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THE JOURNAL OF COMPARATIVE NEUROLOGY 416:451–460 (2000)
Origins of Cholinergic Inputs to the Cell
Bodies of Intestinofugal Neurons in the
Guinea Pig Distal Colon
ALAN E. LOMAX,* JONATHAN Y. ZHANG, AND JOHN B. FURNESS
Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3052,
Victoria, Australia
ABSTRACT
Integration of function between gut regions is mediated by means of hormones and long
neuronal reflex pathways. Intestinofugal neurons, which participate in one of these pathways,
have cell bodies within the myenteric plexus and project their axons from the gut with the
mesenteric nerves. They form excitatory synapses on neurons in prevertebral ganglia that in
turn innervate other gut regions. The aim of the present study was to characterise
immunohistochemically the synaptic input to intestinofugal neurons. The cell bodies of
intestinofugal neurons that project from the distal colon were labelled with Fast Blue that was
injected into the inferior mesenteric ganglia. Varicosities surrounding Fast Blue-labelled
neurons were analysed for immunoreactivity for the vesicular acetylcholine transporter,
vasoactive intestinal peptide, and bombesin. Most intestinofugal neurons were surrounded by
nerve terminals immunoreactive for the vesicular acetylcholine transporter; many of these
terminals also contained vasoactive intestinal peptide and bombesin immunoreactivity. This
combination of markers occurs in axons of descending interneurons. Extrinsic denervation
had no effect on the distribution of cholinergic terminals around intestinofugal neurons. A
decrease in the number of vesicular acetylcholine transporter and vasoactive intestinal
peptide immunoreactive terminals occurred around nerve cells immediately anal, but not
oral, to myotomy operations. Consistent with previous physiological studies, it is concluded
that intestinofugal neurons receive cholinergic synaptic input from other myenteric neurons,
including cholinergic descending interneurons. Thus, intestinofugal neurons are second, or
higher, order neurons in reflex pathways, although physiological data indicate that they also
respond directly to distension of the gut wall. J. Comp. Neurol. 416:451–460, 2000.
r 2000 Wiley-Liss, Inc.
Indexing terms: prevertebral ganglia; enteric nervous system; immunohistochemistry; retrograde
tracing
Physiological and anatomic studies in the 1940s revealed unusual reflexes that bypass the central nervous
system (Kuntz and van Buskirk, 1941; Kuntz and Saccomanno, 1944). These reflexes originate in the alimentary
canal and pass back to the same or more proximal regions
of the digestive tract by way of the abdominal prevertebral
ganglia. The reflexes can be elicited by distension of the
colon and by distension or chemical stimuli applied intraluminally in the small intestine (for review see Szurszewski
and Miller, 1994). Early studies demonstrated that these
intestinointestinal reflexes persist after decentralisation
of the prevertebral ganglia, although the threshold for
activating the reflexes is increased by decentralisation
(Kuntz and Saccomanno, 1944; Semba, 1954; Shapiro and
Woodward, 1959).
r 2000 WILEY-LISS, INC.
Since then, electrophysiological studies have shown that
the cell bodies of sympathetic postganglionic neurons in
prevertebral ganglia receive a prominent excitatory nicotinic synaptic input from axons arising in the viscera
(Crowcroft et al., 1971; Szurszewski and Weems, 1976;
Kreulen and Szurszewski, 1979). Retrograde tracing studies demonstrated that the peripheral inputs to the prever-
Grant sponsor: National Health and Medical Research Council of Australia; Grant number: 963213.
*Correspondence to: Alan Lomax, Department of Anatomy and Cell
Biology, University of Melbourne, Parkville 3052, Victoria, Australia.
E-mail: [email protected]
Received 28 May 1999; Revised 29 September 1999; Accepted 29 September 1999
452
A.E. LOMAX ET AL.
tebral ganglia originate from neurons with cell bodies in
the myenteric plexus (intestinofugal neurons) of all regions of the gastrointestinal tract (Kuramoto and Furness,
1989; Furness et al., 1990; Messenger and Furness, 1991,
1992, 1993; Parr et al., 1993; Mann et al., 1995).
The extent to which intestinofugal neurons are directly
responsive to distension of the colon or are stimulated
indirectly after activation of colonic intrinsic primary
afferent neurons (IPANs) is uncertain. Many physiological
studies have addressed this question without achieving a
complete answer (Crowcroft et al., 1971; Weems and
Szurszewski, 1978; Bywater, 1993; Parkman et al., 1993;
Stebbing and Bornstein, 1993; Miller and Szurszewski,
1997). For these studies, preparations of intestine and
connected prevertebral ganglia were set up in divided
organ baths. By separately superfusing the segment of gut
or the ganglia, comparisons were made of the responses of
intestinofugal neurons to distension of the wall of the gut
before and after synaptic transmission in the gut segment
was blocked by nicotinic antagonists or low calciumcontaining saline. These studies suggested that (1) intestinofugal neurons receive synaptic input from other enteric
neurons that are excited by distension, and (2) intestinofugal neurons also detect distension of the gut. Thus, it
appears that some or all intestinofugal neurons receive
synaptic input from other enteric neurons, and some or all
intestinofugal neurons are also directly distension sensitive. It is possible that there are two populations of
intestinofugal neurons, one that is directly sensitive to
distension and one that is activated by inputs from other
enteric neurons (Szurszewski and Miller, 1994; Sharkey et
al., 1998).
Sharkey et al. (1998) directly investigated whether
intestinofugal neurons receive synaptic input from other
enteric neurons by recording intracellularly from retrogradely labelled intestinofugal neurons that project from
the distal colon to the inferior mesenteric ganglia (IMG) of
the guinea pig. Fast excitatory postsynaptic potentials
(fast EPSPs) were recorded in all intestinofugal neurons in
response to stimulation of internodal strands and some
intestinofugal neurons received on-going spontaneous fast
EPSPs. The morphologies of the intestinofugal neurons
were revealed by intracellular injection of biocytin by
means of the recording electrode. None of the neurons from
which recordings were made had Dogiel type II morphology, which is the morphology of the IPANs of the guinea pig
small intestine, nor did intestinofugal neurons exhibit the
electrophysiological behaviour characteristic of the IPANs
that respond to stretch of the small intestine of the guinea
pig (Kunze et al., 1998, 1999). In the present study, we
have used a combination of retrograde neuronal tracing
with Fast Blue, immunohistochemistry, and surgical interruption of nerve pathways to identify sources of synaptic
input to intestinofugal neurons of the distal colon.
MATERIALS AND METHODS
Retrograde labelling of intestinofugal
neurons
All experimental procedures were in accordance with
the guidelines for animal welfare set out by the National
Health and Medical Research Council of Australia. Experiments were performed on 17 male guinea pigs, each
weighing between 200 and 325g, from the University of
Melbourne colony. The procedure for injection of the retro-
grade neuronal tracer, Fast Blue, was identical for all
guinea pigs and is summarised below. Each animal was
anaesthetized by subcutaneous injection of sodium pentobarbitone (15 mg/kg; Boehringer-Ingelheim, New South
Wales, Australia) 1 hour before the operation and an
intramuscular injection of a mixture of fentanyl citrate
(0.16 mg/kg) and droperidol (8.0 mg/kg) 15 minutes before
the operation. The abdomen was opened by a midline
incision and the IMG was exposed by exteriorising and
retracting the small and large intestines. Fast Blue (Sigma
Chemical Co., St Louis, MO) was dissolved in 10% dimethylsulphoxide (DMSO) in distilled water at 2% weight/
volume and injected into both lobes of the IMG by using a
bevelled glass micropipette of 60–80 µm. An average of two
injections into each lobe (rostral and caudal) was used
(total volume, 1–3 µl).
After injection of the IMG, and return of the intestines to
the body cavity, the peritoneum and abdominal muscles
were sutured and covered with an antibiotic powder,
cicatrin (Wellcome, New South Wales, Australia) and the
skin was stapled together. Each animal received a 0.1 ml
intramuscular injection of terramycin (50 mg/ml; Pfizer
Agricare, New South Wales, Australia), a broad spectrum
antibiotic.
Surgical interruption of nerve pathways
Seven to 12 days after Fast Blue was injected into the
IMG of 10 animals, operations were carried out to surgically ablate specific nerve pathways. In four animals, two
myotomies, separated by 15–70 mm, were performed. In
this procedure, the animals were anaesthetised and opened
in the same manner as before and a pair of parallel
circumferential cuts were made through the external
musculature of the distal colon, thus severing the nerve
pathways in the myenteric plexus (Messenger and Furness, 1990). A loose loop of surgical thread was tied around
the nearest mesenteric blood vessel to mark each operation site. Extrinsic denervations were performed on four
animals. The lumbar colonic nerves were located and
severed as they emerged from the IMG, denervating the
distal colon of sympathetic input and of those sensory
fibers that follow the sympathetic nerves. The surrounding
mesentery was also cut and, to ensure that no fibers in
close proximity to the inferior mesenteric artery remained
intact, the artery was lightly swabbed with cotton wool
soaked in 80% phenol. The site of the operation was
marked by a small piece of surgical thread to enable
identification in dissection. The two remaining animals
underwent combined double myotomy plus extrinsic denervation operations. After these operations, the animals
were left for a further 6–10 days to allow degeneration of
the axons that were severed.
Once enough time was allowed for the Fast Blue to fill
intestinofugal neurons as completely as possible (7–12
days) and the axonal ablations to take effect (a further
6–10 days), animals were stunned by a blow to the back of
the head, then killed by severing the carotid arteries and
the spinal cord. In the animals without myotomies, a 5- to
8-cm-long segment of the distal colon adjacent to the IMG
was removed as a control. Segments of colon were immersed in phosphate-buffered saline (PBS; 0.9% NaCl in
0.01 M sodium phosphate buffer, pH 7.2) containing the
muscle relaxant nicardipine (3 µM, Sigma). The segment
of colon was then cut along the antimesenteric border, as
most of the intestinofugal neurons lie close to the mesen-
SYNAPTIC INPUT TO INTESTINOFUGAL NEURONS
TABLE 1. Characteristics of Primary Antibodies1
453
TABLE 2. Secondary Antibodies or Streptavidin Complexes Used1
Tissue antigen
Host
Dilution
Code and reference
Antibody or streptavidin label
Dilution
Source
Calbindin
Calretinin
VAChT
Bombesin
5-HT (serotonin)
Neural NOS
NPY
Somatostatin
TK (substance P)
TH
VIP
Rabbit
Rabbit
Goat
Mouse
Rat
Rabbit
Rabbit
Mouse
Rat
Mouse
Rabbit
1:1600
1:1000
1:1000
1:100
1:500
1:200
1:1600
1:400
1:200
1:400
1:200
DEMLR 1, Furness et al., 1988
7696, Mann et al., 1997
1624, Li and Furness, 1998
2AII, Costa et al., 1984
3M55, Wardell et al., 1994
N74, Anderson et al., 1995
JM263, Maccarrone and Jarrot, 1985
S895, Buchan et al., 1985
NCI/34HL, Cuello et al., 1979
T341, Boehringer Mannhein
Furness et al., 1981
Biotinylated donkey anti-rabbit IgG
Biotinylated donkey anti-sheep IgG
Biotinylated horse anti-mouse IgG
Biotinylated horse anti-rabbit IgG
Donkey anti-rabbit IgG FITC
Donkey anti-sheep IgG FITC
Donkey anti-rat IgG FITC
Streptavidin-Texas Red
1:100
1:100
1:100
1:100
1:50
1:50
1:100
1:100
Jackson
Jackson
Vector
Jackson
Amersham
Jackson
Jackson
Amersham
1Supply companies: Amersham Pty, Ltd., Melbourne, Australia; Jackson Immunosearch
Laboratories, West Grove, PA; Vector Lab., Burlingame, CA. IgG, immunoglobulin G;
FITC, fluorescein isothiocyanate.
1VAChT, vesicular acetylcholine transporter; NOS, nitric oxide synthase; NPY, neuropeptide Y; TH, tyrosine hydroxylase; VIP, vasoactive intestinal peptide.
teric attachment (Kuramoto and Furness, 1989; Messenger and Furness, 1993), and fecal pellets were removed.
To visualise intestinofugal neurons better, the mesentery was carefully removed and the tissue pinned tautly on
balsa board. The tissue was then fixed by immersion in 2%
formaldehyde plus 0.2% picric acid in 0.1 M sodium
phosphate buffer (pH 7.0) at 4°C overnight and subsequently cleared in DMSO (3 ⫻ 10 minute changes) followed by PBS (3 ⫻ 10 minute changes). The mucosa,
submucosa, and circular muscle were removed, leaving
myenteric plexus and longitudinal muscle whole-mount
preparations.
Immunohistochemistry
Whole-mount preparations of longitudinal muscle with
attached myenteric plexus were incubated for 30 minutes
in 10% normal horse serum in 1% Triton X-100 in PBS, to
limit background staining, before exposure to primary
antisera. Excess serum was then removed, and the preparations were incubated in mixtures of primary antibodies
for 2 nights at room temperature in a humidified chamber
(Table 1). Cell body staining was also carried out on
whole-mount preparations of longitudinal muscle plus
myenteric plexus of guinea pig distal colon that had been
incubated in culture medium containing colchicine under
sterile conditions for 24 hours before fixation (Messenger
and Furness, 1990).
After incubation at room temperature in combinations of
primary antisera, tissue was washed in PBS and then
incubated for 2 hours in a mixture of secondary antibodies
(see Table 2) comprising one antibody linked to biotin and
one directly labelled with fluorescein isothiocyanate (FITC).
The tissue was then washed for 30 minutes in PBS and
incubated with streptavidin-Texas Red for 90 minutes. A
final wash in PBS was made before tissue was mounted in
glycerol buffered with 0.5 M sodium carbonate buffer (pH
8.6).
Preparations were examined on a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Oberkochen, Germany)
equipped with the appropriate filter cubes for discriminating between Fast Blue, FITC, and Texas Red fluorescence.
Preparations were scanned carefully by using the filter
cube to reveal Fast Blue fluorescence. Each successively
located Fast Blue-labelled neuron was examined by switching the filter cube to reveal fluorescence for one of the
immunohistochemical markers of synaptic terminals
within the myenteric plexus. The fluorescence observed for
these immunohistochemical markers, along with the Fast
Blue fluorescence of the intestinofugal neurons, was captured by using a Sensys 12-bit cooled CCD camera (Photometrics, Ltd., Tucson AZ) and V for Windows imaging
software (Digital Optics, Ltd., Auckland, New Zealand).
Contrast and sensitivity adjustment were carried out by
using Corel Photopaint imaging software (Corel Corporation, Dublin, Ireland). Some preparations were examined
by using a confocal scanning laser microscope system
(BioRad MRC 1024 attached to a Zeiss Axioplan fluorescence microscope). The laser was an argon/krypton mixed
gas laser with excitation and emission wavelengths 448
and 522/535 nm, respectively, for FITC and 568 and
605/632 nm for Texas Red. Samples were scanned sequentially to collect light emitted as red or green fluorescence
by using a 100⫻ oil immersion objective and ⫻1.4 zoom.
Optical sections of 0.5 µm thickness were taken in each
case. Images of 768 ⫻ 512 pixels were obtained and
processed by using Confocal Assistant and Corel Photopaint.
Retrogradely labelled neurons were evaluated under the
microscope and in the stored images, to determine whether
they were surrounded by pericellular baskets of immunohistochemically labelled nerve terminals. To do this, we
followed the criteria of Pompolo and Furness (1995) for the
identification of baskets. Pericellular baskets consist of
closely spaced axonal varicosities that are juxtaposed to
cell bodies. They are at a greater density around innervated cells than in the neuropil, or in relation to uninnervated cells, and ultrastructural examination shows that
varicosities in baskets form synapses on the cell bodies
that they surround (Pompolo and Furness 1995; Mann et
al., 1997).
RESULTS
Injection of Fast Blue into the IMG resulted in labelling
of neuronal perikarya in the myenteric plexus of the distal
colon. The staining of these neurons was usually restricted
to the cytoplasm, although occasional nuclear staining was
also observed. The shapes of neurons were not as well
defined as in previous studies, in which tissue was generally taken after 4–10 days. In the present work, 13–22
days elapsed, so that there was time for retrograde accumulation before nerves were severed, and then time for
degeneration of the severed fibers. In this time, much of
the Fast Blue relocated to lysosomes away from the cell
surface and dendrites (e.g., Fig. 1). Retrogradely labelled
nerve cells were distributed around the circumference of
the colon, although they were more numerous at the
mesenteric aspect of the gut, as previously reported (Furness et al., 1990). Neurons whose shapes were revealed fell
into two reasonably distinct morphologic groups. The first
group had many short lamellar dendrites, whereas the
second group had one or more lamellar dendrites but also
had up to five longer dendritic processes that had distinct
irregularities on their surfaces. Both cell types correspond
454
Fig. 1. Fluorescence photomicrographs showing the immunoreactivities of nerve terminals that surround the cell bodies of intestinofugal neurons that were retrogradely labelled with Fast Blue.
A,A8: Images of the same field illuminated to reveal Fast Blue (A) and
vesicular acetylcholine transporter (VAChT) immunoreactive (-IR)
(A8). The image in A is the cell body of an intestinofugal neuron that is
surrounded by a basket-like arrangement of VAChT-IR nerve terminals (image in A8). Most of the Fast Blue is in lysosomes in this neuron.
B,B8,B9: Another field illuminated to reveal Fast Blue (B), VAChT-IR
A.E. LOMAX ET AL.
(B8), and vasoactive intestinal peptide (VIP) -IR (B9). The intestinofugal neuron (B) is surrounded by nerve terminals that are immunoreactive for VAChT (B8) and VIP (B9). Arrows indicate where VAChT-IR
and VIP-IR appear to colocalise within varicosities. C: An intestinofugal neuron that is surrounded by VIP-IR (C8) and bombesin-IR (BN;
C9) terminals. Arrows indicate colocalisation of VIP-IR and bombesin-IR within varicosities. Scale bars ⫽ 10 µm in A,B (apply to
A,A8,B,B8,B9), 20 µm in C (applies to C,C8,C9).
SYNAPTIC INPUT TO INTESTINOFUGAL NEURONS
to the class of neurons designated by Dogiel (1899) as type
I (see Sharkey et al., 1998).
Preparations of longitudinal muscle plus myenteric
plexus containing Fast Blue labelled intestinofugal neurons were stained with a variety of immunohistochemical
markers of nerve terminals of populations of myenteric
neurons. Fast Blue-labelled nerve cell bodies were commonly surrounded by immunoreactive (IR) varicosities
which outlined cell bodies, sometimes forming several
layers of varicosities (Fig. 1A,A8). Such relationships between varicosities and cell bodies, described as pericellular
baskets, have been examined by electron microscopy, and
it had been found that varicosities within these baskets
form synapses with the cell bodies (Pompolo and Furness,
1995). One hundred ninety-nine of 215 nerve cell bodies
(93%) were surrounded by basket-like arrangements of
axon varicosities that were immunoreactive for the vesicular acetylcholine transporter (VAChT), which is a marker
of cholinergic nerve terminals (Fig. 1A,A8,B,B8). The cell
bodies of intestinofugal neurons were also examined for
their relation to vasoactive intestinal peptide (VIP)-IR
and bombesin-IR terminals in preparations stained for
VAChT plus VIP or bombesin plus VIP. One hundred
sixty-nine of 212 intestinofugal neurons (80%) were surrounded by VIP-IR varicosities, whereas 77 of 88 intestinofugal neurons (88%) examined were surrounded by
bombesin-IR terminals (Fig. 1B,B88,C,C88). These doublestaining experiments showed that terminals that were
immunoreactive for each of these markers formed baskets
around intestinofugal neurons and that these markers
were commonly colocalised within individual varicosities
(Fig. 1B8,B88,C8,C8).
Varicose terminals with bombesin-IR were found around
other nerve cells, but some nerve cells were not surrounded by bombesin-IR varicosities. Bombesin-IR varicosities surrounded most bombesin-IR nerve cells (Fig. 3A,B).
Descending inputs
Myotomy and extrinsic denervation operations were
performed to identify the projection patterns of cholinergic
neurons that innervate intestinofugal neurons. In preparations from the animals in which myotomy operations were
performed, there was a decrease in the innervation of
intestinofugal neurons in the 10 mm on the anal sides of
the lesions and quantitative data presented below on the
effects of myotomy operations was obtained from the first
10 mm anal or oral to a myotomy. There was a dramatic
reduction in the number of intestinofugal neurons that
were surrounded by VAChT-IR and VIP-IR varicosities
just anal to a myotomy operation (i.e., where the axons of
neurons with descending (oral to anal) projections had
been severed and allowed to degenerate). In untreated
animals, 93% of intestinofugal neurons examined were
surrounded by VAChT-IR terminals (see above), whereas
after a myotomy operation performed oral to the area
examined 37 of 141 intestinofugal neurons (26%) had
VAChT innervation that was similar to controls; a further
40 of 141 intestinofugal neurons (28%) had few VAChT-IR
terminals close to them, insufficient to be recognised as a
pericellular basket (Fig. 2B,B8), and the remaining 64
neurons (46%) had no VAChT-IR terminals close to the cell
bodies. In areas just anal to a myotomy, 10 of 87 (12%)
intestinofugal neurons had normal innervation by VIP-IR
terminals; a further 18 of 87 (20%) had reduced innervation and 59 of 87 (68%) had no close VIP-IR terminals (Fig.
455
2C,C8). The innervation of intestinofugal neurons by VIP-IR
varicosities returned to normal about 10 mm anal to the
myotomy operation site. This finding is consistent with the
findings of Messenger and Furness (1990), who reported
that VIP fibers in myenteric ganglia in the guinea pig colon
were reduced by oral myotomy operations but that normal
VIP innervation of myenteric ganglia was observed within
approximately 8 mm anal to the site of the myotomy.
Ascending inputs
The effects of myotomy operations that interrupt ascending fibers within the myenteric plexus on the innervation
of intestinofugal neurons by VAChT-IR terminals were
examined. In the 10 mm oral to myotomies, 51 of 60
intestinofugal neurons (85%) had normal VAChT innervation, suggesting that ascending pathways do not contribute significantly to the innervation of intestinofugal neurons by cholinergic terminals (Fig. 2A,A8).
Extrinsic inputs
Extrinsic denervation operations were performed to
determine the contribution of extrinsic neurons to the
innervation of intestinofugal neurons, and immunohistochemistry for tyrosine hydroxylase (TH) was used to
monitor the effectiveness of the denervation. In preparations from unoperated animals, TH-IR fibers formed a
dense network within myenteric ganglia, whereas after
extrinsic denervation, only rare TH-IR fibers could be
seen, consistent with previous findings (Furness, 1970). In
segments of colon that had been extrinsically denervated,
36 of 43 intestinofugal neurons (84%) received normal
VAChT innervation. In the two animals that had undergone both extrinsic denervation and myotomy operations,
there was a reduction in VAChT innervation anal to the
myotomies that recovered at about 1 cm anal as described
above, but further anal between the myotomy operations
no reduction was observed. These results, along with the
finding of only infrequent innervation of intestinofugal
neurons by TH-IR fibers in untreated preparations (5 of
36; 14%, Table 3) suggest that the innervation of intestinofugal neurons originates predominantly within the gut
wall.
Because VAChT, VIP, and bombesin were localised in
many terminals that surrounded intestinofugal neurons,
we investigated whether there were myenteric nerve cell
bodies with the appropriate neurochemistry to be a source
of the terminals. Staining of myenteric neurons of the
guinea pig distal colon that had been pretreated with
colchicine to enhance cell body immunoreactivity for peptides revealed that choline acetyltransferase (ChAT), VIP,
bombesin, and nitric oxide synthase (NOS) immunoreactivities are colocalised in a subset of nerve cells (Fig. 3). Many
of these nerve cells were surrounded by bombesin-IR
terminals. Sixty-six of 100 bombesin-IR myenteric nerve
cell bodies were ChAT-IR, 50 of 56 bombesin-IR were
VIP-IR, and 78 of 79 were NOS-IR.
We also examined whether intestinofugal neurons had
terminals surrounding them that were immunoreactive
for neurochemical markers of subclasses of myenteric
neurons other than VAChT, VIP, and bombesin (Table 3).
No other markers stained terminals that surrounded such
a large proportion of intestinofugal neurons. A complication that made accurate counting of the number of intestinofugal neurons that were surrounded by varicosities
immunoreactive for a particular substance was that the
456
A.E. LOMAX ET AL.
Fig. 2. Paired photomicrographs that show the effects of myotomy
operations on the distribution of labelled nerve terminals around
intestinofugal neurons. A: An intestinofugal neuron in a region within
5 mm oral to a myotomy that caused degeneration of ascending fibers
is surrounded by vesicular acetylcholine transporter (VAChT) –immunoreactive (-IR) terminals (A8). Note that the labelled varicosities
appear to be closely aligned with the surface of the cell body and with
one of its dendrites (arrow). B: An intestinofugal neuron from a region
within 5 mm anal to a myotomy that severed descending axons has a
marked reduction in the number of VAChT-IR terminals that approach
the cell body (B8). No labelled terminals can be seen to contact the cell
body or its processes. C: Another intestinofugal neuron from a region
where descending axons have been severed. In this case, there is an
obvious reduction in the amount of vasoactive intestinal peptide (VIP)
-IR terminals in the ganglion (C8, compare with Fig. 1B9 and Fig. 1C8)
and no immunoreactive terminals can be seen close to the cell body.
Scale bars ⫽ 15 µm in A–C (apply to A–C8).
cell bodies of intestinofugal neurons are immunoreactive
for NOS, bombesin, VIP, calretinin, calbindin, and ChAT
(Furness et al., 1990; Mann et al., 1995). This was not a
problem with bombesin and VIP, because cell body staining of myenteric neurons with these markers is weak
without colchicine treatment, whereas cell body staining
of intestinofugal neurons with calbindin, calretinin, and
NOS is intense and precludes accurate assessment of
whether cell bodies of intestinofugal neurons are surrounded by terminals immunoreactive for these markers.
To determine the proportion of intestinofugal neurons with
inputs from calbindin-, calretinin-, or NOS-IR terminals,
only retrogradely labelled nerve cell bodies that did not
have the same immunohistochemistry were examined. Of
SYNAPTIC INPUT TO INTESTINOFUGAL NEURONS
TABLE 3. Incidence of Pericellular Baskets
Markers
used1
Number
of intestinofugal
neurons examined
Number of
intestinofugal
neurons surrounded
by terminals (%)
88
30
47
69
17
36
77 (88)
7 (23)
6 (13)
15 (23)
12 (70)
5 (14)
215
141
60
43
199 (93)
37 (26)
51 (85)
36 (84)
212
87
169 (80)
10 (12)
Bombesin
Calbindin
Calretinin
NOS
Substance P
TH
VAChT
Normal colon
Anal to myotomy
Oral to myotomy
After extrinsic denervation
VIP
Normal colon
Anal to myotomy
1For
abbreviations, see footnote to Table 1.
the intestinofugal neurons whose cell bodies were not
immunoreactive for calretinin, calbindin or NOS, 6 of 47
(13%) were surrounded by calretinin-IR terminals, 7 of 30
(23%) by calbindin-IR terminals, and 15 of 69 (23%) by
NOS-IR terminals respectively (Table 3).
DISCUSSION
Sources of innervation
We have found that the cell bodies of intestinofugal
neurons that project to the inferior mesenteric ganglia
from the distal colon are surrounded by cholinergic nerve
terminals. The possible sources of these terminals are
intrinsic neurons, extrinsic neurons whose axons reach the
colon by means of the mesenteric nerves, vagal axons that
take an intramural course to reach the colon and branches
of the pelvic nerves that run orally within the gut wall
(intramural pelvic nerves; see Furness and Costa, 1987).
None of the extrinsic sources seem to contribute significantly. When the mesenteric nerves were severed and their
endings allowed to degenerate, there was no detectable
loss of innervation of the cell bodies of intestinofugal
neurons. Moreover, in the region between two circumferential myotomies, which would sever both vagal fibers descending within the gut wall and ascending fibers from the
pelvic nerves, normal innervation persisted in ganglia
toward the anal part of the neurally isolated segment. The
appearance was the same if the two myotomies and the
mesenteric nerve section were performed together. Thus,
the pericellular fibers at the distal part of the segment
between myotomies must arise from neurons with cell
bodies within the neurally isolated segment, and, because
the loss of nerve terminals occurred at the oral part of this
segment only, the neurons must be anally projecting.
However, not all fibers were lost from the oral region. The
remaining fibers are, therefore, of local origin, either from
cell bodies in myenteric ganglia or from cell bodies in
submucosal ganglia. The lack of detectable loss of innervation oral to circumferential lesions of intramural nerve
pathways indicates that ascending fibers do not contribute
significantly to the innervation of the cell bodies of intestinofugal neurons.
We have identified the class of cholinergic descending
interneuron that makes a large contribution to the cholinergic innervation of intestinofugal neurons. VAChT, VIP,
and bombesin all labelled nerve terminals that surround
the majority of intestinofugal neurons. Moreover, by exam-
457
ining the cell bodies of myenteric neurons in preparations
of distal colon that had been pretreated with colchicine, a
class of descending interneurons was identified that was
immunoreactive for ChAT, VIP, and bombesin. In studies
on the myenteric plexus of guinea pig distal colon that
used single label immunohistochemistry, descending interneurons with immunoreactivity for VIP, NOS, and bombesin have been identified (Messenger and Furness 1990;
McConalogue and Furness, 1993; present results). Bombesin/VIP-IR terminals also surrounded many other nerve
cells, including other bombesin-IR neurons, but there were
cells without this innervation; thus, these descending
interneurons evidently make specific connections with
intestinofugal neurons, descending interneurons, and other
classes of neurons, which, by analogy with the small
intestine, probably include inhibitory motor neurons
(Kunze and Furness, 1999).
Relations of labelled nerve terminals
to nerve cell bodies
The inputs to the intestinofugal neurons have been
examined by fluorescence microscopy. Dense baskets of
cholinergic (VAChT-IR) varicosities were closely apposed
to the nerve cells. Electrophysiological studies support the
conclusion that the baskets of VAChT-IR varicosities represent true synapses, because fast EPSPs were recorded
from the intestinofugal neurons, and, although this was
tested in only a few cases, the fast EPSPs were blocked by
an antagonist of cholinergic transmission, hexamethonium (Sharkey et al., 1998). Similar associations of baskets of terminals have been examined by electron microscopy, and in all cases, many of the varicosities were found
to form true synapses (showing presynaptic accumulations
of vesicles and synaptic densities), whereas some varicosities formed close contacts (closely apposed membranes,
presynaptic accumulations of vesicles but no discernible
synaptic densities). The presence of synapses within baskets of immunoreactive terminals in enteric ganglia has
been confirmed by electron microscopy and includes calretinin-IR inputs to calretinin-IR interneurons (Pompolo and
Furness, 1993), somatostatin-IR inputs to somatostatin-IR interneurons (Portbury et al., 1995; Pompolo and
Furness, 1998), NOS-IR inputs to VIP-IR secretomotor
neurons (Li et al., 1995), bombesin-IR inputs to calretinin-IR motor neurons (Pompolo and Furness, 1995),
NOS-IR inputs to NOS-IR motor neurons (Young et al.,
1995), 5-HT-IR inputs to 5-HT-IR interneurons (Young and
Furness, 1995), and somatostatin-IR inputs to NOS-IR cell
bodies (Mann et al., 1997).
The numbers of varicosities seen in apposition to nerve
cells with fluorescence immunohistochemical methods is
likely to be an overestimate of the number of true synaptic
inputs (Mann et al., 1997). This is because some of the
varicosities are separated from the surfaces of nerve cells
by glial cell processes.
Physiological significance
Bywater (1993) made the interesting observation that
when synaptic transmission within the colon was blocked,
the initial response of intestinofugal neurons to distension
was not significantly reduced, whereas the response after
10–15 seconds of sustained distension was almost abolished. This finding suggests that reflexes that pass from
the gut by means of intestinofugal neurons to the prevertebral ganglia have two components: one that is a direct
458
A.E. LOMAX ET AL.
Fig. 3. Paired photomicrographs illustrating the cell body immunoreactivities of the class of descending interneuron that supplies
terminals that surround the cell bodies of intestinofugal neurons
within the myenteric plexus of guinea pig distal colon. A,A8,B,B8: Confocal microscope images, optical thickness 0.7 µm. C,C8: Photomicrographs acquired by using conventional fluorescence illumination.
These descending interneurons are immunoreactive for bombesin
(BN), vasoactive intestinal peptide (VIP), choline acetyltransferase
(ChAT), and nitric oxide synthase (NOS). Bombesin immunoreactivity
(A,B,C), colocalised with VIP-immunoreactive (-IR) (A8), ChAT-IR (B8),
and NOS-IR (C8). These neurons receive bombesin-IR varicose terminals, as can be seen in A and B. Arrows indicate cell bodies where
colocalisation occurs. Scale bars ⫽ 15 µm in A–C (apply to A–C8).
response of intestinofugal neurons to distension and the
other that is a response to synaptic input from other
enteric neurons to intestinofugal neurons (i.e., intestinofugal neurons are indirectly activated by the distension
stimulus). Szurszewski and Miller (1994) have suggested
that intestinofugal neurons may form a homogenous population of neurons that are both directly and indirectly
activated. Evidence in support of this theory was provided
by Sharkey et al. (1998), who found that all intestinofugal
neurons from which intracellular membrane potential
recordings were taken had S-type electrophysiological
characteristics and had Dogiel type I morphology; in other
words, they appear to form a homogenous population.
Experimental evidence also suggests that all intestinofugal neurons have the same primary transmitter, acetylcholine (Szurszweski and Miller, 1994; Mann et al., 1995;
Sharkey et al., 1998). The results of the present study
support the idea that intestinofugal neurons are a population that is homogenous in its most significant characteristics. Ninety-three percent of intestinofugal neurons were
surrounded by basket-like arrangements of VAChT-IR
varicosities. It is not possible to say that the other 7% are
not innervated by cholinergic nerve terminals because the
VAChT-IR terminals may not always form a distinct
pericellular basket and may also contact dendrites of
intestinofugal neurons that are not revealed by Fast Blue
fluorescence. On the other hand, we found that about 25%
of the intestinofugal neurons were surrounded by calbindin terminals. Calbindin is a marker of Dogiel type II
neurons, which are presumed to be IPANs in the colon
(Lomax et al., 1999) but is also contained in other types of
neuron. Thus, it is possible that a minority of intestinofugal neurons receive direct inputs from IPANs.
There is strong experimental evidence that intestinofugal neurons are length sensors (Szurszewski and Miller,
1994), whereas there is good evidence that the IPANs of
the ileum respond to tension generated in the external
muscle in response to stretch rather than the stretch per
se, i.e., they are tension detectors (Kunze et al., 1998,
1999). These IPANs have Dogiel type II morphology and
AH electrophysiological characteristics (Furness et al.,
1998). A recent study has revealed the neurons in the
myenteric plexus of the guinea pig distal colon that have
similar morphologic, immunohistochemical, and electrophysiological characteristics to the IPANs of the ileum
(Lomax et al., 1999). The observations of Bywater (1993)
might be accounted for if intestinofugal neurons respond
to changes in length directly, which would account for the
SYNAPTIC INPUT TO INTESTINOFUGAL NEURONS
initial response to distension that persists after blockade
of synaptic transmission within the gut. The slowly developing increase in tension in response to this distension
might excite the IPANs, but not the intestinofugal neurons
directly. These IPANs then excite each other and provide
input to the descending interneurons, which in turn
provide fast synaptic input to the intestinofugal neurons.
Convergence of fast excitatory postsynaptic potentials
onto the intestinofugal neurons would cause action potential firing in the intestinofugal neurons in response to
distension (by means of the increase in tension) that would
be sensitive to synaptic blockade.
Neurons with similar chemical coding (ChAT, VIP,
bombesin, NOS) to the class of descending interneuron
that provides cholinergic input to intestinofugal neurons
in the distal colon have been examined in the guinea pig
small intestine. Anatomic and pharmacologic studies indicate that this class of interneuron forms descending chains
and that these neurons innervate the inhibitory motor
neurons that relax the circular muscle (Young et al., 1995;
Yuan et al., 1995; Kunze and Furness, 1999), i.e., they are
conduits of local descending reflex pathways in the small
intestine. It is likely that similar descending interneurons
in the distal colon that innervate the intestinofugal neurons are also involved in local (intrinsic) reflex pathways in
the colon because, as discussed above, they innervate other
nerve cells in the myenteric ganglia. This raises the
interesting possibility that the sustained response to distension that is observed in intestinofugal neurons is
controlled by the same class of descending interneuron
that is involved in local descending inhibitory reflexes in
the gut.
ACKNOWLEDGMENTS
We thank Dr. Colin Anderson and Dr. Heather Young for
expert advice on the manuscript and Heather Woodman
and Clare Delaney for excellent technical assistance.
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