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Afferent innervation of gastrointestinal tract smooth muscle by the hepatic branch of the vagus

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Afferent Innervation of Gastrointestinal
Tract Smooth Muscle by the Hepatic
Branch of the Vagus
Purdue University, West Lafayette, Indiana 47907
To survey the vagal hepatic branch afferent projections to and the terminal specializations in the gastrointestinal tract, male Sprague-Dawley rats were given subdiaphragmatic
vagotomies, sparing only the common hepatic branch, and were injected with 3 µl of 8% wheat
germ agglutinin-horseradish peroxidase in the left nodose ganglion. The nodose ganglia, the
stomach, the first 8 cm of duodenum, and the cecum were prepared as wholemounts and were
processed with tetramethyl benzidine. Hepatic afferent innervation of the ventral stomach
consisted of one or more bundles entering at the lower esophageal sphincter and coursing to
the forestomach, where they branched into distinct terminal fields. The only fibers on the
dorsal forestomach were distal branches and terminals that wrapped around the greater
curvature from the ventral side. Hepatic afferents supplied the forestomach with both
intraganglionic laminar endings (IGLEs; putative mechanosensors that coordinate peristalsis) and intramuscular arrays (IMAs; considered tension receptors). IGLEs were located
primarily on the ventral wall of the stomach, whereas IMAs were distributed symmetrically.
Afferents were also supplied to the distal antrum and the pylorus, with pyloric innervation
consisting almost exclusively of IMAs. Innervation of the proximal duodenum was denser in
the first 3 cm and decreased progressively caudally, with only meager innervation after 6 cm.
Cecal innervation consisted of a few fibers at the ileocecal junction. Duodenal and cecal
endings were predominately IGLEs. These results indicate that the hepatic branch carries
sensory information from the forestomach, antrum, pylorus, duodenum, and cecum. Furthermore, the different terminals it supplies suggest that the branch mediates a multiplicity of
gastrointestinal functions. J. Comp. Neurol. 384:248–270, 1997. r 1997 Wiley-Liss, Inc.
Indexing terms: duodenum; liver; stomach; vagotomy; visceral afferents
The vagus nerve contains both sensory and motor axons
that have been implicated in the control of gastrointestinal
physiology and ingestive behavior. Peripherally, the vagi
enter the abdomen as two trunks coursing along the
esophagus. After passing through the diaphragm, these
mixed trunks, the dorsal and ventral, divide into five
distinct branches: paired gastric branches, paired celiac
branches, and a single hepatic branch that originates from
the ventral trunk (Boekelaar, 1985; Prechtl and Powley,
1985, 1987, 1990).
Traditionally, the hepatic branch of the rat vagus was
assumed to be an uncomplicated and coherent bundle
projecting simply, as its name suggests, to the liver.
Indeed, the hepatic branch has been shown to innervate
the liver, but it has only recently been shown to have
projections to the gastrointestinal (GI) tract (Berthoud et
al., 1991, 1992; Prechtl and Powley, 1987).
The hepatic branch ramifies from the ventral vagal
trunk in one to three bundles of fascicles that travel close
together. The branch then runs between the esophagus
and the hepatic artery proper in loose association with the
hepatoesophageal artery. On this path, the bundles that
comprise the branch split into a plexus of fascicles, a few of
which course into the more distal lesser omentum (Boekelaar, 1985; Prechtl and Powley, 1987). Near the junction of
the hepatoesophageal artery and the hepatic artery proper,
the hepatic branch plexus forms into five to seven fascicles,
which organize into two secondary divisions, one that
turns toward the liver hilus (hepatic branch proper) and a
second that turns to course with the common hepatic and
Grant sponsor: NIH; Grant number: DK27627; Grant sponsor: NIMH;
Grant number: MH01023.
*Correspondence to: T.L. Powley, Department of Psychological Sciences,
165 Peirce Hall, Purdue University, West Lafayette, IN 47907.
Received 12 December 1996; Revised 6 March 1997; Accepted 6 March
gastroduodenal arteries (gastroduodenal branch; Berthoud et al., 1992; Prechtl and Powley, 1987). Recognizing
that two divisions separate from the classic hepatic branch
and course to different targets, the classical ‘‘hepatic
branch,’’ as it separates from the ventral vagal trunk and
courses parallel to the hepatoesophageal artery, is now
often more explicitly designated as the common hepatic
branch1. On average, approximately two-thirds of the
afferent fibers contained in the common hepatic branch
bypass the liver hilus on their way to the gastroduodenal
and common hepatic arterial plexuses (Berthoud et al.,
1992). It has also been demonstrated that a portion of the
vagal efferents contained in the common hepatic branch
bypasses the liver hilus and innervates the gastric antrum, pylorus, duodenum, and pancreas (Berthoud et al.,
1991; Berthoud and Powley, 1991).
Although the findings of Berthoud et al. (1991) provide
evidence for hepatic motor innervation of the GI tract, they
may still underestimate the projection field of the common
hepatic branch due to their sampling of efferents, which
are only 7% of the fibers in the common hepatic branch (for
fiber counts, see Prechtl and Powley, 1987). Furthermore,
the findings of Berthoud and coworkers may also have
underestimated the projection field, because the somata of
the common hepatic branch efferents are widely distributed within the dorsal motor nucleus (Fox and Powley,
1985), and it is difficult to obtain complete injections in
this highly elongated nucleus. Reinforcing the concern
that the Berthoud et al. efferent study underestimated the
extent, and perhaps the breadth, of the common hepatic
branch(es) GI projection field, a recent survey of the
spatial location of vagal afferent innervation of the GI tract
(Powley and Wang, 1995; Wang, 1995; Wang and Powley,
1994) found that the left nodose ganglion (the source of
common hepatic branch afferents) provided significantly
more afferents to the duodenum than did the right nodose
ganglion. This pattern led the authors to speculate that
the difference was due to a heavy contribution from the
common hepatic branch.
In addition to the issue of distribution, another question
concerns the types of afferent terminal specializations or
endings the common hepatic branch of the vagus supplies
to its different targets. Although terminal specializations
or endings provided by hepatic branch afferents to the liver
have been characterized (Berthoud et al., 1992, 1995;
Prechtl and Powley, 1987), such information has not been
available for the corresponding projections to the GI tract.
The characterization of the hepatic branch distribution
based on efferents does not speak to the question, of
course, and previous experiments characterizing vagal
afferent terminals in the GI tract (see, e.g., Berthoud and
Powley, 1992; Berthoud et al., 1995; Kressel et al., 1994;
Powley et al., 1994; Wang, 1995) have not distinguished
hepatic branch afferents from other vagal afferents. These
experiments, however, have distinguished two terminal
specializations [intraganglionic laminar endings (IGLEs)
and intramuscular arrays (IMAs)] in the smooth muscle
wall of the GI tract, either or both of which could conceiv-
1In this report, the classic term hepatic branch and the more explicit
expression common hepatic branch are used synonymously to refer to the
initial segment of the branch that leaves the ventral trunk and courses with
the hepatoesophageal artery. The more distal secondary divisions of this
branch are consistently referred to as the hepatic branch proper and the
gastroduodenal branch.
ably be found among hepatic branch afferents. The goals of
the present study were 1) to describe both qualitatively
and quantitatively the innervation pattern of the common
hepatic branch of the vagus along the GI tract by using a
protocol that would delineate this pattern in detail and 2)
to provide a survey of the types of afferent endings within
the projection field.
Male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN; n 5 25) weighing 300–350 g (ranging from 13
to 15 weeks old) at the time of first surgery were housed
individually and maintained on a 12:12 hour light:dark schedule at 23°C. Animals had ad libitum access to tap water
and 17-hours-per-day (1700–1000 hours) access to Isocal
(Mead Johnson, Evansville, IN), a nutritionally complete
liquid diet (1.05 kcal/ml). All procedures were conducted in
accordance with American Association for Accreditation of
Laboratory Animal Care guidelines and were approved by
the Purdue University Animal Care and Use Committee.
Surgeries and tracers
Vagotomy. Rats were anesthetized by using sodium
pentobarbital (60 mg/kg, i.p.) and then treated with atropine (1.0 mg/kg, s.c.). Each animal was then laparotomized
and selectively vagotomized, as previously described (Fox
and Powley, 1985; Powley et al., 1987). To eliminate the
vagal innervation of the GI tract, except for that supplied
by the common hepatic branch of the vagus, all animals
were given partial subdiaphragmatic vagotomies, consisting of a transection of 1) the posterior trunk above the
point where it bifurcates into the gastric and celiac
branches as well as both 2) the accessory celiac and 3) the
anterior gastric branches below the point where the common hepatic branch separates from the ventral trunk. The
appropriate branch was isolated by dissection, supported
by a microdissecting hook, and cauterized with a hightemperature ophthalmic cautery; the proximal stump was
then recauterized, effectively sealing the exposed nerve end.
Fluoro-Gold injection. To verify the sparing of the
common hepatic branch, we used a fluorescent tracer
strategy that effectively retrogradely labels autonomic as
well as other peripheral axon terminals that lack complete
blood-nerve barriers (Fox and Powley, 1985; Powley et al.,
1987). Eight days postvagotomy, each animal received an
i.p. injection of 1 mg/1 ml saline of Fluoro-Gold (Fluorochrome, Inc., Englewood, CO) to label the cell bodies of
intact efferent fibers.
Wheat germ agglutinin-horseradish peroxidase injection. To label the afferent innervation of the GI tract
supplied by the common hepatic branch, an adaptation of
the wheat germ agglutinin-horseradish peroxidase (WGAHRP) protocol introduced by Aldskogius and coworkers
(1986), specifically for its success in labeling visceral
afferents, was used. In addition, Robertson et al. (1992)
have shown that all nodose ganglion neurons appear to
incorporate WGA-HRP. Three days prior to perfusion, each
rat was reanesthetized and placed in a supine position,
and a ventral midline incision was then made in the neck.
The left vagus, including the nodose ganglion, was exposed
by blunt dissection, and WGA-HRP (3.0 µl; 8% in phosphate-buffered saline; Sigma, St. Louis, MO) was pressure
injected (PicoSpritzer II, General Valve Corporation, Fair-
field, NJ) through a glass micropipette (I.D. 25 µm) into
the ganglion. The incision was then closed and treated
with nitrofurazone, and the animal was allowed to recover
on a heating pad and then returned to its cage.
Tissue preparation
Two weeks after the vagotomy and 3 days after the
WGA-HRP injection, each animal was deeply anesthetized
with a lethal dose of pentobarbital. When it was completely unresponsive to nociceptive stimuli, the animal
was injected in the left ventricle with 0.1 ml Heparin
(Elkins-Sinn, Inc., Cherry Hill, NJ) (1,000 units/ml) to
prevent coagulation and 0.1 ml propranolol (Ayerst Laboratories, Inc., Philadelphia, PA) to produce vasodilation and
was then perfused transcardially with 500 ml of 0.9%
saline at 40°C. To produce matched stomach and duodenal
sizes across animals, the jejunum was clamped during this
saline perfusion, and the stomach and area proximal to the
clamp site were filled with 0.9% saline (15 6 5 ml; volume
varied depending on quantity of stomach contents) at
40°C. Once the organs were filled with saline, the tissues
were fixed by transcardial perfusion of 3% paraformaldehyde and 0.4% glutaraldehyde in 0.1 M sodium phosphate
buffer, pH 7.4, at 4°C, and, finally, 10% sucrose in the same
buffer at 4°C.
The brainstem was removed and cryoprotected in 15%
sucrose in phosphate buffer. The medulla was then blocked,
sectioned coronally at 56 µm with a cryotome, and prepared to verify both the vagotomy (Fluoro-Gold: threefourths of the sections) and the nodose injection (WGAHRP: one-fourth of the sections). The nodose ganglia, the
entire stomach, the first 8 cm of the duodenum, and the
cecum were prepared as wholemounts, as described previously by this laboratory (Powley et al., 1994). Briefly, the
whole GI tract was rinsed with cold tap water and then
separated into the areas of interest. The stomach and
cecum were divided into dorsal and ventral halves by
cutting along the greater and lesser curvatures. The
duodenum was divided into two 4-cm lengths and was
opened with a longitudinal cut along the mesenteric
attachment. Wholemounts were then processed with tetramethyl benzidine (TMB) according to the protocol of Mesulam (1978). Sections for the Fluoro-Gold analysis were
thaw-mounted on slides, air dried, dehydrated in alcohol,
cleared in xylene, and coverslipped with DPX (Aldrich,
Milwaukee, WI). Sections and wholemounts for WGA-HRP
analysis were cleared in xylene and coverslipped with DPX.
Medulla sections, nodose ganglia, and GI tract wholemounts processed for WGA-HRP were examined with
light- and darkfield illumination, and all animals were
screened for the presence of labeled fibers in the nucleus of
the solitary tract and left nodose ganglion. Counts of the
different elements were conducted as described below.
Specific stereological techniques were not employed, because 1) only relative comparisons were used, 2) the
observations were to be compared with earlier relative
estimates that did not employ a director strategy, and 3)
most of the tissue was examined in wholemounts, not in
serial sections.
Verification of hepatic-spared vagotomies
To verify the vagal surgery, the Fluoro-Gold test of
selective vagotomies (Powley et al., 1987) based on the
characteristic distributions of efferent somata within the
dorsal motor nucleus of the vagus (dmnX) was employed
(no comparably sensitive protocol is available specifically
for afferents). In this procedure, Fluoro-Gold-labeled neurons in the dmnX were counted, and their distributions
were analyzed by an observer, who was blind to each
animal’s identity and WGA-HRP profiles. Because only
three-fourths of the coronal sections of the brainstem were
used for Fluoro-Gold analyses, the total cell counts were
corrected (34/3). Because any ‘‘split-cell’’ counting bias
would have inflated the resultant counts toward an overestimate of the number of dmnX neurons spared and a
correction was problematic owing to the sequence of histological sections counted, no split-cell correction was used.
A priori, brainstem Fluoro-Gold patterns had to satisfy
four criteria for an animal to be classified as a common
hepatic branch-spared case with no extraneous label: 1)
Numerically, in terms of left vs. right dmnX distributions,
labeled cells had to be found almost exclusively on the left
side (i.e., the side occupied by the hepatic branch column).
To be considered successful, verified cases had to have less
than 20 labeled cells on the right and, then, only in the
caudal part of the nucleus, where the left and right
half-nuclei are fused on the midline. 2) Also numerically,
the total of labeled preganglionic somata on the left side
had to be less than or equal to the total number of hepatic
motor neurons estimated in the quantitative description of
the hepatic branch column by Fox and Powley (1985). 3)
Spatially, the distribution of the labeled neurons in the left
dmnX had to agree with the earlier characterizations of
the column (Fox and Powley, 1985; Norgren and Smith,
1988; Powley et al., 1987). 4) Conversely, in contrast to the
last criterion, the selective vagotomy was considered inappropriate if the distribution of Fluoro-Gold-labeled cells
was organized in a pattern similar to the distinctive
columns associated with either of the other two branches,
i.e., the accessory celiac and ventral gastric branches, also
organized in the left dmnX.
Evaluation and screening of wholemounts
A need for intact (required to preserve spatial information), low-artifact (required to permit counts of fine fibers
and terminals) wholemounts made it necessary to screen
specimens and eliminate those that could not be reliably
quantified. In particular, it was found that the physical
integrity of some wholemounts was compromised by dissection damage resulting from surgically induced tissue adhesions, and others were compromised during the removal of
the mucosa from the muscle wall. In terms of the staining
protocol, strong reactions of mast cells and perhaps other
TMB-reactive nonneuronal cells, such as granulocytes and
intravasal granular leukocytes associated with the surgery and related scarring, produced excessive artifact,
which masked axon staining in some specimens.
Before quantitative analyses were undertaken, each
wholemount of each organ was evaluated. Those specimens that were judged excessively damaged or potentially
masked by artifact were excluded from formal quantitative analyses. All judgments were made blind, prior to the
Fluoro-Gold verification assessments.
Quantification of fibers and terminals
Counting of all intact, low-artifact wholemounts processed with WGA-HRP was then done without knowledge
of the results of the verification procedure for the selective
Criteria for counting. Based on the findings of Wang
(1995), four main vagal afferent elements are found in the
muscle layers of the rat GI tract and needed to be counted
to provide a description. The first two are bundles and
fibers. Bundles were defined as two or more axons running
together (see Fig. 1A), and fibers were defined as individual axons (see Fig. 1A). The third and fourth elements
are terminal specializations, which are found on vagal
afferents innervating the GI tract (Berthoud and Powley,
1992; Powley and Wang, 1995; Rodrigo et al., 1982; Wang,
1995; Wang and Powley, 1994). These consist of IGLEs and
IMAs. An ending was determined to be, and was counted
as, an IGLE if it consisted of fine terminal puncta within
the neuropil of a myenteric ganglion, effectively encapsulating all or part of the ganglion (see Fig. 1B; see also
Neuhuber, 1987; Neuhuber et al., 1995; Rodrigo et al.,
1975, 1982). Finally, an ending was counted as an IMA
(Berthoud and Powley, 1992; Wang, 1995) if it consisted of
an array of parallel axonal telodendria in close proximity,
interconnected by bridging axonal elements, and was
located in either the circular or the longitudinal muscle
layer (see Fig. 1C).
Counting grid. To facilitate a quantitative reconstruction of the innervation of the GI tract by the common
hepatic branch of the vagus (as well as to facilitate
comparison across experiments), the sampling procedure
of Wang (1995) was used. Two different reticles, a counting
grid and a sampling grid, were employed. Briefly, at each
sampling point (see below), a camera lucida drawing arm
was used to merge an image of a 1.7 3 1.7 mm counting
grid, consisting of 9 3 9 equidistant lines (forming 64
squares), with the tissue image in the microscope. Counting was done at a final magnification of 350 (eyepiece 38,
objective 36.3).
Before counts were taken at a sampling point, the grid
was rotated, so that one set of lines paralleled the circular
muscle, and the second set paralleled the longitudinal
muscle of the organ wall. Each of the four elements
(bundles, fibers, IGLEs, and IMAs) were then counted in a
similar manner by using the counting grid. This grid was
visually scanned from left to right/top to bottom, and the
innervation pattern was then recorded. For bundles and
fibers, the total number of intersections within the grid
was the characteristic counted. For example, a single fiber
(or bundle) traveling the width of the grid in a longitudinal
direction would intersect with nine of the lines that make
up the counting grid, so the fiber (or bundle) would be
counted as nine longitudinal intersections. Similarly, a
case in which two fibers (or bundles) were traveling in a
similar fashion would yield a count of 18 longitudinal
intersections, and so on. Due to the fact that distinguishing between an IMA and several IMAs is problematic, we
used a modification of the counting criteria for IMAs. The
same sampling grid was used, and direction of innervation
was determined; but, instead of total number of intersections being counted, total number of squares in a counting
grid that contained IMAs was recorded. So, a sampled area
could not have more than 64 squares innervated. In the
case of IGLEs, total number of squares containing IGLEs
was counted. If an IGLE was contained entirely within one
square, then it was counted as one; but, if the IGLE
overlapped into an adjacent square, then that square was
also counted. However, even if a single square contained
two or more separate IGLEs, it was still only recorded as
In composite, the counts of the four elements (bundles,
fibers, IGLEs, and IMAs) as they intersected the grid lines
oriented parallel to the two different muscle layers pro-
vided useful profiles of innervation patterns. For example,
a region with a number of individual fibers as well as
coherent bundles would presumably be an area in which
axons were dispersing from connectives to form terminal
fields, whereas a region with bundles but no fibers would
represent an area that the vagus was merely traversing.
For another example, a region in which most axons (or
IMAs) intersected grid lines parallel to the circular muscle
and few fibers (or terminals) intersected the grid lines
parallel to the longitudinal muscle would be an area in
which fibers (or endings) were traveling longitudinally.
Sampling algorithms: Stomach and pylorus. Because
of the morphological differences among the organs surveyed, the counting grid was positioned according to
different sampling algorithms in the different organs. In
the case of the stomach, the wholemounts of the dorsal and
ventral halves were each sampled at 45 similar areas by
the use of a sampling grid. To minimize variability resulting from differences in stomach size or distention, the
tissue was first normalized with this sliding-scale sampling grid. For this purpose, each half of the stomach was
measured and fitted with grid dimensions that provided
ten equidistant intervals in the cranial-to-anal direction
and six similar intervals from the lesser to the greater
curvature. The survey generated by then positioning the
counting grid at each of the 45 intersections within the
sampling grid included 10% of the area of each wholemount.
In addition to quantification of the gastric innervation
with the counting and sampling grid strategy, the distribution pattern of the vagal bundles coursing into the stomach
was also mapped graphically to facilitate describing the
innervation of the stomach in terms of the different regions
(e.g., forestomach, corpus, antrum). For these latter maps,
each stomach wholemount was placed in an enlarger, and
the specimen was projected onto a sheet of graph paper.
The contour of the stomach was traced, and WGA-HRPlabeled bundles were then drawn onto the organ outline by
using a camera lucida strategy (see Figs. 3A,B, 6A,B).
The radial or circumferential extent of the pylorus that
was innervated by common hepatic axons, the types of
endings, and the location of the innervation were also
determined through the use of the same counting grid. The
entire pylorus was successively blanketed by the counting
grid, and the total number of squares containing innervation and location of the innervation was recorded. Types of
endings and location of the endings were also recorded.
Sampling algorithm: Duodenum. The first 8 cm of
the duodenum were sampled in a manner similar to the
strategy for the stomach, allowing for the differences in the
nature of the tissues. Specifically, the duodenal wholemount was counted in transverse sampling bands situated
every 3.2 mm along its entire length. Distance was measured aborally from the pylorus. The circumferential midpoint of the transverse band (i.e., the antimesentery pole,
given the dissection procedure) was located by measuring
from the edges of the longitudinal tissue cut, and the
counting grid was centered at that point, with the grid
lines oriented parallel to the fibers of the two muscle
sheets. Bundles, fibers, IGLEs, and IMAs within the
counting grid were then recorded exactly as for the stomach. After the antimesenteric pole was counted in this
manner, the entire circumferential band of duodenum was
sampled by moving the counting grid in both radial
directions in successive 1.7-mm steps. After the entire
Fig. 1. Darkfield photomicrographs illustrating vagal elements
that were quantified. A: Bundles and fibers. A bundle of axons (solid
arrow) with several individual fibers (open arrows) separating from
the main bundle. B: Intraganglionic laminar ending (IGLE). A single
afferent vagal fiber ending in an IGLE. Notice the distinct punctate
pattern of the ending as it encapsulates portions of a myenteric
ganglion. C: Intramuscular array (IMA). A bundle of afferent axons
wrapping around from the ventral forestomach and terminating as
several IMAs (arrows), which end in the longitudinal muscle. The
diffusely distributed white spots are labeled mast cells that are not
part of the ending. Mast cells and perhaps other granulocytes are
labeled due to the reaction of tetramethyl benzidine (TMB) with the
endogenous peroxidase. Scale bars 5 200 µm.
transverse band had been counted, the counting grid was
then relocated at the antimesenteric point 3.2 mm aboral
to the band just counted, and the procedure was repeated.
By using this procedure, approximately 53% of the tissue
area was sampled.
Sampling algorithm: Cecum. Due to the sparse innervation of the cecum by hepatic vagal fibers, the entire
organ was systematically scanned, and the total numbers
of fibers and types of endings were counted and described
without the use of the counting grid.
Graphical display of data. Graphpad Prism (version
2.0; Graphpad Software, Inc., San Diego, CA) software was
used for line graphs; three-dimensional topographic and
surface plots were created with Surfer Version 6.0 (Golden
Software, Inc., Golden, CO).
Control for possible regeneration
of vagal branches
Recently, it has been reported that vagal branches
exhibit plasticity after vagotomy (Phillips et al., 1996;
Powley et al., 1996). This plasticity involves regeneration
of cauterized axons and possibly reorganization of any
spared axons. Changes in the pattern of vagal fibers are
found from 6 to 18 weeks after selective vagotomy. To
address the possibility that the patterns of innervation
observed in the present experiment might reflect some
axonal reorganization, three animals were treated as
described in Materials and Methods, except that, instead
of a common hepatic-spared vagotomy, they received a
complete subdiaphragmatic vagotomy, including the common hepatic branch. These animals were then examined
for possible indications of vagal plasticity, such as the
presence of bundles, fibers, and growth cone profiles
(Phillips et al., 1996; Powley et al., 1996).
Verification of hepatic-spared vagotomies
with retrograde transport of Fluoro-Gold
Twenty-five rats satisfied the criteria used to establish
that the selective vagotomy procedure had eliminated all
vagal abdominal branches except the common hepatic
branch. In terms of the four specific criteria employed: 1)
Labeled hepatic preganglionics were essentially confined
to the left dmnX, with a mean (6S.E.M.) of only 8 (60.8)
Fluoro-Gold cells per animal observed in the right dmnX.
2) Compared with animals with all vagal branches and
their corresponding dmnX columns intact (cf. Fig. 2A,
control section), hepatic-spared animals had only a few
Fluoro-Gold-labeled motor neurons distributed sparsely
through the left dmnX (mean 6 S.E.M. 5 112 6 8; cf. Fig.
2B; for comparison, the other two branches of the ventral
trunk are represented by approximately 3,000 somata in
the left dmnX; Fox and Powley, 1985). 3) In all animals
judged to have successful hepatic-spared vagotomies, these
labeled preganglionic neurons were distributed throughout the left dmnX in the sparse columnar pattern characteristic of the hepatic column (cf. Fox and Powley, 1985;
Norgren and Smith, 1988; Powley et al., 1987). This
pattern included a distinct and consistent ‘‘arch’’ or ‘‘horseshoe’’ of preganglionic somata at the level of the area
postrema (Fig. 2C). The arch gets its appearance because
preganglionic neurons of the common hepatic branch
column form a thin dorsal plate of cells spanning through
Fig. 2. Photomicrographs of frontal sections of dorsal medulla at
level of area postrema showing Fluoro-Gold label in the dorsal motor
nucleus of the vagus (dmnX). With this protocol, neurons of the dmnX
with intact projections to viscera in the peritoneal space label strongly
with Fluoro-Gold (‘‘primary labeling’’), whereas neurons and other
tissue associated with heavily vascularized sites without fully effective
blood-nerve barriers (cf. the hypoglossal neurons and area postrema)
sequester some Fluoro-Gold that is circulated systemically after i.p.
injections (‘‘secondary label’’). The dmnX neurons axotomized by
cauterization contain only secondary Fluoro-Gold labeling and appear
no brighter than neurons in the hypoglossal nucleus. Also note that
tissue autofluorescence at background was high in the present tissue,
because the glutaraldehyde used in the wheat germ agglutininhorseradish peroxidase (WGA-HRP) protocol induces autofluorescence. A: Control animal with no previous surgery. The left and right
dmnX can be seen clearly with the gastric (solid arrows) and celiac
(open arrows) poles intact. B: Two hepatic motor cells with primary
Fluoro-Gold labeling (arrows) can be seen in the region of the left
gastric pole. The number of labeled motor cells is unequivocally less in
the hepatic-spared case compared with the control case. C: A distinct
arch pattern of cells with primary Fluoro-Gold labeling (arrows) can be
seen in the left dmnX. This pattern was found to occur when scanning
through the sections starting at the area postrema and moving distally
through the medulla. Almost all animals with hepatic-spared vagotomies had this distinct pattern. Scale bar 5 200 µm.
the central part of the nucleus (viewed in transverse
sections), with the more medially and laterally situated
cells dispersed from dorsal to ventral. 4) None of the
animals judged to have successful hepatic-spared surgeries had condensations of preganglionic neurons in either
the distinctive ventral gastric column or the compact celiac
column of the left dmnX.
WGA-HRP-labeled fibers and terminals
in the stomach and pylorus
General pattern. Wholemounts of 11 complete (dorsal
and ventral halves) stomachs from the sample of verified
animals met the criteria for analysis (i.e., the specimens
were intact and low in artifact) and were mapped, quantified, and photographed. These 11 specimens were representative of the larger set of 25 animals, because their mean
dmnX verification profiles (left dmnX 5 114, right dmnX 5
7) were similar to those of the other 14, which were not
suitable for counting (left dmnX 5 110, right dmnX 5 8) as
well as the profile for the entire set of 25 animals (left
dmnX 5 112, right dmnX 5 8).
Stomach bundles and fibers. When the normalized
sampling grid was fitted to the individual stomachs and
used to determine regions for counting, each animal
exhibited a similar pattern (for discussion of exceptions,
see below) of innervation across the different points
sampled. The hepatic afferents were predominately distributed within the ventral wall of the stomach (Figs. 3A,C,E,
4A,C), with the dorsal gastric wall receiving only 27% of
the total innervation (Figs. 3B,D,F, 4B,D). Innervation of
the ventral stomach consisted of one or more large bundles
that entered at the lower esophageal sphincter and traveled through the upper portion of the forestomach (close to
the lesser curvature) toward the greater curvature, where
it wrapped around to the dorsal side and terminated (Figs.
3A,B, 6C). As the bundles coursed through the ventral
forestomach, they branched into smaller bundles and
individual fibers, some of which also wrapped around to
the dorsal forestomach (Fig. 3C–F). Seventy three percent
of the bundles (Fig. 3C,E) and 73% of the fibers (Fig. 4A,C)
were found to innervate the ventral stomach.
Neither bundles nor fibers evidenced a preferential
direction of innervation (i.e., paralleled one of the muscle
fiber axes) on either side of the stomach. On the ventral
side, 47% of the bundles (Fig. 3C) and 44% of the fibers
(Fig. 4A) traveled in the longitudinal direction. A similar
pattern was determined for the dorsal side, with 52% of
the bundles (Fig. 3D) and 52% of the fibers (Fig. 4B)
coursing in the longitudinal direction. Bundles and fibers
were also found to innervate both sides of the stomach in
the region of the antrum (almost always circular in
direction), but these bundles and fibers usually terminated
quickly upon entering the stomach wall from the lesser
Stomach terminals. Both types of afferent terminals
that have been observed in the gastric wall (i.e., IGLEs
and IMAs) were formed in the forestomach by common
hepatic branch afferents. IGLEs were diffusely distributed
throughout the rostral portion of the ventral and dorsal
forestomach, with 70% of the terminals located on the
ventral side (Fig. 4E,F). Relatively speaking, IMAs were
symmetrically distributed on both sides of the stomach,
with 59% of the terminals on the ventral side and 41% on
the dorsal. Whereas IGLEs were distributed diffusely
throughout the upper forestomach, IMAs were concentrated in a particular location. This locus was on the
greater curvature of the ventral and dorsal forestomach
(Fig. 5A,B), with IMAs forming large arrays of axons
running parallel to each other (Figs. 1C, 5C). In addition to
being concentrated at one location, IMAs were also found
to innervate principally longitudinal muscle, with 93% of
the endings running in the longitudinal direction (Figs.
1C, 5C).
Range of stomach innervation patterns. The pattern of innervation described above was the typical pattern
observed in most of the stomachs counted (n 5 8) and was
validated as the characteristic pattern when the average
for the eight cases were compared with averages for all 11
stomachs. However, this description does not completely
describe the range of patterns produced by the hepatic
innervation of the stomach in some animals. In one of the
stomachs studied, the projection consisted of bundles and
fibers that traveled along the limiting ridge of the ventral
forestomach to wrap around the greater curvature of the
dorsal forestomach at a site somewhat more distal than
the typical location. Finally, the remaining two stomachs
were found to be densely innervated compared with the
typical stomach, with bundles and fibers encompassing the
whole ventral forestomach, including extensive wraparounds to the dorsal side (Fig. 6A,B).
Pylorus. The innervation of the pylorus by the common hepatic branch was limited to a few fibers ending
exclusively in IMAs. In 10 of 12 stomach wholemounts
counted (five ventral and dorsal pairs), the IMAs were
localized within a small region of the pylorus (33% of the
total area of the pylorus) on the greater curvature side of
the stomach (Fig. 7). In the remaining two stomach
specimens counted (one ventral and dorsal pair), the IMAs
were not localized to one discrete area but were distributed
more diffusely throughout the entire pylorus. This less
typical pyloric pattern was not correlated with the less
typical gastric innervation patterns.
Duodenum and cecum
General pattern. A total of 16 duodena were analyzed.
These 16 specimens were representative of the larger set of
25 animals, because their average dmnX verification profiles (left dmnX 5 111, right dmnX 5 7) were similar to
those of the other nine, which were not suitable for
counting (left dmnX 5 112, right dmnX 5 8), as well as the
profile for the entire set of 25 animals (left dmnX 5 112,
right dmnX 5 8). The number of transverse sampling
bands that could be counted per specimen ranged from 3 to
13, with a mean of 11 bands for the first wholemount of the
duodenum and 7 bands for the remaining wholemount.
Duodenal bundles and fibers. Afferent bundles entered the duodenum from the mesentery at about 3.2 mm
distal to the pylorus and densely innervated the tissue for
the next 3.0 cm, falling off to almost no innervation by 5.0
cm (Figs. 8A, 10A, 11A). Bundles were also seen to
innervate the first 3.2 mm (see 0 mm, Figs. 8A, 10A, 11A),
but the innervation consisted primarily of fascicles that
branched from larger bundles at more distal sites and
traveled toward the pylorus. In addition, a few bundles
were found to enter the most proximal duodenum from the
mesenteric attachment, but hepatic afferent bundles were
never seen crossing the pylorus from the stomach. Bundles
ran preferentially in the longitudinal direction parallel to
the mesentery for approximately the first 3.5 cm (Figs. 8A,
11). Rarely were bundles at the antimesentery found to be
traveling in the longitudinal direction (Fig. 11B,C). Bundles
traversing the tissue in the circular direction (Figs. 8A, 9,
10) were also denser along the mesenteric attachment and
were progressively sparser moving toward the antimesentery (Fig. 10B,C). At all points, however, fewer bundles ran
in the circular orientation than in the longitudinal direction.
The quantitative survey of fibers revealed patterns
similar to those of the bundles. The fibers entered the
duodenum from the mesentery at about 3.2 mm and
densely innervated the tissue for the first 3 cm, then
progressively declined in density more distally, until there
Fig. 3. Map and plots of the hepatic branch innervation of the
ventral and dorsal stomach by bundles. For ease in comparing
symmetrical sites from the two half stomachs, the ventral gastric
wholemount has been ‘‘flipped over.’’ In effect, the viewer is examining
the ventral gastric wholemount from the mucosal side and the dorsal
wholemount from the conventional serosal side. This same convention
is used throughout this report for all maps, plots, and drawings. A,B:
Camera lucida tracing of the ventral (A) and dorsal (B) stomach from
the same rat. This map illustrates the typical pattern of innervation
by bundles in the fundus, although the one bundle on the ventral
stomach that branches from the main trunk and crosses over the
corpus to terminate in the antrum is atypical. C–F: Surface and
contour plots of counts and direction of innervation by hepatic
bundles. A silhouette of a stomach wholemount (see A for orientation
and regions) is superimposed on the contour plots to highlight the
location of the innervation. The x-axis is the sampling grid column
sampled from, the y-axis is the sampling grid row sampled from, and
the z-axis is the total number of intersections by bundles per counting
grid averaged across the 11 stomachs sampled. Scale bars 5 2 mm.
Fig. 4. A–D: Plots of the same type shown Figure 3C–F, except the
plots are of the fibers counted. E,F: Plots of the same type shown in
Figure 3C–F, except the plots illustrate the location of IGLEs in the
stomach. Notice the concentration of IGLEs in the region of the
antrum. Antral fibers ended primarily in IGLEs. The x- and y-axes are
the same as previous plots, but the z-axis now represents the density
of IGLEs per counting grid averaged across stomachs sampled.
Fig. 5. A,B: Plots of IMA terminal fields. The x- and y-axes are the
same as the plots in Figures 3C–F and 4A–D, but the z-axis now
represents the number of squares containing IMAs per counting grid
averaged across stomachs sampled. The IMAs are unequivocally and
solely focused on the greater curvature of the forestomach. C: Darkfield image of IMAs (arrows) running in the longitudinal muscle. Scale
bar 5 200 µm.
Fig. 6. A,B: Camera lucida tracing of hepatic bundles in the
ventral (A) and dorsal (B) stomach from the same rat. These maps
illustrate the extensiveness of the innervation by the hepatic branch of
some animals (n 5 2). Bundles and their branches are contained in the
region of the ventral forestomach, with some wrapping around to the
dorsal forestomach where they terminate. None is seen to cross the
limiting ridge into the corpus of the stomach, and only one bundle was
found in the antrum. C: Darkfield image of a bundle (solid arrows) in
the ventral forestomach branching into a smaller bundle and several
individual fibers (open arrows) as it courses toward the greater
curvature. Scale bars 5 2 mm in A,B, 200 µm in C.
Fig. 8. Graphs of the bundles (A), fibers (B), and IGLEs (C)
counted in the duodenum. Error bars are 6S.E.M. There are two main
findings conveyed by the three graphs. First, for all three graphs, the
innervation is denser in the second 3.2-mm circular band sampled
compared with the first circular band, which was sampled from the
region adjacent to the pylorus. Finally, the error bars become relatively small to nonexistent at about 50 mm, indicating a consistent
diminution of hepatic branch afferent innervation at this distance.
Fig. 7. Darkfield photomicrograph demonstrating the typical innervation seen in the rat pylorus by vagal hepatic afferent fibers. The
dominate pattern was for bundles, fibers, and IMAs to be concentrated
in the greater curvature side of the pylorus. The long arrow indicates a
bundle that projects to the pylorus, and the short arrows delineate the
region of the pylorus that is innervated by IMAs. Scale bar 5 200 µm.
Fig. 9. Darkfield image taken from the 1st cm of the duodenum. A
bundle (solid arrow) can be seen entering from the mesentery (not
shown) and traveling in a circular direction (vertically in the photograph) toward the antimesentery. Numerous fibers separate from the
bundle, forming a meshwork of fine fibers, which travel in both
circular and longitudinal directions. A multitude of IGLEs are distributed across the duodenal tissue. A typical fiber (open arrow) and IGLE
(open arrowhead) are indicated. Scale bar 5 200 µm.
Fig. 10. A: Surface and contour maps of bundles running in a
circular direction of the duodenum. The x-axis is the distance anal
from the pylorus that was sampled, the y-axis is the width of the tissue
sampled (with 0 mm indicating the center of the antimesentery), and
the z-axis is the total number of intersections per counting grid
averaged across the duodena sampled. B,C: Head-on views of the
surface map from the proximal end (B) and the distal end (C). Notice
the paucity of innervation of the antimesentery. D: Darkfield image
taken from the 2nd cm of the duodenum. Bundles (arrows) can clearly
be seen entering from the mesenteric border (top) and traveling
toward the antimesentery. Scale bar 5 200 µm.
Fig. 11. A: Surface and contour maps (for conventions, see Fig. 10)
of duodenal bundles running in a longitudinal direction. B,C: Head-on
views of the surface map from the proximal end (B) and the distal end
(C). Notice how the bundles are densest along the sides where the
duodenum is attached to the mesentery, and note the sparseness of
innervation of the antimesentery. D: Darkfield image taken from the
3rd cm of the duodenum. Large longitudinal bundles (arrows) run
parallel to the mesenteric border (bottom of picture; not shown). Scale
bar 5 200 µm.
was little innervation at about 6 cm (Figs. 8B, 12A, 13A).
In the same vein as innervation by bundles, fibers were
also found in the first 3.2 mm (see 0 mm, Figs. 8B, 12A,
13A), but not to the same extent as more distal sites. These
fibers branched from more distal bundles at 3.2 mm and
traveled toward the pylorus. In the case of fibers, antrumto-duodenum transpyloric crossings did occur in a small
percentage of the duodena surveyed. This similarity between the patterns of bundles and fibers partially reflects
the fact that fibers separated from the bundles (Figs. 9,
12D, 13D), so that they would naturally parallel, at least to
some extent, the innervation pattern of the bundles.
Where fibers differed from bundles was in the distribution
radially throughout the tissue. Fibers had no predominant
orientation and distributed around the full circumference
of the tissue (Figs. 8B, 12B,C, 13B,C), creating a meshwork (or web-like) pattern across the entire width of the
tissue from the mesentery to the antimesentery (Figs. 9,
12D, 13D).
Duodenal terminals. Hepatic afferent duodenal terminal specializations consisted almost exclusively of IGLEs,
with IMAs comprising only about 0.5% of the terminals
counted. IGLEs followed a distribution pattern similar to
that of duodenal fibers. The densest area of terminals
occurred in the first 3 cm of the duodenum and progressively declined in density moving distal to the pylorus
(Figs. 8C, 14A). IGLEs were scattered evenly and transversely around the tissue, although a slightly greater
concentration was seen at the mesenteric border (Fig.
14A–C). IGLEs were generally similar in appearance from
region to region, although some differences were seen
based upon the density of innervation along the length of
the duodenum. In the first 3 cm, where the terminals were
densest, several fibers entered or continued from a single
IGLE (Figs. 9, 14F), and an individual IGLE would
frequently be situated in close proximity to neighboring
IGLEs (Figs. 9, 14D). In contrast, from 4 cm distal to the
pylorus on, IGLEs were associated with fewer (typically
one) fibers (Fig. 14E) and were sparsely distributed in the
tissue. At this distance from the pylorus, IGLEs were also
usually located in close proximity to the mesenteric border
(Fig. 14D). The small subset of hepatic duodenal afferents
ending in IMAs (Fig. 15A) always consisted of very fine
axons running exclusively in the circular direction (Fig.
15A) adjacent to the mesenteric border.
Cecum. Seven cecal wholemounts met the criteria to
be counted and photographed. The cecum was very sparsely
innervated by common hepatic branch afferents. The
projection usually consisted of 2 to 15 fibers (mean 5 8)
entering at the ileocecal junction and terminating close to
that site. Terminals consisted exclusively of IGLEs (Fig.
15B), with an average of four IGLEs per cecum.
Control for regeneration
No indication of regeneration was found to occur by the
2-week sampling point in the three control animals that
received complete subdiaphragmatic vagotomies, thus verifying that the innervation seen in the hepatic-spared
animals was the normal or ‘‘intact’’ hepatic branch pattern
and was not contaminated by regeneration of vagal axons
transected by selective vagotomy. Two weeks after the
complete vagotomy procedure, as verified by the lack of
Fluoro-Gold-labeled cell bodies in the dmnX (mean 6
S.E.M.; left dmnX 5 6 6 2, right dmnX 5 3 6 1 labeled cell
bodies), not a single axon was found in the stomachs and
ceca of the three control animals. On the other hand, a
mean of one axon was found to innervate the first 8 cm of
the small intestine. Because only a mean of one axon per
animal (with one of the three cases having no axons) was
found, the pattern presumably reflects either the existence
of a stray minor fascicle that was missed when the vagal
bundles were cauterized at surgery or else the earliest
(still inconsequential) stage of vagal plasticity occurring at
2 weeks postvagotomy.
The present results show that hepatic afferents not only
innervate the duodenum, pylorus, and antrum, as implied
by previous studies of efferents (Berthoud et al., 1991), but
that afferents from the branch also project to the forestomach and cecum. In addition to describing this regional
specificity, we also demonstrate that the hepatic branch
has distinct patterns of terminal specializations, varying
by region or organ (e.g., the combination of both IGLEs and
IMAs in the forestomach or the preponderance of IGLEs in
the duodenum). Furthermore, the patterns and densities
of hepatic terminals represent discrete, organized, and
localized concentrations that cannot be explained as random and proportional subsets of the overall vagal truncal
afferent pattern or of the density of myenteric ganglia
(Gabella, 1987; Powley and Wang, 1995; Wang, 1995;
Wang and Powley, 1994). Before considering the results for
the different organs in detail, however, two critical elements of the protocol should be considered.
Effectiveness of selective vagotomy
verification and nodose injections
The interpretations of the present results hinge on the
vagotomy verification and the afferent labeling strategy.
Two critical questions arise. Were we able to accurately
determine a selective hepatic-spared vagotomy with the
Fluoro-Gold test, and were only vagal afferents, and not
efferent fibers of passage, labeled by nodose injection of
Three relevant observations on the use of the FluoroGold verification test indicate that hepatic-spared cases
were effectively distinguished. First, the vast majority
(96%) of the preganglionic somata in the dorsal motor
nucleus of the vagus are organized into two symmetrical
pairs of coherent subnuclei or columns, which issue the
efferent axons of the two gastric (ventral and dorsal) and
the two celiac (ventral or ‘‘accessory’’ and dorsal) branches
of the vagus, respectively (Fox and Powley, 1985). If a
fraction of one of these gastric or celiac branches was
inadvertently spared, then the nonaxotomized neurons
would have been detected both as a substantial increase in
the counts of labeled cells in the dmnX and as a remaining
fragment of one of the subnuclei (for an example of
incomplete branch cuts, see Powley et al., 1987). Second,
the common hepatic branch also contains the axons of a
distinctive column of preganglionic neurons within the left
dmnX (Fox and Powley, 1985; Powley et al., 1987). This
hepatic column pattern includes a distinguishing ‘‘arch’’ of
cells at the level of the area postrema that is noticeable in
the figures of previous reports (see Fox and Powley, 1985;
in particular, for a good example, also see Fig. 2D in
Berthoud et al., 1990a). Furthermore, the distinctive
distribution of this column at different longitudinal levels
constitutes a series of signature features that would be all
Fig. 12. A: Surface and contour maps (for conventions, see Fig. 10)
of circular fibers distributed evenly across the width of the duodenum.
B,C: Head-on views of the surface map from the proximal end (B) and
the distal end (C). Note how the fibers are located in the antimesentery
as well as in the mesentery. D: Darkfield photograph taken from the
first 4 cm of the duodenum. The fibers (open arrows) form a characteristic meshwork pattern that consists of fibers running in the longitudinal and circular directions. Scale bar 5 200 µm.
Fig. 13. A: Surface and contour maps (for conventions, see Fig. 10)
of longitudinal fibers uniformly distributed across the width of the
duodenum. B,C: Head-on views of the surface map from the proximal
end (B) and the distal end (C). Note how, unlike vagal bundles, the
fibers are located in both the antimesentery and the mesentery. D:
Darkfield photograph of longitudinal fibers (arrows) ending in IGLEs.
Scale bar 5 200 µm.
Fig. 14. IGLEs were evenly distributed across the width of the
duodenum and progressively diminished in density, moving distal to
the pylorus. A: Surface and contour maps (for conventions, see Fig. 10)
of IGLEs in the duodenum. The x- and y-axes are the same as in
previous plots, but the z-axis now represents the density of IGLEs per
counting grid averaged across duodena sampled. B,C: Head-on views
of the surface map from the proximal end (B) and the distal end (C). D:
A single afferent bundle (solid arrow) can be seen entering from the
mesentery (top) and terminating in several IGLEs (open arrowheads).
This was standard for the innervation of the duodenum at about 5–8
cm distal from the pylorus. E: A single fiber can be seen in the
antimesentery running in the longitudinal direction and ending in an
IGLE. F: Several fibers are seen innervating a single IGLE. Scale
bars 5 200 µm.
Fig. 15. A: Darkfield image taken from the duodenum, 5 cm distal
from the pylorus. Several fibers (open arrow) can be seen separating
from a bundle (long solid arrow) and ending in several IGLEs
(arrowhead) and IMAs (short solid arrow). IMAs were rarely found to
occur in the duodenum in this study. B: A single afferent vagal fiber
(arrows) entering the cecum at the ileocecal junction and ending in two
IGLEs (arrowheads). IGLEs were the only type of ending found on
hepatic afferent fibers innervating the cecum. Scale bars 5 200 µm.
but impossible to duplicate by randomly sparing minor
elements of the other branches of the abdominal vagus.
Third, the dmnX cell counts of the present experiment
made it possible to compare the present hepatic branch
preganglionic population with that previously estimated
(Fox and Powley, 1985). The somewhat lower counts in the
present experiment (112 6 8 vs. 215 for the left dmnX)
probably represent different counting criteria—particularly a higher threshold for cell recognition produced by
the higher background autofluorescence yielded by the
fixation protocol of the present experiment. Alternatively,
if one were to argue that the lower cell counts in the
present experiment resulted from partial surgical damage
to the hepatic branch, then it would follow that the present
survey of the distribution of hepatic branch projections,
despite its finding more extensive patterns than were
previously recognized, would be a conservative underestimate of the full projections.
Concerning the second methodological point above,
namely, the issue of selectivity of afferent labeling, it is
important that 1) the method of labeling used (WGA-HRP
into the nodose ganglion) produced at least relatively
complete labeling of vagal afferent projections to the GI
tract and 2) did not also label efferent fibers of passage. In
terms of the labeling of vagal afferents, it should be noted
that all afferent neurons in the nodose ganglion appear to
bind and transport WGA-HRP (Robertson et al., 1992). In
addition, the volume of the injections employed in the
present study was determined, on the basis of pilot work,
to infiltrate the entire ganglion with tracer and to produce
asymptotic labeling. It should also be noted that any
incompleteness of labeling would have caused us to underestimate the full extent of hepatic afferent labeling. Because the present results identified all of the characteristic
projections to the intestines and antrum suggested by the
earlier work on hepatic efferents as well as described
additional discrete fields of projections in the gut, a major
(and systematic across-animals) underestimation seems
Several observations also suggest that the innervation
patterns reported here did not result from inadvertently
labeling efferent fibers of passage. Perhaps most tellingly,
the types of hepatic branch endings observed in the
present analysis corresponded to the afferent endings
described in previous work that employed selective motor
and sensory vagotomies to distinguish unequivocally the
distinctively different types of endings in the gut (Berthoud and Powley, 1992; Berthoud et al., 1991; Powley et
al., 1994; Wang, 1995). In addition, the mechanism of
WGA-HRP labeling assures that, as demonstrated previously (Neuhuber, 1987; Pugh and Kalia, 1982), nodose
injections of the conjugate preferentially label vagal afferents. This relative selectivity for afferents results from the
combined facts that WGA-HRP binds to membrane sites
that are internalized by endocytosis and that endocytosis
typically occurs at the cell body, not at the axon. Similarly,
nodose ganglion injections of other tracers [e.g., 1,18dioctadecyl-3,3,38,38-tetramethylindo-carbocyanine perchlorate (DiI) injected in vivo; Berthoud and Powley, 1992;
Berthoud et al., 1992; Kressel et al., 1994] relying on
endocytosis also do not generally label fibers of passage.
Finally, these various tracer experiments suggest that only
damaged fibers of passage would take up and possibly
transport the marker. With such damage, retrograde, not
anterograde, labeling has typically been observed. These
arguments are consistent with the fact that we did not
observe classical efferent profiles, i.e., varicose processes
terminating in myenteric ganglia (Berthoud et al., 1990b;
Holst et al., 1997), in the present series.
In summary, then, by a variety of criteria, the WGAHRP labeling in the present experiment was both relatively complete and selective. Furthermore, sampling biases would have led to some underestimation of what is a
dense, although regionally restricted, set of projections to
the GI tract. These projections are considered below.
Stomach and pylorus
Previous studies (Powley and Wang, 1995; Sugitani et
al., 1993; Wang, 1995; Wang and Powley, 1994) have shown
that the vagal innervation of the esophagus and stomach is
predominately lateralized, with the left trunk innervating
the ventral side of the stomach and the right trunk
innervating the dorsal side of the stomach (although, for
an experiment claiming the projections are bilateral, see
Hudson, 1989). The present observations refine and extend this idea. We found that the elements of the hepatic
branch (bundles, fibers, IGLEs) of the left vagus predominately innervated the ventral side of the stomach. In
particular, it is instructive that the hepatic afferents that
originate from the left nodose express this innervation
selectivity for the ventral (or ‘‘ipsilateral’’) side of the
stomach, even though the afferent axons of the hepatic
branch reach the stomach by a very different trajectory
than the axons that stay in the ventral vagal trunk on the
esophagus and that form the ventral gastric branch.
The distribution of hepatic branch IMAs in the stomach
seems to be in apparent contrast to the pattern just
described. Unlike the distributions for bundles, individual
axons, and IGLEs, the IMA terminal specializations were
essentially evenly distributed on both sides. This discrepancy, however, may reflect the fact that the IMAs of
hepatic branch origin were located predominately on the
greater curvature, where they innervated longitudinal
muscle. In effect, these IMAs were situated on the midline
rather than throughout either side of the stomach. Consistent with the general pattern of a predominant distribution of the hepatic afferents on the ventral side, these
IMAs typically originated from axons traveling in bundles
in the ventral wall of the stomach.
The findings that hepatic IMAs are located primarily in
one gastric locus (greater curvature of the forestomach)
and that they innervate almost exclusively the longitudinal muscle layer raise the possibility that this branch is
important for the detection of a particular type of stimulus
localized to a restricted area of the organ. The type of
stimulus (possibly distention; Berthoud and Powley, 1992)
and the role of the hepatic branch in the detection of this
stimulus have yet to be elucidated. However, the fact that
overall hepatic gastric innervation was contained primarily in the forestomach indicates that this branch may play
an important role in the reservoir function (Gabella, 1987)
that is associated with this region. In addition, by supplying the forestomach with two morphologically distinct
terminal specializations, hepatic afferents may detect more
than one gastric cue. Consistent with these ideas, it is
worth noting that animals with the hepatic branch spared
but with all other abdominal vagal branches cut are still
capable of detecting gastric distension (Gonzalez and
Deutsch, 1981; Phillips and Powley, unpublished observations).
Kressel et al. (1994) found that DiI-labeled vagal afferents innervating the circular muscle of the pylorus ended
exclusively in IMAs. Due to the structural similarity of
these endings to previously described in-series tension
receptors, the authors proposed a possible functional role
for these endings in the detection of passive distention and
sphincter constriction. In agreement, we found that hepatic afferents innervated the circular muscle of the
pylorus and ended exclusively in IMAs. Although the
Kressel and coworkers experiment describes the afferent
innervation of the pylorus by either the ventral or the
dorsal trunk of the vagus, our findings take the next step
by determining the innervation of the pylorus by an
individual branch of the vagus. The fact that the hepatic
branch is only one of five vagal subdiaphragmatic branches
and yet it innervates a discrete region (about one-third) of
the pyloric torus may indicate a prominent role for this
branch in pyloric function. It should also be taken into
account that the present study only looked at the innervation of the pylorus in a superficial manner, so additional
studies looking at the innervation of the pylorus by the
hepatic branch are needed before any strong claims about
hepatic primacy can be made.
Duodenum and cecum
The first 8 cm of the duodenum were chosen for study,
because this proximal region, including the duodenal bulb,
appears to be particularly important in the feedback
control of gastric activity as well as feeding behavior.
Anatomically, this is the region of the small intestine that
receives bile duct secretory products, is attached to the
ligament of Treitz, and receives overlapping efferent projections from the gastric, celiac, and hepatic branches of the
vagus (Berthoud et al., 1991). Examination of the region
also made it possible to compare the present afferent
results with the earlier efferent projection results. Physiologically, the proximal duodenum receives the initial rush
of chyme from the stomach and has been shown to play an
important role in the detection of nutrients (Walls et al.,
1995a,b), distension, (Hölzer and Raybould, 1992) and
hormones (Smith et al., 1995).
Wang (1995) found that the left nodose ganglion provided significantly more vagal afferent innervation to the
duodenum compared with the right nodose ganglion. This
pattern led him to speculate that this difference was due to
the additional contribution of the hepatic branch. Although it is not possible to determine directly from the
present findings the percentage of the total innervation of
the duodenum contributed by the hepatic branch, through
comparisons between the present study and the Wang
(1995) study, we can make some inferences about the
contribution of the common hepatic branch to the duodenal field. It appears that the hepatic branch provides the
majority of the vagal innervation to the first 3 cm of the
small intestine. Due to the importance of this region in GI
physiology, we can only speculate that the hepatic branch,
as the prominent source of sensory feedback in this region,
must play a key role in ingestive behavior and GI physiology.
Even though the innervation of the cecum by hepatic
afferents was sparse, the present results still provide
direct evidence of hepatic vagal involvement in this organ.
In addition, one may be able to extrapolate from the
finding of hepatic afferents in the cecum as well as the
duodenum to the innervation of the intestines, which we
did not directly examine. It would be surprising to find
that the hepatic branch only innervated the small length
of intestine that we examined. A more parsimonious
picture of its innervation pattern would be that the hepatic
branch innervates the duodenum heavily at its initial site
of contact and then travels through the mesentery toward
the cecum, innervating the small intestine periodically
along its length. However, we cannot rule out the possibility that there may be other areas of the small intestine
(i.e., ileum and jejunum) that are heavily innervated by
hepatic afferents.
Implications of IMA and IGLE distributions
The present results establish that the IMAs and IGLEs
supplied by the vagal hepatic branch to the GI tract have
quite different distributions, as has been reported more
generally by Wang in his survey of all subdiaphragmatic
vagal afferents projections (Wang, 1995; Wang and Powley,
1994). Indeed, some regions innervated by the hepatic
branch (e.g., the pylorus) contain only IMAs, whereas
other regions (e.g., the duodenum and cecum) have almost
exclusively IGLEs. This spatial segregation of putative
receptors offers a useful perspective on the earlier observa-
tions of Berthoud and Powley (1992) indicating that single
afferent axons of the vagus in the forestomach could
terminate polymorphically, with some collaterals forming
IMAs and others forming IGLEs. The present results
illustrate that the overall distributions of IMAs and IGLEs
are not highly correlated and that, therefore, the polymorphic character of some fibers in the forestomach is by no
means a universal pattern of vagal afferent termination
within the muscle wall of the GI tract.
Overall, the more extensive distributions of IGLEs and
the relatively restricted regional concentrations of IMAs
reinforces the speculation that the two endings are different types of receptors. The widespread distribution of
IGLEs is consistent with the hypothesis that they serve as
mechanoreceptors that detect the shearing action of muscle
layers during movement of the wall of the GI tract
(Neuhuber, 1987; Neuhuber et al., 1995) and, even more
specifically, as receptors (as well as perhaps axon reflex
elements) involved in the coordination of peristalsis and
other intrinsic motility patterns of the gut (Wang, 1995;
Wang and Powley, 1994). In contrast the more localized
concentrations of IMAs, particularly in sphincters and in
the forestomach, suggest that these endings may operate
at strategic GI sites to detect distension provided by the
passage of ingesta and the presence of intraluminal contents (Wang, 1995).
In conclusion, the results of this study as well as others
on the hepatic branch (Berthoud et al., 1992; Prechtl and
Powley, 1987) depict a complex nerve that serves as a
pathway for fibers with numerous destinations. Not only
are these destinations spread extensively across a single
organ, they are also distributed across several abdominal
organs from the liver to the cecum. These projections
suggest that a single vagal afferent branch has the capability to detect stimuli from multiple areas that play key roles
in the maintenance of GI function and feeding behavior.
The unique anatomic innervation pattern displayed by the
hepatic branch makes it a likely candidate for orchestrating a finely tuned system of interorgan communication and
cross-organ coordination to facilitate the maintenance of
body homeostasis.
We thank M.-C. Holst and F.B. Wang for graciously
demonstrating the WGA-HRP technique as well as J. Kelly
(photography) and J. Mitchell (art work) for their expert
technical assistance. In addition, we thank Drs. Robert
Meisel, Susan Swithers, Zixi Chang, and Elwood Walls as
well as Mike Jarvanin, Fred Martinson, and Wyatt Wollmann for their comments on earlier drafts of this paper. A
preliminary report of the present findings was given in
abstract at the Annual Meeting of the Society for Neuroscience (1995). The research was performed in partial fulfillment of the Master’s degree to R.J.P.
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muscle, smooth, gastrointestinal, trace, hepatica, branch, afferent, vagus, innervation
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