Origins of cholinergic inputs to the cell bodies of intestinofugal neurons in the guinea pig distal colon

Origins of Cholinergic Inputs to the CellBodies 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

ABSTRACTIntegration 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 themesenteric nerves. They form excitatory synapses on neurons in prevertebral ganglia that inturn innervate other gut regions. The aim of the present study was to characteriseimmunohistochemically the synaptic input to intestinofugal neurons. The cell bodies ofintestinofugal neurons that project from the distal colon were labelled with Fast Blue that wasinjected into the inferior mesenteric ganglia. Varicosities surrounding Fast Blue-labelledneurons were analysed for immunoreactivity for the vesicular acetylcholine transporter,vasoactive intestinal peptide, and bombesin. Most intestinofugal neurons were surrounded bynerve terminals immunoreactive for the vesicular acetylcholine transporter; many of theseterminals also contained vasoactive intestinal peptide and bombesin immunoreactivity. Thiscombination of markers occurs in axons of descending interneurons. Extrinsic denervationhad no effect on the distribution of cholinergic terminals around intestinofugal neurons. Adecrease in the number of vesicular acetylcholine transporter and vasoactive intestinalpeptide immunoreactive terminals occurred around nerve cells immediately anal, but notoral, to myotomy operations. Consistent with previous physiological studies, it is concludedthat intestinofugal neurons receive cholinergic synaptic input from other myenteric neurons,including cholinergic descending interneurons. Thus, intestinofugal neurons are second, orhigher, order neurons in reflex pathways, although physiological data indicate that they alsorespond 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 re-vealed unusual reflexes that bypass the central nervoussystem (Kuntz and van Buskirk, 1941; Kuntz and Sacco-manno, 1944). These reflexes originate in the alimentarycanal and pass back to the same or more proximal regionsof the digestive tract by way of the abdominal prevertebralganglia. The reflexes can be elicited by distension of thecolon and by distension or chemical stimuli applied intralu-minally in the small intestine (for review see Szurszewskiand Miller, 1994). Early studies demonstrated that theseintestinointestinal reflexes persist after decentralisationof the prevertebral ganglia, although the threshold foractivating the reflexes is increased by decentralisation(Kuntz and Saccomanno, 1944; Semba, 1954; Shapiro andWoodward, 1959).

Since then, electrophysiological studies have shown thatthe cell bodies of sympathetic postganglionic neurons inprevertebral ganglia receive a prominent excitatory nico-tinic synaptic input from axons arising in the viscera(Crowcroft et al., 1971; Szurszewski and Weems, 1976;Kreulen and Szurszewski, 1979). Retrograde tracing stud-ies demonstrated that the peripheral inputs to the prever-

Grant sponsor: National Health and Medical Research Council of Austra-lia; Grant number: 963213.

*Correspondence to: Alan Lomax, Department of Anatomy and CellBiology, University of Melbourne, Parkville 3052, Victoria, Australia.E-mail: [email protected]

Received 28 May 1999; Revised 29 September 1999; Accepted 29 Septem-ber 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 416:451–460 (2000)

r 2000 WILEY-LISS, INC.

tebral ganglia originate from neurons with cell bodies inthe myenteric plexus (intestinofugal neurons) of all re-gions 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 directlyresponsive to distension of the colon or are stimulatedindirectly after activation of colonic intrinsic primaryafferent neurons (IPANs) is uncertain. Many physiologicalstudies have addressed this question without achieving acomplete answer (Crowcroft et al., 1971; Weems andSzurszewski, 1978; Bywater, 1993; Parkman et al., 1993;Stebbing and Bornstein, 1993; Miller and Szurszewski,1997). For these studies, preparations of intestine andconnected prevertebral ganglia were set up in dividedorgan baths. By separately superfusing the segment of gutor the ganglia, comparisons were made of the responses ofintestinofugal neurons to distension of the wall of the gutbefore and after synaptic transmission in the gut segmentwas blocked by nicotinic antagonists or low calcium-containing saline. These studies suggested that (1) intesti-nofugal neurons receive synaptic input from other entericneurons that are excited by distension, and (2) intestinofu-gal neurons also detect distension of the gut. Thus, itappears that some or all intestinofugal neurons receivesynaptic input from other enteric neurons, and some or allintestinofugal neurons are also directly distension sensi-tive. It is possible that there are two populations ofintestinofugal neurons, one that is directly sensitive todistension and one that is activated by inputs from otherenteric neurons (Szurszewski and Miller, 1994; Sharkey etal., 1998).

Sharkey et al. (1998) directly investigated whetherintestinofugal neurons receive synaptic input from otherenteric neurons by recording intracellularly from retro-gradely labelled intestinofugal neurons that project fromthe distal colon to the inferior mesenteric ganglia (IMG) ofthe guinea pig. Fast excitatory postsynaptic potentials(fast EPSPs) were recorded in all intestinofugal neurons inresponse to stimulation of internodal strands and someintestinofugal neurons received on-going spontaneous fastEPSPs. The morphologies of the intestinofugal neuronswere revealed by intracellular injection of biocytin bymeans of the recording electrode. None of the neurons fromwhich recordings were made had Dogiel type II morphol-ogy, which is the morphology of the IPANs of the guinea pigsmall intestine, nor did intestinofugal neurons exhibit theelectrophysiological behaviour characteristic of the IPANsthat respond to stretch of the small intestine of the guineapig (Kunze et al., 1998, 1999). In the present study, wehave used a combination of retrograde neuronal tracingwith Fast Blue, immunohistochemistry, and surgical inter-ruption of nerve pathways to identify sources of synapticinput to intestinofugal neurons of the distal colon.

MATERIALS AND METHODS

Retrograde labelling of intestinofugalneurons

All experimental procedures were in accordance withthe guidelines for animal welfare set out by the NationalHealth and Medical Research Council of Australia. Experi-ments were performed on 17 male guinea pigs, eachweighing between 200 and 325g, from the University ofMelbourne colony. The procedure for injection of the retro-

grade neuronal tracer, Fast Blue, was identical for allguinea pigs and is summarised below. Each animal wasanaesthetized by subcutaneous injection of sodium pento-barbitone (15 mg/kg; Boehringer-Ingelheim, New SouthWales, Australia) 1 hour before the operation and anintramuscular injection of a mixture of fentanyl citrate(0.16 mg/kg) and droperidol (8.0 mg/kg) 15 minutes beforethe operation. The abdomen was opened by a midlineincision and the IMG was exposed by exteriorising andretracting the small and large intestines. Fast Blue (SigmaChemical Co., St Louis, MO) was dissolved in 10% dimeth-ylsulphoxide (DMSO) in distilled water at 2% weight/volume and injected into both lobes of the IMG by using abevelled glass micropipette of 60–80 µm. An average of twoinjections into each lobe (rostral and caudal) was used(total volume, 1–3 µl).

After injection of the IMG, and return of the intestines tothe body cavity, the peritoneum and abdominal muscleswere sutured and covered with an antibiotic powder,cicatrin (Wellcome, New South Wales, Australia) and theskin was stapled together. Each animal received a 0.1 mlintramuscular injection of terramycin (50 mg/ml; PfizerAgricare, New South Wales, Australia), a broad spectrumantibiotic.

Surgical interruption of nerve pathways

Seven to 12 days after Fast Blue was injected into theIMG of 10 animals, operations were carried out to surgi-cally ablate specific nerve pathways. In four animals, twomyotomies, separated by 15–70 mm, were performed. Inthis procedure, the animals were anaesthetised and openedin the same manner as before and a pair of parallelcircumferential cuts were made through the externalmusculature of the distal colon, thus severing the nervepathways in the myenteric plexus (Messenger and Fur-ness, 1990). A loose loop of surgical thread was tied aroundthe nearest mesenteric blood vessel to mark each opera-tion site. Extrinsic denervations were performed on fouranimals. The lumbar colonic nerves were located andsevered as they emerged from the IMG, denervating thedistal colon of sympathetic input and of those sensoryfibers that follow the sympathetic nerves. The surroundingmesentery was also cut and, to ensure that no fibers inclose proximity to the inferior mesenteric artery remainedintact, the artery was lightly swabbed with cotton woolsoaked in 80% phenol. The site of the operation wasmarked by a small piece of surgical thread to enableidentification in dissection. The two remaining animalsunderwent combined double myotomy plus extrinsic dener-vation operations. After these operations, the animalswere left for a further 6–10 days to allow degeneration ofthe axons that were severed.

Once enough time was allowed for the Fast Blue to fillintestinofugal neurons as completely as possible (7–12days) and the axonal ablations to take effect (a further6–10 days), animals were stunned by a blow to the back ofthe head, then killed by severing the carotid arteries andthe spinal cord. In the animals without myotomies, a 5- to8-cm-long segment of the distal colon adjacent to the IMGwas removed as a control. Segments of colon were im-mersed in phosphate-buffered saline (PBS; 0.9% NaCl in0.01 M sodium phosphate buffer, pH 7.2) containing themuscle relaxant nicardipine (3 µM, Sigma). The segmentof colon was then cut along the antimesenteric border, asmost of the intestinofugal neurons lie close to the mesen-

452 A.E. LOMAX ET AL.

teric attachment (Kuramoto and Furness, 1989; Messen-ger and Furness, 1993), and fecal pellets were removed.

To visualise intestinofugal neurons better, the mesen-tery was carefully removed and the tissue pinned tautly onbalsa board. The tissue was then fixed by immersion in 2%formaldehyde plus 0.2% picric acid in 0.1 M sodiumphosphate buffer (pH 7.0) at 4°C overnight and subse-quently cleared in DMSO (3 3 10 minute changes) fol-lowed by PBS (3 3 10 minute changes). The mucosa,submucosa, and circular muscle were removed, leavingmyenteric plexus and longitudinal muscle whole-mountpreparations.

Immunohistochemistry

Whole-mount preparations of longitudinal muscle withattached myenteric plexus were incubated for 30 minutesin 10% normal horse serum in 1% Triton X-100 in PBS, tolimit background staining, before exposure to primaryantisera. Excess serum was then removed, and the prepa-rations were incubated in mixtures of primary antibodiesfor 2 nights at room temperature in a humidified chamber(Table 1). Cell body staining was also carried out onwhole-mount preparations of longitudinal muscle plusmyenteric plexus of guinea pig distal colon that had beenincubated in culture medium containing colchicine understerile conditions for 24 hours before fixation (Messengerand Furness, 1990).

After incubation at room temperature in combinations ofprimary antisera, tissue was washed in PBS and thenincubated for 2 hours in a mixture of secondary antibodies(see Table 2) comprising one antibody linked to biotin andone directly labelled with fluorescein isothiocyanate (FITC).The tissue was then washed for 30 minutes in PBS andincubated with streptavidin-Texas Red for 90 minutes. Afinal wash in PBS was made before tissue was mounted inglycerol buffered with 0.5 M sodium carbonate buffer (pH8.6).

Preparations were examined on a Zeiss Axioplan fluores-cence microscope (Carl Zeiss, Oberkochen, Germany)equipped with the appropriate filter cubes for discriminat-ing between Fast Blue, FITC, and Texas Red fluorescence.Preparations were scanned carefully by using the filtercube to reveal Fast Blue fluorescence. Each successivelylocated Fast Blue-labelled neuron was examined by switch-ing the filter cube to reveal fluorescence for one of theimmunohistochemical markers of synaptic terminalswithin the myenteric plexus. The fluorescence observed forthese immunohistochemical markers, along with the FastBlue fluorescence of the intestinofugal neurons, was cap-tured by using a Sensys 12-bit cooled CCD camera (Photo-metrics, Ltd., Tucson AZ) and V for Windows imaging

software (Digital Optics, Ltd., Auckland, New Zealand).Contrast and sensitivity adjustment were carried out byusing Corel Photopaint imaging software (Corel Corpora-tion, Dublin, Ireland). Some preparations were examinedby using a confocal scanning laser microscope system(BioRad MRC 1024 attached to a Zeiss Axioplan fluores-cence microscope). The laser was an argon/krypton mixedgas laser with excitation and emission wavelengths 448and 522/535 nm, respectively, for FITC and 568 and605/632 nm for Texas Red. Samples were scanned sequen-tially to collect light emitted as red or green fluorescenceby using a 1003 oil immersion objective and 31.4 zoom.Optical sections of 0.5 µm thickness were taken in eachcase. Images of 768 3 512 pixels were obtained andprocessed by using Confocal Assistant and Corel Photo-paint.

Retrogradely labelled neurons were evaluated under themicroscope and in the stored images, to determine whetherthey were surrounded by pericellular baskets of immuno-histochemically labelled nerve terminals. To do this, wefollowed the criteria of Pompolo and Furness (1995) for theidentification of baskets. Pericellular baskets consist ofclosely spaced axonal varicosities that are juxtaposed tocell bodies. They are at a greater density around inner-vated cells than in the neuropil, or in relation to uninner-vated cells, and ultrastructural examination shows thatvaricosities in baskets form synapses on the cell bodiesthat they surround (Pompolo and Furness 1995; Mann etal., 1997).

RESULTS

Injection of Fast Blue into the IMG resulted in labellingof neuronal perikarya in the myenteric plexus of the distalcolon. The staining of these neurons was usually restrictedto the cytoplasm, although occasional nuclear staining wasalso observed. The shapes of neurons were not as welldefined as in previous studies, in which tissue was gener-ally taken after 4–10 days. In the present work, 13–22days elapsed, so that there was time for retrograde accumu-lation before nerves were severed, and then time fordegeneration of the severed fibers. In this time, much ofthe Fast Blue relocated to lysosomes away from the cellsurface and dendrites (e.g., Fig. 1). Retrogradely labellednerve cells were distributed around the circumference ofthe colon, although they were more numerous at themesenteric aspect of the gut, as previously reported (Fur-ness et al., 1990). Neurons whose shapes were revealed fellinto two reasonably distinct morphologic groups. The firstgroup had many short lamellar dendrites, whereas thesecond group had one or more lamellar dendrites but alsohad up to five longer dendritic processes that had distinctirregularities on their surfaces. Both cell types correspond

TABLE 2. Secondary Antibodies or Streptavidin Complexes Used1

Antibody or streptavidin label Dilution Source

Biotinylated donkey anti-rabbit IgG 1:100 JacksonBiotinylated donkey anti-sheep IgG 1:100 JacksonBiotinylated horse anti-mouse IgG 1:100 VectorBiotinylated horse anti-rabbit IgG 1:100 JacksonDonkey anti-rabbit IgG FITC 1:50 AmershamDonkey anti-sheep IgG FITC 1:50 JacksonDonkey anti-rat IgG FITC 1:100 JacksonStreptavidin-Texas Red 1:100 Amersham

1Supply companies: Amersham Pty, Ltd., Melbourne, Australia; Jackson ImmunosearchLaboratories, West Grove, PA; Vector Lab., Burlingame, CA. IgG, immunoglobulin G;FITC, fluorescein isothiocyanate.

TABLE 1. Characteristics of Primary Antibodies1

Tissue antigen Host Dilution Code and reference

Calbindin Rabbit 1:1600 DEMLR 1, Furness et al., 1988Calretinin Rabbit 1:1000 7696, Mann et al., 1997VAChT Goat 1:1000 1624, Li and Furness, 1998Bombesin Mouse 1:100 2AII, Costa et al., 19845-HT (serotonin) Rat 1:500 3M55, Wardell et al., 1994Neural NOS Rabbit 1:200 N74, Anderson et al., 1995NPY Rabbit 1:1600 JM263, Maccarrone and Jarrot, 1985Somatostatin Mouse 1:400 S895, Buchan et al., 1985TK (substance P) Rat 1:200 NCI/34HL, Cuello et al., 1979TH Mouse 1:400 T341, Boehringer MannheinVIP Rabbit 1:200 Furness et al., 1981

1VAChT, vesicular acetylcholine transporter; NOS, nitric oxide synthase; NPY, neuropep-tide Y; TH, tyrosine hydroxylase; VIP, vasoactive intestinal peptide.

SYNAPTIC INPUT TO INTESTINOFUGAL NEURONS 453

Fig. 1. Fluorescence photomicrographs showing the immunoreac-tivities of nerve terminals that surround the cell bodies of intestinofu-gal neurons that were retrogradely labelled with Fast Blue.A,A8: Images of the same field illuminated to reveal Fast Blue (A) andvesicular acetylcholine transporter (VAChT) immunoreactive (-IR)(A8). The image in A is the cell body of an intestinofugal neuron that issurrounded by a basket-like arrangement of VAChT-IR nerve termi-nals (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

(B8), and vasoactive intestinal peptide (VIP) -IR (B9). The intestinofu-gal neuron (B) is surrounded by nerve terminals that are immunoreac-tive for VAChT (B8) and VIP (B9). Arrows indicate where VAChT-IRand VIP-IR appear to colocalise within varicosities. C: An intestinofu-gal neuron that is surrounded by VIP-IR (C8) and bombesin-IR (BN;C9) terminals. Arrows indicate colocalisation of VIP-IR and bombe-sin-IR within varicosities. Scale bars 5 10 µm in A,B (apply toA,A8,B,B8,B9), 20 µm in C (applies to C,C8,C9).


to the class of neurons designated by Dogiel (1899) as typeI (see Sharkey et al., 1998).

Preparations of longitudinal muscle plus myentericplexus containing Fast Blue labelled intestinofugal neu-rons were stained with a variety of immunohistochemicalmarkers of nerve terminals of populations of myentericneurons. Fast Blue-labelled nerve cell bodies were com-monly surrounded by immunoreactive (IR) varicositieswhich outlined cell bodies, sometimes forming severallayers of varicosities (Fig. 1A,A8). Such relationships be-tween varicosities and cell bodies, described as pericellularbaskets, have been examined by electron microscopy, andit had been found that varicosities within these basketsform 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 ofaxon varicosities that were immunoreactive for the vesicu-lar acetylcholine transporter (VAChT), which is a markerof cholinergic nerve terminals (Fig. 1A,A8,B,B8). The cellbodies of intestinofugal neurons were also examined fortheir relation to vasoactive intestinal peptide (VIP)-IRand bombesin-IR terminals in preparations stained forVAChT plus VIP or bombesin plus VIP. One hundredsixty-nine of 212 intestinofugal neurons (80%) were sur-rounded by VIP-IR varicosities, whereas 77 of 88 intesti-nofugal neurons (88%) examined were surrounded bybombesin-IR terminals (Fig. 1B,B88,C,C88). These double-staining experiments showed that terminals that wereimmunoreactive for each of these markers formed basketsaround intestinofugal neurons and that these markerswere commonly colocalised within individual varicosities(Fig. 1B8,B88,C8,C8).

Varicose terminals with bombesin-IR were found aroundother nerve cells, but some nerve cells were not sur-rounded by bombesin-IR varicosities. Bombesin-IR varicosi-ties surrounded most bombesin-IR nerve cells (Fig. 3A,B).

Descending inputs

Myotomy and extrinsic denervation operations wereperformed to identify the projection patterns of cholinergicneurons that innervate intestinofugal neurons. In prepara-tions from the animals in which myotomy operations wereperformed, there was a decrease in the innervation ofintestinofugal neurons in the 10 mm on the anal sides ofthe lesions and quantitative data presented below on theeffects of myotomy operations was obtained from the first10 mm anal or oral to a myotomy. There was a dramaticreduction in the number of intestinofugal neurons thatwere surrounded by VAChT-IR and VIP-IR varicositiesjust anal to a myotomy operation (i.e., where the axons ofneurons with descending (oral to anal) projections hadbeen severed and allowed to degenerate). In untreatedanimals, 93% of intestinofugal neurons examined weresurrounded by VAChT-IR terminals (see above), whereasafter a myotomy operation performed oral to the areaexamined 37 of 141 intestinofugal neurons (26%) hadVAChT innervation that was similar to controls; a further40 of 141 intestinofugal neurons (28%) had few VAChT-IRterminals close to them, insufficient to be recognised as apericellular basket (Fig. 2B,B8), and the remaining 64neurons (46%) had no VAChT-IR terminals close to the cellbodies. In areas just anal to a myotomy, 10 of 87 (12%)intestinofugal neurons had normal innervation by VIP-IRterminals; a further 18 of 87 (20%) had reduced innerva-tion and 59 of 87 (68%) had no close VIP-IR terminals (Fig.

2C,C8). The innervation of intestinofugal neurons by VIP-IRvaricosities returned to normal about 10 mm anal to themyotomy operation site. This finding is consistent with thefindings of Messenger and Furness (1990), who reportedthat VIP fibers in myenteric ganglia in the guinea pig colonwere reduced by oral myotomy operations but that normalVIP innervation of myenteric ganglia was observed withinapproximately 8 mm anal to the site of the myotomy.

Ascending inputs

The effects of myotomy operations that interrupt ascend-ing fibers within the myenteric plexus on the innervationof intestinofugal neurons by VAChT-IR terminals wereexamined. In the 10 mm oral to myotomies, 51 of 60intestinofugal neurons (85%) had normal VAChT innerva-tion, suggesting that ascending pathways do not contrib-ute significantly to the innervation of intestinofugal neu-rons by cholinergic terminals (Fig. 2A,A8).

Extrinsic inputs

Extrinsic denervation operations were performed todetermine the contribution of extrinsic neurons to theinnervation of intestinofugal neurons, and immunohisto-chemistry for tyrosine hydroxylase (TH) was used tomonitor the effectiveness of the denervation. In prepara-tions from unoperated animals, TH-IR fibers formed adense network within myenteric ganglia, whereas afterextrinsic denervation, only rare TH-IR fibers could beseen, consistent with previous findings (Furness, 1970). Insegments of colon that had been extrinsically denervated,36 of 43 intestinofugal neurons (84%) received normalVAChT innervation. In the two animals that had under-gone both extrinsic denervation and myotomy operations,there was a reduction in VAChT innervation anal to themyotomies that recovered at about 1 cm anal as describedabove, but further anal between the myotomy operationsno reduction was observed. These results, along with thefinding of only infrequent innervation of intestinofugalneurons by TH-IR fibers in untreated preparations (5 of36; 14%, Table 3) suggest that the innervation of intesti-nofugal neurons originates predominantly within the gutwall.

Because VAChT, VIP, and bombesin were localised inmany terminals that surrounded intestinofugal neurons,we investigated whether there were myenteric nerve cellbodies with the appropriate neurochemistry to be a sourceof the terminals. Staining of myenteric neurons of theguinea pig distal colon that had been pretreated withcolchicine to enhance cell body immunoreactivity for pep-tides revealed that choline acetyltransferase (ChAT), VIP,bombesin, and nitric oxide synthase (NOS) immunoreactivi-ties are colocalised in a subset of nerve cells (Fig. 3). Manyof these nerve cells were surrounded by bombesin-IRterminals. Sixty-six of 100 bombesin-IR myenteric nervecell bodies were ChAT-IR, 50 of 56 bombesin-IR wereVIP-IR, and 78 of 79 were NOS-IR.

We also examined whether intestinofugal neurons hadterminals surrounding them that were immunoreactivefor neurochemical markers of subclasses of myentericneurons other than VAChT, VIP, and bombesin (Table 3).No other markers stained terminals that surrounded sucha large proportion of intestinofugal neurons. A complica-tion that made accurate counting of the number of intesti-nofugal neurons that were surrounded by varicositiesimmunoreactive for a particular substance was that the


cell bodies of intestinofugal neurons are immunoreactivefor NOS, bombesin, VIP, calretinin, calbindin, and ChAT(Furness et al., 1990; Mann et al., 1995). This was not aproblem with bombesin and VIP, because cell body stain-ing of myenteric neurons with these markers is weakwithout colchicine treatment, whereas cell body stainingof intestinofugal neurons with calbindin, calretinin, and

NOS is intense and precludes accurate assessment ofwhether cell bodies of intestinofugal neurons are sur-rounded by terminals immunoreactive for these markers.To determine the proportion of intestinofugal neurons withinputs from calbindin-, calretinin-, or NOS-IR terminals,only retrogradely labelled nerve cell bodies that did nothave the same immunohistochemistry were examined. Of

Fig. 2. Paired photomicrographs that show the effects of myotomyoperations on the distribution of labelled nerve terminals aroundintestinofugal neurons. A: An intestinofugal neuron in a region within5 mm oral to a myotomy that caused degeneration of ascending fibersis surrounded by vesicular acetylcholine transporter (VAChT) –immu-noreactive (-IR) terminals (A8). Note that the labelled varicositiesappear to be closely aligned with the surface of the cell body and withone of its dendrites (arrow). B: An intestinofugal neuron from a regionwithin 5 mm anal to a myotomy that severed descending axons has a

marked reduction in the number of VAChT-IR terminals that approachthe cell body (B8). No labelled terminals can be seen to contact the cellbody or its processes. C: Another intestinofugal neuron from a regionwhere descending axons have been severed. In this case, there is anobvious 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 5 15 µm in A–C (apply to A–C8).


the intestinofugal neurons whose cell bodies were notimmunoreactive 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%) byNOS-IR terminals respectively (Table 3).

DISCUSSION

Sources of innervation

We have found that the cell bodies of intestinofugalneurons that project to the inferior mesenteric gangliafrom the distal colon are surrounded by cholinergic nerveterminals. The possible sources of these terminals areintrinsic neurons, extrinsic neurons whose axons reach thecolon by means of the mesenteric nerves, vagal axons thattake an intramural course to reach the colon and branchesof 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 signifi-cantly. When the mesenteric nerves were severed and theirendings allowed to degenerate, there was no detectableloss of innervation of the cell bodies of intestinofugalneurons. Moreover, in the region between two circumferen-tial myotomies, which would sever both vagal fibers de-scending within the gut wall and ascending fibers from thepelvic nerves, normal innervation persisted in gangliatoward the anal part of the neurally isolated segment. Theappearance was the same if the two myotomies and themesenteric nerve section were performed together. Thus,the pericellular fibers at the distal part of the segmentbetween myotomies must arise from neurons with cellbodies within the neurally isolated segment, and, becausethe loss of nerve terminals occurred at the oral part of thissegment only, the neurons must be anally projecting.However, not all fibers were lost from the oral region. Theremaining fibers are, therefore, of local origin, either fromcell bodies in myenteric ganglia or from cell bodies insubmucosal ganglia. The lack of detectable loss of innerva-tion oral to circumferential lesions of intramural nervepathways indicates that ascending fibers do not contributesignificantly to the innervation of the cell bodies of intesti-nofugal neurons.

We have identified the class of cholinergic descendinginterneuron that makes a large contribution to the cholin-ergic innervation of intestinofugal neurons. VAChT, VIP,and bombesin all labelled nerve terminals that surroundthe majority of intestinofugal neurons. Moreover, by exam-

ining the cell bodies of myenteric neurons in preparationsof distal colon that had been pretreated with colchicine, aclass of descending interneurons was identified that wasimmunoreactive for ChAT, VIP, and bombesin. In studieson the myenteric plexus of guinea pig distal colon thatused single label immunohistochemistry, descending inter-neurons with immunoreactivity for VIP, NOS, and bombe-sin have been identified (Messenger and Furness 1990;McConalogue and Furness, 1993; present results). Bombe-sin/VIP-IR terminals also surrounded many other nervecells, including other bombesin-IR neurons, but there werecells without this innervation; thus, these descendinginterneurons evidently make specific connections withintestinofugal neurons, descending interneurons, and otherclasses of neurons, which, by analogy with the smallintestine, probably include inhibitory motor neurons(Kunze and Furness, 1999).

Relations of labelled nerve terminalsto nerve cell bodies

The inputs to the intestinofugal neurons have beenexamined by fluorescence microscopy. Dense baskets ofcholinergic (VAChT-IR) varicosities were closely apposedto the nerve cells. Electrophysiological studies support theconclusion that the baskets of VAChT-IR varicosities repre-sent true synapses, because fast EPSPs were recordedfrom the intestinofugal neurons, and, although this wastested in only a few cases, the fast EPSPs were blocked byan antagonist of cholinergic transmission, hexametho-nium (Sharkey et al., 1998). Similar associations of bas-kets of terminals have been examined by electron micros-copy, and in all cases, many of the varicosities were foundto form true synapses (showing presynaptic accumulationsof vesicles and synaptic densities), whereas some varicosi-ties formed close contacts (closely apposed membranes,presynaptic accumulations of vesicles but no discerniblesynaptic densities). The presence of synapses within bas-kets of immunoreactive terminals in enteric ganglia hasbeen confirmed by electron microscopy and includes calreti-nin-IR inputs to calretinin-IR interneurons (Pompolo andFurness, 1993), somatostatin-IR inputs to somatosta-tin-IR interneurons (Portbury et al., 1995; Pompolo andFurness, 1998), NOS-IR inputs to VIP-IR secretomotorneurons (Li et al., 1995), bombesin-IR inputs to calreti-nin-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 andFurness, 1995), and somatostatin-IR inputs to NOS-IR cellbodies (Mann et al., 1997).

The numbers of varicosities seen in apposition to nervecells with fluorescence immunohistochemical methods islikely to be an overestimate of the number of true synapticinputs (Mann et al., 1997). This is because some of thevaricosities are separated from the surfaces of nerve cellsby glial cell processes.

Physiological significance

Bywater (1993) made the interesting observation thatwhen synaptic transmission within the colon was blocked,the initial response of intestinofugal neurons to distensionwas not significantly reduced, whereas the response after10–15 seconds of sustained distension was almost abol-ished. This finding suggests that reflexes that pass fromthe gut by means of intestinofugal neurons to the preverte-bral ganglia have two components: one that is a direct

TABLE 3. Incidence of Pericellular Baskets

Markers used1

Numberof intestinofugal

neurons examined

Number ofintestinofugal

neurons surroundedby terminals (%)

Bombesin 88 77 (88)Calbindin 30 7 (23)Calretinin 47 6 (13)NOS 69 15 (23)Substance P 17 12 (70)TH 36 5 (14)VAChT

Normal colon 215 199 (93)Anal to myotomy 141 37 (26)Oral to myotomy 60 51 (85)After extrinsic denervation 43 36 (84)

VIPNormal colon 212 169 (80)Anal to myotomy 87 10 (12)

1For abbreviations, see footnote to Table 1.


response of intestinofugal neurons to distension and theother that is a response to synaptic input from otherenteric neurons to intestinofugal neurons (i.e., intestinofu-gal neurons are indirectly activated by the distensionstimulus). Szurszewski and Miller (1994) have suggestedthat intestinofugal neurons may form a homogenous popu-lation of neurons that are both directly and indirectlyactivated. Evidence in support of this theory was providedby Sharkey et al. (1998), who found that all intestinofugalneurons from which intracellular membrane potentialrecordings were taken had S-type electrophysiologicalcharacteristics and had Dogiel type I morphology; in otherwords, they appear to form a homogenous population.Experimental evidence also suggests that all intestinofu-gal neurons have the same primary transmitter, acetylcho-line (Szurszweski and Miller, 1994; Mann et al., 1995;Sharkey et al., 1998). The results of the present studysupport the idea that intestinofugal neurons are a popula-tion that is homogenous in its most significant characteris-tics. Ninety-three percent of intestinofugal neurons weresurrounded by basket-like arrangements of VAChT-IRvaricosities. It is not possible to say that the other 7% arenot innervated by cholinergic nerve terminals because theVAChT-IR terminals may not always form a distinct

pericellular basket and may also contact dendrites ofintestinofugal neurons that are not revealed by Fast Bluefluorescence. On the other hand, we found that about 25%of the intestinofugal neurons were surrounded by calbin-din terminals. Calbindin is a marker of Dogiel type IIneurons, which are presumed to be IPANs in the colon(Lomax et al., 1999) but is also contained in other types ofneuron. Thus, it is possible that a minority of intestinofu-gal neurons receive direct inputs from IPANs.

There is strong experimental evidence that intestinofu-gal neurons are length sensors (Szurszewski and Miller,1994), whereas there is good evidence that the IPANs ofthe ileum respond to tension generated in the externalmuscle in response to stretch rather than the stretch perse, i.e., they are tension detectors (Kunze et al., 1998,1999). These IPANs have Dogiel type II morphology andAH electrophysiological characteristics (Furness et al.,1998). A recent study has revealed the neurons in themyenteric plexus of the guinea pig distal colon that havesimilar morphologic, immunohistochemical, and electro-physiological characteristics to the IPANs of the ileum(Lomax et al., 1999). The observations of Bywater (1993)might be accounted for if intestinofugal neurons respondto changes in length directly, which would account for the

Fig. 3. Paired photomicrographs illustrating the cell body immuno-reactivities of the class of descending interneuron that suppliesterminals that surround the cell bodies of intestinofugal neuronswithin the myenteric plexus of guinea pig distal colon. A,A8,B,B8: Con-focal microscope images, optical thickness 0.7 µm. C,C8: Photomicro-graphs 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 termi-nals, as can be seen in A and B. Arrows indicate cell bodies wherecolocalisation occurs. Scale bars 5 15 µm in A–C (apply to A–C8).


initial response to distension that persists after blockadeof synaptic transmission within the gut. The slowly devel-oping increase in tension in response to this distensionmight excite the IPANs, but not the intestinofugal neuronsdirectly. These IPANs then excite each other and provideinput to the descending interneurons, which in turnprovide fast synaptic input to the intestinofugal neurons.Convergence of fast excitatory postsynaptic potentialsonto the intestinofugal neurons would cause action poten-tial firing in the intestinofugal neurons in response todistension (by means of the increase in tension) that wouldbe sensitive to synaptic blockade.

Neurons with similar chemical coding (ChAT, VIP,bombesin, NOS) to the class of descending interneuronthat provides cholinergic input to intestinofugal neuronsin the distal colon have been examined in the guinea pigsmall intestine. Anatomic and pharmacologic studies indi-cate that this class of interneuron forms descending chainsand that these neurons innervate the inhibitory motorneurons that relax the circular muscle (Young et al., 1995;Yuan et al., 1995; Kunze and Furness, 1999), i.e., they areconduits of local descending reflex pathways in the smallintestine. It is likely that similar descending interneuronsin the distal colon that innervate the intestinofugal neu-rons are also involved in local (intrinsic) reflex pathways inthe colon because, as discussed above, they innervate othernerve cells in the myenteric ganglia. This raises theinteresting possibility that the sustained response to dis-tension that is observed in intestinofugal neurons iscontrolled by the same class of descending interneuronthat is involved in local descending inhibitory reflexes inthe gut.

ACKNOWLEDGMENTS

We thank Dr. Colin Anderson and Dr. Heather Young forexpert advice on the manuscript and Heather Woodmanand Clare Delaney for excellent technical assistance.

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Origins of cholinergic inputs to the cell bodies of intestinofugal neurons in the guinea pig distal colon