Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig

Summary Neuromedin U immunoreactivity was located histochemically in the guinea-pig small intestine. Projections of immunoreactive neurons were determined by analysing patterns of degeneration following nerve lesions. The co-localization of neuromedin U immunoreactivity with immunoreactivity for substance P, neuropeptide Y, vasoactive intestinal peptide and calbindin was also investigated. Neuromedin U immunoreactivity was found in nerve cells in the myenteric and submucous plexuses and in nerve fibres in these ganglionated plexuses, around submucous arterioles and in the mucosa. Reactive fibres did not supply the muscle layers. Most reactive nerve cells in the myenteric ganglia had Dogiel type-II morphology and in many there was co-localization of calbindin, although some Dogiel type-II neuromedin U neurons were calbindin negative. Lesion studies suggest that these myenteric neurons project circumferentially to local myenteric ganglia. Projections from myenteric neurons also run anally in the myenteric plexus, while other projections extend to submucous ganglia, and still further projections run from the intestine to provide terminals in the coeliac ganglia. In the submucous ganglia neuromedin U was co-localized in three populations of nerve cells: (i) those with vasoactive intestinal peptide immunoreactivity, (ii) neurons containing neuropeptide Y, and (iii) neurons containing substance P. Each of these populations sends nerve fibres to the mucosa. Neuromedin U immunoreactivity is thus located in a variety of neurons serving different functions in the intestine and therefore probably does not have a single role in intestinal physiology.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunhistochemistry using a nerve cell body antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 m. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, nerve cell body antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105±10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17300 nerve cells/cm².     

Projections and chemistry of Dogiel type II neurons in the mouse colon

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X₂ receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.These studies were funded by the National Health and Medical Research Council (Australia)     

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

Summary The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm² of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.     

Primary afferent neurons intrinsic to the guinea-pig intestine,like primary afferent neurons of spinal and cranial sensory ganglia,bind the lectin,IB4

The plant lectin, IB4, binds to the surfaces of primary afferent neurons of the dorsal root and trigeminal ganglia and is documented to be selective for nociceptive neurons. Physiological data suggest that the intrinsic primary afferent neurons within the intestine are also nociceptors. In this study, we have compared IB4 binding to each of these neuron types in the guinea-pig. The only neurons in the intestine to be readily revealed by IB4 binding have Dogiel-type-II morphology; these neurons have been previously identified as intrinsic primary afferent neurons. Most of the neurons that are IB4-positive in the myenteric plexus are calbindin-immunoreactive, whereas those in the submucosal ganglia are immunoreactive for NeuN. The neurons that bind IB4 strongly have a similar appearance in enteric, dorsal root and trigeminal ganglia. Binding is to the cell surface, to the first part of axons and to cytoplasmic organelles. A low level of binding was found in the extracellular matrix. A few other neurons in all ganglia exhibit faint staining with IB4. Strongly reactive neurons are absent from the gastric corpus. Thus, IB4 binding reveals primary afferent neurons with similar morphologies, patterns of binding and physiological roles in enteric, dorsal root and trigeminal ganglia.This work was supported by a grant from the National Health and Medical Council of Australia.     

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release. Work in this study was funded by the National Health and Medical Research Council (grant numbers: 114215 and 216704; Senior Research Fellowship to B.S.), a Melbourne University Research Scholarship and the Murdoch Children’s Research Institute.     

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.     

Expression of sodium channel Nav1.6 in cholinergic myenteric neurons of guinea pig proximal colon

We wished to establish the functional identity of Na_v1.6-expressing myenteric neurons of the guinea pig proximal colon by determining the extent of colocalization of Na_v1.6 and selected neurochemical markers. Na_v1.6-like immunoreactivity (-li) was primarily localized to the hillock and initial segments of myenteric neurons located near junctions with internodal fiber tracts. Immunoreactivity for Na_v1.6 was co-localized with choline-acetyltransferase-li, representing 96% of Na_v1.6-immunoreactive neurons; about 5% of these neurons showed co-localization with calretinin-li, but none with substance-P-li. Cholinergic neurons expressing Na_v1.6 were amongst the smallest (somal area <300 μm²) of all cholinergic myenteric neurons observed. Only three of 234 Na_v1.6-immunoreactive neurons exhibited nNOS-li, and none co-localized with calbindin-li. These data suggest that Na_v1.6 is expressed in a small uniform population of cholinergic myenteric neurons that lie within the guinea pig proximal colon and that are likely to function as excitatory motor neurons.This work was supported in part by grants from the Autzen Endowment and Cadeau Foundation. A.C. Bartoo was supported by a grant from the Poncin Foundation.     

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa. This study was supported by a Waseda University Grant for Special Research Projects (2008A-889).     

Intramural neurons in the urinary bladder of the guinea-pig

Summary The urinary bladder of adult female guinea-pigs was stained histochemically to detect the presence of intramural ganglion neurons. Counts on wholemount preparations of entire bladders revealed the presence of 2000–2500 neurons per bladder, either as individual nerve cells or, more often, as ganglia containing up to 40 neurons. Both ganglia and single neurons lie along nerve trunks and are interconnected to form a plexus. Ganglia occur in every part of the bladder; they are more numerous on the dorsal than on the ventral wall, and they are especially abundant in an area within a radius of 800 m from the point of entry into the bladder wall of ureters and urinary arteries. The ganglia are located inside the muscle coat and close to muscle bundles; they usually lie nearer the mucosa than the serosa. Ultrastructurally, each ganglion is surrounded by a capsule; in addition to neurons and glial cells, the ganglia contain capillaries, collagen fibrils and fibroblasts; ganglion neurons are individually wrapped by glial cells and are separated from one another by connective tissue.     

Morphology of VIP/nNOS-immunoreactive myenteric neurons in the human gut

In this study, we characterized human myenteric neurons co-immunoreactive for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) by their morphology and their proportion as related to the putative entire myenteric neuronal population. Nine wholemounts (small and large intestinal samples) from nine patients were triple-stained for VIP, neurofilaments (NF) and nNOS. Most neurons immunoreactive for all three markers displayed radially emanating, partly branching dendrites with spiny endings. These neurons were called spiny neurons. The spiny character of their dendrites was more pronounced in the small intestinal specimens and differed markedly from enkephalinergic stubby neurons described earlier. Exclusively in the duodenum, some neurons displayed prominent main dendrites with spiny side branches. Of the axons which could be followed from the ganglion of origin within primary strands of the myenteric plexus beyond the next ganglion (70 out of 140 traced neurons), 94.3% run anally and 5.7% orally. Very few neurons reactive for both VIP and nNOS could not be morphologically classified due to weak or absent NF-immunoreactivity. Another six wholemounts were triple-stained for VIP, nNOS and Hu proteins (HU). The proportion of VIP/nNOS-coreactive neurons in relation to the number of HU-reactive neurons was between 5.8 and 11.5% in the small and between 10.6 and 17.5% in the large intestinal specimens. We conclude that human myenteric spiny neurons co-immunoreactive for VIP and nNOS represent either inhibitory motor or descending interneurons.     

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons

Summary Immunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.     

Identification of neurons that express 5-hydroxytryptamine4 receptors in intestine

5-Hydroxytryptamine (5-HT) is an endogenous stimulant of intestinal propulsive reflexes. It exerts its effects partly through 5-HT₄ receptors; 5-HT₄ receptor agonists that are stimulants of intestinal transit are in clinical use. Both pharmacological and recent immunohistochemical studies indicate that 5-HT₄ receptors are present on enteric neurons but the specific neurons that express the receptors have not been determined. In the present work, we describe the characterization of an anti-5-HT₄ receptor antiserum that reveals immunoreactivity for enteric neurons and other cell types in the gastrointestinal tract. With this antiserum, 5-HT₄ receptor immunoreactivity has been found in the muscularis mucosae of the rat oesophagus, a standard assay tissue for 5-HT₄ receptors. It is also present in the muscularis mucosae of the guinea-pig and mouse oesophagus. In guinea-pig small intestine and rat and mouse colon, 5-HT₄ receptor immunoreactivity occurs in subpopulations of enteric neurons, including prominent large neurons. Double-staining has shown that these large neurons in the guinea-pig small intestine are also immunoreactive for two markers of intrinsic primary afferent neurons, cytoplasmic NeuN and calbindin. Some muscle motor neurons in the myenteric ganglia are immunoreactive for this receptor, whereas it is rarely expressed by secretomotor neurons. Immunoreactivity also occurs in the interstitial cells of Cajal but is faint in the external muscle. Expression of the protein and mRNA has been confirmed in extracts containing enteric neurons. The observations suggest that one site of action of 5-HT₄ receptor agonists is the intrinsic primary afferent neurons.This work was supported by the National Health and Medical Research Council of Australia and Pfizer Pharmaceuticals, Japan.     

The source of the nerve fibres forming the deep muscular and circular muscle plexuses in the small intestine of the guinea-pig

Summary A quantitative ultrastructural study was made of the neuntes forming the deep muscular and circular muscle plexuses of the guinea-pig small intestine following microsurgical lesions designed to interrupt intrinsic and extrinsic nerve pathways within the intestinal wall. Removal of a collar of longitudinal muscle with attached myenteric plexus from the circumference of a segment of small intestine resulted in the subsequent disappearance of 99.3% of neurites in the underlying circular muscle. The few surviving neurites in the deep muscular plexus and circular muscle disappeared completely from lesioned segments that were, in addition, extrinsically denervated surgically. These results indicate that the majority of nerve fibres in the deep muscular and circular muscle plexuses of the guinea-pig small intestine is intrinsic to the intestine and originates from nerve cell bodies located in the overlying myenteric plexus. At the light-microscopic level, nerve bundles were traced from the myenteric plexus to the circular muscle.     

Projections of nitric oxide synthesizing neurons in the guinea-pig colon

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.     

High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

Previous studies have demonstrated that neurofilament proteins are expressed by type II neurons in the enteric plexuses of a range of species from mouse to human. However, two previous studies have failed to reveal this association in the guinea-pig. Furthermore, immunohistochemistry for neurofilaments has revealed neurons with a single axon and spiny dendrites in human and pig but this morphology has not been described in the guinea-pig or other species. We have used antibodies against high- and medium-weight neurofilament proteins (NF-H and NF-M) to re-examine enteric neurons in the guinea-pig. NF-H immunoreactivity occurred in all type II neurons (identified by their IB4 binding) but these neurons were never NF-M-immunoreactive. On the other hand, 17% of myenteric neurons expressed NF-M. Many of these were uni-axonal neurons with spiny dendrites and nitric oxide synthase (NOS) immunoreactivity. NOS immunoreactivity occurred in surface expansions of the cytoplasm that did not contain neurofilament immunoreactivity. Thus, because of their NOS immunoreactivity, spiny neurons had the appearance of type I neurons. This indicates that the apparent morphologies and the morphological classifications of these neurons are dependent on the methods used to reveal them. We conclude that spiny type I NOS-immunoreactive neurons have similar morphologies in human and guinea-pig and that many of these are inhibitory motor neurons. Both type II and neuropeptide-Y-immunoreactive neurons in the submucosal ganglia exhibit NF-H immunoreactivity. NF-M has been observed in nerve fibres, but not in nerve cell bodies, in the submucosa. This work was supported by a grant from the National Health and Medical Council of Australia (grant number 400020).