muscle, sacral afferent fibers with a
perpendicular and oblique orientation may course through the
circular layer to innervate mucosa or submucosa. Vagal
afferent fibers which penetrate circular muscle to enter
submucosal and mucosa regions have been described for cat
esophagus (Clerc and Condamin, 1987).

Sacral afferent fibers were not traced from myenteric
plexus to the longitudinal muscle layer nor were they vis-
ualized as intramuscular fiber bundles. Also, a paucity of
sacral afferent fibers was detected in interganglionic fiber
tracts within the submucosal plexus and none were detected

in submucosal ganglia and colonic mucosa. An absence of

105

vagal afferent fibers in longitudinal muscle has been re-
ported for rabbit stomach and duodenum (Sato and Kayono,
1987). For rat stomach and duodenum, few vagal afferent
fibers were visualized in submucosa and none in mucosa
(Berthoud et al., 1990).

For most regions of urinary bladder (dome, trigone and
dorsal, ventral and lateral walls), a paucity of afferent
fibers was detected in smooth muscle layers and they were
absent in mucosa and submucosa. For urinary bladder neck,
afferent fibers were detected in the smooth muscle layer of
bladder wall and submucosa.

The abscence or paucity of sacral afferent fibers to
mucosa and submucosa, respectively, of colon and urinary
bladder and the paucity detected in smooth muscle layers of
urinary bladder is not understood. It may be related to
difficulty in visualizing a paucity of small diameter fibers
with the anterograde transport of WGA-HRP or unconjugated
HRP. This appears unlikely because afferent fibers were
detected at a greater density in urethral mucosa/submucosa
and were visualized in the proximal colon at long conduction
distances (14 to 15 cm).

Our data show that sacral visceral afferent fibers

106

contained within colonic fiber bundles at the level of the
mid colon enter the sacral spinal cord primarily through
bilateral second (77%) and third (20%) sacral dorsal roots.
An average of 945 sacral sensory neurons with small soma
diameters were visualized. These results confirm earlier
data that show that a majority of cat afferent fibers in
bilateral pelvic nerves (Morgan et al. 1981) and distal
colon (Kawatani et al., 1985: de Groat, 1987) have their
cell bodies in the $2 and 82 - S3 dorsal root ganglia,
respectively.

The total number of neurons located in sacral dorsal
root ganglia which provide sensory innervation to effector
structures in the large intestine is yet to be determined.
However, it is possible to estimate this number. The total
number of neurons in bilateral pelvic nerves of the cat is
approximately 7300 (Morgan et al., 1981). We speculate that
one-third provide innervation to the large intestine (ap-
proximately 2400 neurons) and that the remainder innervate
urinary bladder, urethra and sexual organs. If this assump-
tion is correct, one thousand neurons innervate the distal
colon (Kawatani et al., 1985: de Groat, 1987) and nearly

another thousand innervate mid and proximal colon (present

107

study). The remainder, we surmise, innervate the rectum-
anal canal.

Within parasympathetic colonic ganglia, pelvic plexus
ganglia, urinary bladder ganglia and ganglia of the myen-
teric plexus, sacral afferent fibers were nonvaricose and
visualized as a linear arrangement of reaction product
granules of uniform diameter. Some fibers in myenteric
plexus ganglia were varicose. Afferent fibers were detected
as bundles to ganglion borders and central regions and as
dispersed fibers in proximity to neuronal soma. 100% of
labeled parasympathetic colonic ganglia and pelvic plexus
ganglia and 97% of labeled urinary bladder ganglia had
sacral afferent fibers in proximity to neuronal soma. Also,
in myenteric plexus there were dense arborization patterns
present. We speculate that some may be mechanoreceptors or
collaterals of sacral afferents which mediate axon reflex-
es. A similar anatomical proposal has been made for vagal
afferent fibers in cat esophagus (Rodrigo et al., 1982),
rabbit stomach and duodenum (Sato and Kayono, 1987) and rat
stomach, small and large intestine (Berthoud et al., 1990).
Data from recent electron microscopic studies suggest that

vagal afferent fibers form synaptic contacts with neurons in

108

rat esophageal myenteric plexus (Neuhuber, 1987).

Afferent collaterals may release neurotransmitter
substances on enteric neurons to modulate local circuits
(Delbro and Lissander, 1980: Delbro et al., 1981: de Groat
et al., 1987). Electrical stimulation of afferent fibers in
sympathetic (lumbar splanchnic nerves) and parasympathetic
(vagus nerve) nerve trunks cause non-nicotinic, non-adren-
ergic contractions of cat colon and stomach, respectively
(Delbro et al., 1983; Fandriks and Delbro, 1985; Delbro and
Lisander, 1980; Fandriks et al., 1985; Delbro et. al.,
1981). These contractions were unaffected by ganglionic and
adrenergic antagonists, but were reduced by substance-P
antagonists and atropine. This suggests that the contra-
ctions involved substance-P containing afferent fibers and
cholinergic fibers. It is likely that afferent fibers de-
tected in proximity to neurons in myenteric plexus and
prevertebral ganglia in the present study may be afferent
collaterals involved in local reflex mechanisms.

The data show that some neurons in sacral dorsal root
ganglia also project peripheral afferent fibers to the
urinary bladder, urethra and external urethral sphincter

through pelvic and pudendal nerves. Neurons in unilateral

109

sacral dorsal root ganglia project peripheral sensory fibers
to the ipsilateral pelvic and pudendal nerves, but bilater-
ally to urinary bladder, urethra and external urethral
sphincter.

The data show that sacral afferent fibers were sparse
to all regions of the urinary bladder. They passed through
the pelvic nerve and were located predominantly on smooth
muscle fibers. A paucity of sacral afferent fibers were
previously detected when degeneration (Uemura et al., 1973)
and retrograde tracing (Downie et al., 1984) techniques were
used. Degenerating terminals comprised less than 5% of
total axon terminals detected in cat urinary bladder (Uemura
et al., 1973). In a retrograde tracing study of cat urinary
bladder and urethra, a small number of neurons was located
in sacral dorsal root ganglia. The mean number of afferent
neurons was: urethra, 81; detrussor smooth muscle, 159; and
bladder base, 178 (Downie et al., 1984). This represents
less than 6% of the estimated 7300 sacral dorsal root gan-
glion neurons that project peripheral processes to cat
pelvic nerve (Morgan et al., 1981).

Afferent fibers in the present study may correpond to

mechanoreceptive afferent fibers described in cat urinary

110

bladder. Mechanoreceptive afferent fibers are both myelinat-
ed (mean conduction velocity = 10 m/s) and non-myelinated
(mean conduction velocity = 1.0 m/s) fibers (Habler et al.,
1990; de Groat et al., 1981: Bahns et al., 1987). Myelinat-
ed fibers are predominantly small diameter fibers with low
thresholds and firing frequencies that rise with increasing
intraluminal pressure during passive distension and isovolu—
metric active contractions (Bahns et al., 1987; Iggo, 1955).

Approximately 98% of non-myelinated afferent fibers in
pelvic nerve did not respond to innocuous or noxious in-
creases in intravesicular pressure (Habler et al., 1990).
However, when the bladder was inflamed by injection of
mustard oil, previously silent non-myelinated fibers ini-
tiated an action potential discharge to both the irritant
and increases in intravesicular pressure. This suggests that
these silent non-myelinated fibers may be chemoreceptors and
mechanoreceptors (Habler et al., 1990). They may also be
involved in mediating the sensation of pain (Habler et al.,
1990).

Sacral afferent fibers to urethra occurred in serosal
fiber bundles, external smooth muscle layers, submucosa,

mucosa and striated muscle fibers of distal urethra and

111

external urethral sphincter. Afferent fibers which projected
through pelvic nerves were detected in all effector struc-
tures of mid and proximal urethra and submucosa of distal
urethra (approximately 5 mm rostral to external urethral
sphincter). Afferent fibers which projected through pudendal
nerves innervate effector structures of distal urethra and
submucosa of distal, mid and proximal urethra. This repre-
sents a partial overlap of innervation by sacral afferent
fibers via pelvic and pudendal nerves to the distal, mid and
proximal urethra.

For external smooth muscle layers, sacral afferent
fibers were detected as small bundles and fibers oriented in
the long axis of smooth muscle fibers. The mean percentage
of labeled sections was approximately 39% and 46%, respec-
tively, for longitudinal and circular smooth muscle.

The greatest number of afferent fibers in urethra
occurred in the submucosa. Approximately 86% of labeled
urethral sections had afferent fibers in submucosa. They
were detected as dispersed fibers and arborizations in all
regions of submucosa. In addition, approximately 47% of
labeled urethral sections had afferent fibers in all regions

of mucosa.

112

The distribution of sacral afferent fibers to urethral
mucosa/submucosa may have functional significance. Electro-
physiological studies demonstrate that urethral afferent
units in sacral dorsal roots respond to mechanical stimula-
tion of the urethral mucosa, but do not respond to passive
distension or active contraction of cat urinary bladder
(Bahns et al., 1987). These afferents may be responsible
for the sensations of impending micturition and urinary
flow attributed to human urethra (Nathan, 1956).

Afferent fibers to urinary bladder and urethra may also
be thermoreceptors. When cold water was introduced into cat
urinary bladder or urethra, a reflex contraction of urinary
bladder occurred (Fall et al., 1990). The bladder to bladder
cooling reflex was unaffected by acute sectioning of hypo-
gastric nerves but abolished by acute sectioning of pelvic
nerves. The urethra to bladder cooling reflex was abolished
by sectioning pudendal nerves. It is possible that sacral
afferent fibers, identified anatomically in urinary bladder
and urethra in the present study, may be thermoreceptors
involved in reflex contraction of urinary bladder.

The present study confirms that sacral afferent fibers

113

cross the midline to innervate contralateral urinary blad-
der, urethra and pelvic plexus and bladder ganglia. Afferent
fibers which project through pudendal nerve to external
urethral sphincter and urethra also show peripheral crosso-
ver. In cat urinary bladder, one third of degenerating
sacral afferent fibers cross the midline to innervate the
contralateral side (Uemura et al., 1973: Uemura et al.,
1975). In retrograde tracing studies, neurons in sacral
dorsal root ganglia project afferent fibers across the
midline in cat urethra and rat and cat urinary bladder
(Downie et al., 1984: Applebaum et al., 1980). The function-
al significance of this bilateral innervation is unknown.
This report describes the distribution of sacral affer-
ent fibers through pelvic nerves to the large intestine,
colonic ganglia, pelvic ganglia, urinary bladder ganglia,
urinary bladder, urethra and external urethral sphincter. It
also describes the distribution of sacral afferent fibers
through pudendal nerves to urethra and external urethral
sphincter. A dense innervation of afferent fibers occurred
in urethral submucosa, colon circular muscle, and a small
percentage of myenteric ganglia. These afferent fibers may

correspond in part to the substance-P (SP), calcitonin gene-

114

related peptide (CGRP) and vasoactive intestinal polypeptide
(VIP) containing somas previously demonstrated with immuno-
histochemical techniques in sacral dorsal root ganglia (de
Groat et al., 1983; Kawatani et al. 1985; de Groat, 1987,

Keast and de Groat, 1992).

115

CHAPTER 2

INTRINSIC NEURAL PATHWAYS OF THE COLON

Introduction

Colonic fiber bundles originate within the pelvic plexus
and pass to the colon, rectum and anal canal. Their distri-
bution has been reported for humans (Kumar and Phillips,
1989) and experimental animals (Christensen and Rick, 1985:
Christensen and Rick, 1987: Christensen et al., 1983; Chris-
tensen et al., 1984). For the cat, usually 6 to 8 fiber
bundles ascend orad beneath the serosal surface of the
distal colon and between the external muscle layers (de
Groat and Krier, 1976: de Groat and Krier, 1978: Krier and
Hartman, 1984: Langley and Anderson, 1895). They project
considerable distances to the mid and proximal regions of
the colon. Branches of the pelvic plexus also descend as
rectal fiber bundles to the rectum and anal canal. There
are branch points of both myelinated and non myelinated

colonic fiber bundles in relation to neurons and cell pro-

116

cesses of the myenteric plexus (Christensen and Rick, 1987).

Colonic fiber bundles contain sacral parasympathetic as
well as lumbar and sacral sympathetic pathways. The sacral
parasympathetic pathway regulates colonic (de Groat and
Krier, 1976: de Groat and Krier, 1978; Fukai and Fukada,
1984) and rectal motility. It originates in the sacral
autonomic nucleus and extramural colonic ganglia and pro-
jects through the pelvic nerve and colonic fiber bundles (de
Groat and Krier, 1976; de Groat and Krier, 1978; Kennedy and
Krier, 1987: Krier and Hartman, 1984). The sacral sympa-
thetic pathway originates from neurons in sacral paraverte-
bral ganglia and projects through the pelvic nerve and
colonic fiber bundles (Kuo et al., 1984). The lumbar sympa-
thetic pathway originates from neurons in the inferior
mesenteric ganglia and distributes fibers through the hypo-
gastric nerve and colonic fiber bundles (de Groat and Krier,
1976).

Although the anatomical organization of these fiber
bundles is well known for several species, it remains to be
determined whether they function as another intrinsic con-
nection for myenteric neurons. Some neurons in the distal

colon may project orad to mid-colon whereas others in the

117

proximal and mid-colon may project aborad to mid and distal
colon, respectively. This dissertation identifies the origin
of myenteric fibers in colonic fiber bundles using tech-

niques for retrograde axonal transport.

METHODS

Retrograde tracing techniques

Experiments were performed on 10 anesthetized cats. A
midline abdominal incision was made exposing the large
intestine and five millimeter segments of 2 to 6 colonic
fiber bundles were dissected from the serosal surface of
the mid colon at the level of the inferior mesenteric
artery or at the distal colon 1.0 cm orad to the pelvic brim
(Fig.2). Colonic fiber bundles were cut at the level of the
inferior mesenteric artery and the central or peripheral
ends were labeled to determine orad and aborad projections,
respectively, of myenteric neurons. Colonic fiber bundles
were cut at the level of the distal colon, 1 cm orad to
pelvic brim and peripheral ends were labeled to determine

the aborad projections of myenteric neurons. Central and

118

peripheral cut ends of colonic fiber bundles were placed
into small chambers containing 10 pl of 15% horseradish
peroxidase (HRP) solution (Sigma Type VI)(6 s) or 10 pl of
8% fast blue (Sigma)(4 5). After a 90 minute incubation
period, the nerves were removed from the chambers and the
abdomen was closed. After 36 to 96 hours, animals were re-
anesthetized and perfused. HRP-labeled neurons were visual-
ized by incubating the tissue with TMB/DAB. Whole—mount
sections were viewed under light- and dark-field microscopy
using magnifications between 100 and 400 X. Fast blue-la-
beled neurons were visualized by fluorescence microscopy at
100 and 250x. For each whole-mount section, the distribution
of myenteric neurons in relation to colonic fiber bundles
was determined by counting neurons in the axis of both
circular and longitudinal muscle fiber layers. Neurons were
counted in 700 pm wide serial rows oriented in the axis of

longitudinal muscle fibers.

RESULTS

Ascending axial projections of myenteric neurons

When central cut ends of 2 or 3 colonic fiber bundles

were placed in an HRP solution, cell bodies and

119

processes were traced to the myenteric plexus of the mid and
distal colon. As shown in Figure 24, somas were grouped
within ganglia and their cell processes extended within
interganglionic fiber tracts to branch points of colonic
fiber bundles (Fig.24B). No somas or processes were labeled
when axonal transport was interrupted by damaging colonic
fiber bundles.

The axial distribution (distribution along the axis of
colonic fiber bundles) of myenteric neurons is shown in
Figure 25. Cell bodies were in the mid and distal colon at
axial distances from 10 to 45 mm (maximum distance: 34 to 45
mm: mean 1 SE, 42 1 1; n = 5). The total labeled cells were
from 82 to 393 (mean 1 SE, 192 1 48; n = 5). These data
show that myenteric neurons project orad through colonic

fiber bundles.

120

Figure 24. A: Camera lucida drawing of colonic fiber
bundle with fiber connections to ganglia in the myen-
teric plexus of distal colon. Stippled areas in ganglia
show location of myenteric neurons retrogradely labeled
with HRP. B: fluorescent photomicrograph of colonic
fiber bundle with branch point indicated in A. C:
Bright-field photomicrograph of myenteric neurons in
ganglion indicated in A. Double arrow indicates long
axis of longitudinal muscle fibers. Horizontal bar in A
equals 1mm. Horizontal bar in C equals 100 pm and also

refers to B.

121

Figure 24

COLON! C

FIBER
/ arson.

 

 

122

Descending axial projections of myenteric neurons

Other colonic fiber bundles were cut 1 cm orad to the
pelvic brim (Figure 2). When peripheral cut ends of 4 or 5
colonic fiber bundles were placed in an HRP solution, cell
bodies and processes were detected in the myenteric plexus
of the mid and distal colon (Fig.26A, 268) at axial distanc-
es ranging from 10 to 59 mm (maximum: 49 to 59 mm; mean 1
SE, 54.0 1 1.5). The total labeled cells were from 147 to
375 (mean 1 SE, 220 1 33: n = 4). In one animal, the periph-
eral cut ends of three colonic fiber bundles in distal colon
(1 cm orad to pelvic brim) and two in mid-colon (1 cm orad
to inferior mesenteric artery) were placed in a fast blue
solution. There were cell bodies in the distal and mid—
colon (Fig. 26C) and mid and proximal colon (Fig. 26D) at
maximal distances of 49 and 44 mm, respectively. The total
number of neurons labeled was 194 and 175, respectively.
These data show that a population of myenteric neurons in
both proximal and mid colon project aborad through colonic

fiber bundles.

123

Figure 25. Distribution of myenteric neurons in distal
colon in relation to axial distance from labeled colo-
nic fiber bundles. Bars in each panel show consecutive
wholemount segments. Each panel shows data from a

different animal. Axial distance reported is limited to

 

labeling site of colonic fiber bundle.

124

 

Figure 25

 

mcotaoz Co tonEaz

istance (mm)

D

 

125

Figure 26. Distance of myenteric neurons in mid-distal
colon (A-C) and mid-proximal colon (D) in relation to
axial distance from labeled colonic fiber bundles. Bars
in each panel show consecutive wholemount segments.

A,B: Neurons labeled using retrograde transport of HRP.
C,D: Neurons labeled using retrograde transport of Fast
Blue. A and B show data from different animals. C and D
show data from the same animal. Axial distance reported

is limited to labeling site of colonic fiber bundle.

126

 

Figure 26

 

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127

Lateral distribution of myenteric neurons

Myenteric neurons not only project fibers along the axis
of colonic fiber bundles, they also lie at different dis-
tances beside them (axis of circular muscle fibers, Figure
28). For neurons in mid and distal colon which project orad
(Fig.27 A-C), 65% (577 of 756) were within 2.8 mm of a
labeled colonic fiber bundle. The remainder (179 of 756)
were between 2.8 mm and 7.0 mm of a labeled colonic fiber
bundle. For neurons in mid and distal colon which project
aborad (Fig.27D-F), 85% (329 of 385) were within 2.8 mm of a
labeled colonic fiber bundle. The remainder (56 of 385)
were between 2.8 and 4.9 mm of a labeled colonic fiber
bundle. These data indicate that the majority of neurons
which project axons in either an orad or aborad axial direc-
tion have soma which lie within 2.8 mm of a colonic fiber

bundle.

Morphological characteristics of myenteric neurons

Neurons were divided into two types using morphological

criteria. One type had a rough somal surface (mean soma

128

Figure 27. Distribution of myenteric neurons lateral
to labeled colonic fiber bundles. Center vertical line
is position of colonic fiber bundle. Wholemount conse-
cutive sections obtained at axial distances from label-
ing site. A, 10-21mm; B, 21-32mm: C, 32-43mm; D, 10-
24mm: E, 24-45mm: F, 45-70mm. A-C, histograms obtained
from four animals; D-F, histograms obtained from one

animal.

129

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Distance (mm)

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diameter 1 SE, 40.5 1 0.6 pm) and was filled with a brown,
agranular reaction product. It also had multiple short,
broad processes which radiated from the soma and it had one
long process (Figure 28). It was similar to myenteric cells
in guinea-pig ileum (Furness et al., 1988) and opossum
rectum (Christensen, 1988)(Dogiel type I cells; Dogiel,
1899). The other type had a smooth oval or rectangular soma
(mean soma diameter 1 SE, 26.4 1 0.3 pm) which was filled
with a black granular reaction product. It had a blank
nucleus and few if any short fine processes (Figure 29). It
was similar to neuronal somas in the myenteric plexus
(Dogiel type II or Dogiel type III)(Dogiel, 1899: Furness et
al., 1988: Furness et al., 1985). Because of the long
axonal projection to colonic fiber bundles, these cells are
considered to be Dogiel type III. For neurons which project
orad, 62% (369 of 598) were classified as Dogiel type III,
the remainder (229 of 598) were classified as Dogiel type I.
For neurons which project aborad 70% (653 of 939) were
Dogiel type III and the remainder (286 of 939) were Dogiel

type I.

131

Figure 28. Bright-field photomicrograph of Dogiel type
I neurons which show short, broad processes and one
long process. Long processes of neurons in B, C, D, F
and G project to colonic fiber bundle at distances
ranging from 2.9 to 6.0 mm. Long process of neuron in A
was traced to an interganglionic fiber tract at a
distance of 1.1 mm. Long process of neuron in E re-
mained within ganglion at a distance of 0.14 mm. Bar

equals 50 pm.

132

 

 

 

Figure 28

 

.
T.
l" a

133

Figure 29. A-L: Bright-field photomicrographs of
Dogiel type III neurons which show a nucleus and a soma
partially filled with black granular reaction product.

Bar equals 50 pm.

134

 

 

 

 

Figure 29

 

135

DISCUSSION

This study shows that some myenteric neurons in the cat
colon project processes orad or aborad through colonic fiber
bundles from at least 5 to 59 mm. Neurons in the distal
colon project to mid-colon at the level of the inferior
mesenteric artery and those in proximal colon and mid-colon
extend to mid-colon and distal colon, respectively. Neurons
were in ganglia adjacent to colonic fiber bundles, about 73%
of which were within 2.8 mm and none was beyond 7.7 mm.

The inference is that colonic fiber bundles are another
intrinsic fiber connection for neurons in the myenteric
plexus.

The distribution of neurons that project through colonic
fiber bundles described here may have functional signifi-
cance. They may innervate other myenteric or submucosal
neurons, mucosa-submucosa or extrinsic smooth muscle layers
and blood vessels to regulate reflexly trans-epithelial
fluid transport, blood flow and muscle contractions.

Several reflexes have been shown to be dependent on the

integrity of colonic fiber bundles (Fukai and Fukada, 1984).

136

Colonic contractions induced by rectal distension (recto-
colonic) or mechanical stimulation of the anus (ano-colonic)
remain after ligation of the myenteric plexus/muscularis
externa, but are abolished by colonic fiber bundle transec-
tion (Fukai and Fukada, 1984). Ascending contractions and
descending relaxations demonstrated in response to a bolus
in an isolated guinea-pig distal colon were abolished by
interuption of the myenteric plexus, presumably also in-
terupting the colonic fiber bundles in the same plane (Costa
and Furness, 1976: Christensen et al., 1984). This would
suggest that myenteric neurons projecting processes orad and
aborad over long axial distances in colonic fiber bundles
may play a role in the propagation of reflex contraction
and/or relaxation of intestinal smooth muscle.

Myenteric neurons which project orad and aborad through
colonic fiber bundles are Dogiel type I and type III.
Dogiel type I (Christensen, 1988; Furness et al., 1988) had
a rough somal surface, few if any short, broad dendrites and
one long process which often extended to a branch point of
an adjacent colonic fiber bundle. The reaction product in
this cell type was an agranular brown precipitate that

filled the soma and obscured the nucleus. Dogiel type III

137

neurons had a smooth somal surface and few if any fine
dendrites. They had one long process which projected
through colonic fiber bundles. The reaction product in this
cell type was a black granular precipitate that left the
nucleus blank.

Neurons in the cat colon may be similar to myenteric
(Bornstein et al., 1984; Furness et al., 1988: Furness et
al., 1985; Iyer et al., 1988) and submucosal neurons (Fur-
ness et al., 1985) of guinea-pig small intestine and taenia
coli (Furness et al., 1981). Dogiel type I cells in the
present study may be primarily "8" type (cholinergic, fast
EPSP's which discharge repetitively during depolarizing
current pulses; Hirst et al., 1974: Iyer et al., 1988;
Tamura and Wood, 1989) and are perhaps immunoreactive for
enkephalin (Bornstein et al., 1984: Krier and Hartman,
1984), 5-hydroxytryptamine (Costa et al., 1982) or VIP
(Furness et al., 1981: Katayama et al., 1986).

Neurons similar to Dogiel I morphology and immunoreac-
tive for VIP (Furness et al., 1981) projected axons to the
external longitudinal smooth muscle layer of guinea-pig
taenia coli. VIP immunoreactive neurons also project pro-

cesses aborad 2 to 10 mm to other myenteric neurons and up

138

to 15mm aborad to submucoal neurons.

Neurons similar in morphology to Dogiel I and immunor-
eactive for enkephalin were also "S" type neurons, recieved
a cholinergic input from other enteric neurons and were
predicted to project to the circular muscle layer and orally
to other myenteric ganglia (Bornstein et al., 1984; Furness
and Costa, 1980). These results suggest that some Dogiel
type I cells in the small intestine which are immunoreactive
for enkephalins and VIP may have a motor function.

Dogiel type III neurons identified in the present study
compare to those in guinea pig taenia coli that are immunor-
eactive for VIP (Furness et al., 1981). VIP has been sug-
gested to play a role in descending smooth muscle relaxation
associated with peristalsis (Farhrenkrug et al., 1978:
Furness and Costa, 1979). VIP may also play a role in intes-
tinal vasodilation (Costa et al., 1980: Fahrenkrug et al.,
1978) and increased transepithelial transport of water and
electrolytes (Costa and Furness, 1983).

Cat Dogiel type III neurons are also comparable to a
population of Dogiel type III myenteric and submucosal
neurons in guinea pig small intestine that project to the

underlying mucosa (Furness et al., 1985). These neurons are

139

immunoreactive for several peptides, including CGRP. CGRP is
a potent vasodilator in rabbit skin (Brain et al., 1985) and
rat mesentery (Han et al., 1990a: Han et al., 1990b) and
inhibits smooth muscle contraction in mouse vas deferens
(Al-Kazwini et al., 1986). Some of the small intestinal
neurons in the myenteric and submucosal plexuses with Dogiel
type III morphology innervate mucosa, suggesting a secretory
and/or vasomotor function.

In summary, this chapter describes observations that
show that myenteric neurons in cat colon project axons both
orad and aborad over relatively long distances through
colonic fiber bundles and have cell bodies within only a few
millimeters of them. There might also be a similar organi-
zation of myenteric neurons in the stomach and esophagus for
which serosal fiber bundles have been described (Christensen

and Rick, 1985: Kumar and Phillips, 1989).

140

Chapter 3

Sacral Parasympathetic and Lumbar Sympathetic

Postganglionic Pathways

Introduction

Preganglionic fibers which originate from neurons in
sacral spinal cord provide input to neurons in parasympa-
thetic colonic ganglia (de Groat and Krier, 1976; Krier and
Hartman, 1984; Kennedy and Krier, 1987). Neurons in parasym-
pathetic colonic ganglia project postganglionic fibers
through colonic fiber bundles to distal colon (Krier and
Hartman, 1984). In the present study I used retrograde
tracing techniques to estimate the number of neurons in
parasympathetic colonic ganglia that project postganglionic
fibers to at least mid colon regions.

The distribution of neurons in lumbar sympathetic chain
ganglia (paravertebral) and inferior mesenteric ganglia
(prevertebral) that project processes to hypogastric and
lumbar colonic nerve trunks has been studied with retrograde

tracing technique (Baron et al., 1985, I: Baron et a1, 1985

141

II: Baron et al., 1985 III). The subpopulation of sympathet-
ic neurons within prevertebral and paravertebral ganglia
that project processes to colon via hypogastric nerves and
colonic fiber bundles is unknown. I determined a subpopula-
tion of sympathetic prevertebral and paravertebral neurons
that project processes through hypogastric nerves and colo-
nic fiber bundles to at least the mid-colon region utilizing

retrograde tracing techniques.

Methods

Retograde Labeling techniques

Experiments were performed on 10 anesthetized cats. A
midline abdominal incision was made exposing the large
intestine and 2 to 6 colonic fiber bundles were isolated and
labeled at the level of the inferior mesenteric artery
(arrows)(Fig. 30) with either 15% HRP or 4% fast blue.
Lumbar colonic nerves were chronically sectioned (n = 4).
This procedure interupted the postganglionic fiber projec-
tions of sympathetic neurons located in inferior mesenteric

ganglion (IMG), superior mesenteric ganglion (SMG), coeliac

142

ganglion, and lumbar sympathetic chain ganglia (Baron et
al., 1985 III). After 36 to 48 hours (HRP) or 72 to 96 hours
(fast blue), animals were reanesthetized and perfused. The
parasympathetic colonic ganglia, inferior mesenteric ganglia
(IMG), superior mesenteric ganglia (SMG), coeliac ganglia
and lumbar sympathetic chain ganglia were removed and pro-
cessed for HRP or fast blue as previously described. The IMG
has four lobes distributed around the inferior mesenteric
artery: left caudal (LC), right caudal (RC), left rostral
(LR), and right rostral (RR). HRP labeled sections were
viewed under light- and dark-field microscopy using magnifi-
cations between 100 and 400 X. Fast blue labeled neurons
were visualized by fluorescence microscopy at 100 and 250x.
In each horizontal section, counts of neurons were cor-
rected for double counting (Abercombie, 1946). The mean
soma diameter was computed as being one half of the sum of

the long and short axis.

Results
Parasympathetic colonic ganglia: retrograde axonal tracing
with HRP and Fast Blue

Neurons in colonic ganglia that project processes orad

143

within colonic fiber bundles to at least mid-colon regions
(range 70 - 90 mm: mean 1 SE = 83 1 3.7 mm: n = 9) were
retrogradely labeled. Horseradish peroxidase (HRP)(n = 5) or
fast blue (n = 4) was placed on the central cut ends of 2 to
6 colonic fiber bundles at mid-colon (level of the inferior
mesenteric artery)(Fig. 30). The retrogradely labeled soma
were completely filled with black granular reaction product
(HRP) with few or no processes (Fig. 31) or partially filled
with blue fluorescent granules (fast blue), with no process-
es and a blank nucleus. Their mean soma diameter (1 SE) was

32.4 1 0.2 pm (n

253 cells). The number of soma per
experiment (mean 1 SE = 233.1 1 49.0: n = 9) is shown in
table 6.
Sympathetic Prevertebral and Paravertebral Ganglia: Retro-
grade Tracing with Fast Blue

When fast blue was placed on central cut ends of 5 to 6
colonic fiber bundles (n=4) at mid-colon (level of inferior
mesenteric artery)(Fig. 30), somas were detected in inferior
mesenteric ganglion, superior mesenteric ganglion, coeliac
ganglion and lumbar sympathetic chain ganglia (Fig. 32).
They were partially filled with blue granular

reaction product with a blank nucleus and no processes.

144

Figure 30. Drawing of cat colon, pelvic nerve, colonic
ganglion (CG), paravertebral sympathetic chain ganglia,
inferior mesenteric ganglion (IMG), superior mesenteric
ganglion (SMG) and coeliac ganglion. Colonic fiber
bundles were labeled with either HRP or Fast Blue at

the level of the inferior mesenteric artery (arrows).

145

 

 

 

Figure 30

_ , L.
FELVIC New:
FELVIC prams
a:

PARAV’BTBRAL SYMPA‘I'HEI'IC GANGLIA

   

COELIAC
GANGLZON

 

 

146

Figure 31. Bright-field photomicrograph of horizontal
section of colonic ganglion at low (A) and high (B)
magnification. Colonic fiber bundles were labeled with
HRP solution. Somas are completely filled and short
processes are occasionally observed. Neurons projected
long processes orad through colonic fiber bundles at

least 60 to 90 mm. Bar in A = 80 pm, in B = 40 pm.

147

 

 

Figure 31

 

 

148

Table 6

Distribution of Neurons in Parasympathetic Colonic Ganglia that
Project to Colonic Fiber Bundles

 

Right Colonic Ganglia Left Colonic GangliaTotal

1) 31 24 55
2) 95 11 106
3) 132 51 183
4) 27 17 44
5) 168 133 301
6) 47 272 309
7) 152 106 258
8) 228 249 477
9) 104 252 356

 

(n = 9, corrected for double counting by method of Aber-
combe, 1 - 5 = HRP, 6 - 9 = fast blue).
Inferior mesenteric ganglion

Somas were detected in the four lobes of the inferior
mesenteric ganglion (IMG)(Fig. 32A). The mean soma diameter
(1 SE) was 29.2 1 1.0 pm (n = 252 cells). Their distribution
is shown in table 7. The mean (1 SE) number of neurons
retrogradely labeled per experiment was 2755 1 660 (n = 4).
Their distribution within individual lobes was: LC, 36.6%
(3473/9481): RC, 45% (4267/9481): LR, 9.2% (874/9481): and

RR, 9.1% (867/9481).

149

Figure 32. Fluorescent photomicrographs of horizontal
sections of IMG (A), SMG (B), coeliac ganglion (C) and
lumbar paravertebral chain ganglion (L3)(D). Colonic
fiber bundles were labeled with fast blue at the level
of the IMA. Horizontal bar in D equals 300 pm for A

and 50 pm for B-D.

150

 

Figure 32

 

151

Superior Mesenteric Ganglion

Somas were detected in the superior mesenteric ganglion
(Fig. 32B). The mean soma diameter (1 SE) was 31.3 1 1.6 pm
(n = 163 cells). Their distribution is shown in table 7. The
mean (1 SE) number of soma per experiment was 356 1 130.7
(n = 4).
Coeliac Ganglion

Soma were detected in both right and left lobes of the
coeliac ganglion. The mean soma diameter (1 SE) is 33.6 1
1.3 pm (n = 201 cells). Their distribution is shown in table
7.
The mean number of soma (1 SE) per animal was 1415 1 874.3
(n = 4). The distribution of soma per lobe was: left lobe,

52.5% (2976/5663); and right lobe, 47.4% (2687/5663).

Sympathetic Lumbar Chain Ganglia

Somas were detected in lumbar sympathetic chain ganglia
(Fig. 32D). The mean soma diameter (1 SE) was 25.9 1 1.1 pm
(n = 193 cells). Their distribution is shown in table 8. The
mean number (1 SE) of soma labeled per experiment was 779.8
1 313.9 (n = 4). The distribution of neurons in lumbar

sympathetic chain gangia is: L1, 13% (409/3119): L2, 29.7%

152

(926/3119): L3, 31.6% (986/3119); L4, 24.8% (773/3119); and

L5, 0.8% (25/3119).

TABLE 7

DISTRIBUTION OF NEURONS IN SYMPATHETIC PREVERTEBRAL GANGLIA

THAT PROJECT TO COLONIC FIBER BUNDLES

 

 

IMG SMG Coeliac Ganglia

Left Right Left Right Left Right
Caudal Caudal Rostral Rostral

l) 1142 1235 329 320 502 508 457

2) 922 526 200 309 593 320 408

3) 288 1021 109 121 O 3 l

4) 1121 1485 236 117 329 2145 1821

(n = 4: IMG, inferior mesenteric ganglia: SMG, superior mesenter-

ic ganglia: corrected for double counting by the method of Aber-
combie)

153

'lel. 8

DISTRIBUTION OF NEURONS IN LUMBAR SYMPATHETIC CHAIN GANGLIA

THAT PROJECT TO COLONIC FIBER BUNDLES

 

Left
L1

1) 0
2) 51
3) 0
4) 201

Right Left
L1 L2
18 0
74 71

0 0
65 397

Right

334

Left Right Left Right Left Right
L3 L3 L4 L4 L5 L5

279 270 268 313 - -

94 91 13 27 5 20
0 0 0 O - -

125 127 26 126 - -

 

(n = 4: corrected for double counting by the method of Abercom-

bie)

Discussion

The present study shows that some neurons in parasympa-

thetic prevertebral and sympathetic prevertebral and para-

vertebral ganglia project processes through colonic fiber

bundles to at least the mid colon region. Neurons in para-

sympathetic

colonic ganglia projected processes approx-

154

imately 83 mm orad in colonic fiber bundles. Neurons in
sympathetic prevertebral and paravertebral ganglia projected
processes over considerably longer distances to reach the
same mid colon regions via hypogastric nerves and colonic
fiber bundles.

For parasympathetic colonic gangia, approximately 233
neurons were retrogradely labeled per animal. This anatomi-
cal data supports a previous electrophysiological study
(Krier and Hartman, 1984). Electrical stimulation of colonic
fiber bundles elicited antidromic potentials in 62% of
neurons tested in parasympathetic colonic ganglia. Approx-
imately 95% of these postganglionic fibers were nonmyelinat-
ed (conduction velocity range: 0.4 to 2.0 m/s)(Krier and
Hartman, 1984). The previous electrophysiological study and
the present study show that neurons in parasympathetic
colonic ganglia project postganglionic processes through
colonic fiber bundles to at least distal colon and at least
mid colon, respectively.

My data shows that approximately 2,755 neurons in IMG
send processes through bilateral hypogastric nerves and
colonic fiber bundles to at least mid-colon. Approximately

82% of neurons were located in caudal lobes of the IMG. The

155

remainder were in rostral lobes. When the ipsilateral hypo-
gastric nerve was retrogradely labeled, approximately 16,150
neurons were detected primarily in the ipsilateral caudal
lobe of the IMG (Baron et al., 1985 I). After correction for
double counting (Abercombie, 1946), it was estimated that
approximately 21,544 neurons in IMG project processes
through bilateral hypogastric nerves. The subpopulation of
neurons in IMG that project processes to at least mid colon
through colonic fiber bundles (present study) represents
approximately 13% (2,755/21,544).

The data show that approximately 780 neurons in lumbar
sympathetic chain ganglia project processes through bilater-
al hypogastric nerves and colonic fiber bundles to at least
mid-colon. The majority of these neurons (86%) were in
bilateral LZ-L4 ganglia. The distribution of neurons in the
present study is similar to a previous study (Baron et al.,
1985 I) where the ipsilateral hypogastric nerve was retro-
gradely labeled. Approximately 350 neurons were detected in
lumbar sympathetic chain ganglia, predominantly in the
ipsilateral L4 ganglion (Baron et al., 1985 I). After cor-
rection for double counting (Abercombie, 1946), I estimated

that approximately 400 neurons in lumbar sympathetic chain

156

ganglia project processes through bilateral hypogastric
nerves. These anatomical studies suggest that almost twice
as many neurons (780/400) in lumbar sympathetic chain gan-
glia project processes to colonic fiber bundles compared to
those with processes in hypogastric nerves. This may be due
to a difference in the percentage of fibers labeled by fast
blue (present study) and HRP (previous study).

Our data shows that neurons in SMG (mean number 1 SE,
356 1 130) and bilateral coeliac ganglia (mean number 1 SE,
1415 1 874) project processes to at least mid colon through
colonic fiber bundles. For neurons in the SMG, this appears
to be the major projection pathway to colonic effector
structures in view of the paucity of neurons that project
their proCesses through lumbar colonic nerves (Baron et al.,
1985 I). A sympathetic pathway originating from neurons in
coeliac ganglia and projecting through hypogastric nerves
and colonic fiber bundles, described in the present study,
was not previously identified.

This report shows that some neurons in parasympathetic
colonic ganglia project postganglionic processes over long
axial distances to at least mid colon. They may provide

synaptic input to myenteric and/or submucosal plexus neu-

157

 

rons and/or directly innervate colonic effector structures
(de Groat and Krier, 1976). This report also identifies a
subpopulation of neurons in sympathatic prevetebral and
paravertebral ganglia that project long postganglionic
processes through hypogastric nerves and colonic fiber
bundles to at least mid-colon. For neurons in SMG and coeli-
ac ganglia, this represents a previously unidentified pro-
jection pathway to colonic effector structures.

The functional significance of this anatomical arrange-
ment is unknown. It is likely these neurons are noradrener-
gic and innervate blood vessels, myenteric plexus ganglia
and intestinal smooth muscle (Furness and Costa, 1974). In
myenteric plexus ganglia they may modulate cholinergic and
non-adrenergic, non-cholinergic transmission by activating

prejunctional adrenoceptors.

158

SUMMARY

In summary, this disssertation has described the dis-
tribution of sacral afferent fibers in effector structures
of the colon, urinary bladder and urethra using anterograde
tracing techniques. Previous anatomical studies of this
distribution were limited to degeneration techniques, which
visualize short fragments of degenerating fibers. The anter-
ograde tracing techniques used in the present study demon-
strated continuous long fibers and fiber arborizations
within ganglia and tissue layers that were not seen with
degeneration.

These sacral afferent fibers may serve as mechanorecep-
tors, chemoreceptors and/or thermoreceptors. They may also
be activated by inflamation and be involved in sensations of
pain. Sacral afferent fibers have also been described in
proximity to neurons in colon myenteric plexus, urinary
bladder, pelvic plexus and colonic ganglia. These afferent
fibers may be collateral branches of fibers innervating
effector structures. Orthodromic activation of the sensory
fiber on the effector structure could initiate antidromic
activation of these afferent collaterals, causing the re-

lease neurotransmitter substances in proximity to neurons.

159

This could represent a feedback mechanism involving sacral
afferent fibers for modulating local neuronal circuits.

This dissertation has also described a population of
neurons in the colon myenteric plexus that project processes
over long axial distances orad and aborad through colonic
fiber bundles. This suggests that intrinsic neurons utilize
colonic fiber bundles to communicate with distant colonic
regions. These long ascending and descending pathways may be
involved in the ano-colonic, recto-colonic and cola-colonic
reflex contractions demonstrated in response to mechanical
stimulation of anal mucosa and passive distension of the
rectum and colon, respectively.

Finally, this dissertation has described the distribu-
tion of a subpopulation of neurons in parasympathetic pre-
vertebral and sympathetic prevertebral and paravertebral
ganglia that project postganglionic processes through colo-
nic fiber bundles to at least mid-colon. For the superior
mesenteric and coeliac ganglia, this represented a peripher-
al pathway to the colon that had not been previously identi-
fied. The functional significance of this projection pathway

is yet to be determined.

160

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