Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern

Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm² and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis. Received: 6 August 1997/Accepted: 8 October 1997     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract,with particular reference to the mucosa

Summary The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions.VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers.These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine

This study aimed at estimating the proportion of human myenteric Dogiel type II neurons, putative intrinsic primary afferent neurons (IPANs), in relation to the entire myenteric neuron population. Since, at present, there is no known single marker, which specifically labels these neurons, we tried to identify the most appropriate marker combination based on the results of an earlier study. For this purpose, 10 wholemounts derived from human small intestinal segments were immunohistochemically triple-stained for calretinin (CALR), somatostatin (SOM) and neurofilaments (NF) and 9 were stained for substance P (SP), SOM and NF. In each wholemount, 15 ganglia selected randomly were evaluated. On the basis of their NF-reactivity, neurons reactive for one or co-reative for both of the other two markers, respectively, were morphologically classified as type II or non-type II neurons. We found that the majorities of neurons co-reactive for CALR/SOM and SP/SOM, respectively, were type II neurons whereas this was not the case for neurons, which were reactive for only one of the two markers. One of the statistical parameters estimated was the positive predictive value, the probability that a neuron displaying CALR/SOM- or SP/SOM-co-reactivity, respectively, is a type II neuron. This value was 97% in case of CALR/SOM- and 95% in case of SP/SOM-co-staining. Although the difference of the statistical parameters between the two stainings was not significant, CALR and SOM were used to estimate indirectly the proportion of type II neurons, in wholemounts co-stained with the pan-neuronal marker neuronal protein HuC/HuD (HU). In these wholemounts, altogether 9.1% of neurons were coreactive for CALR/SOM. We suggest that the proportion of myenteric type II neurons in the human small intestine is related to the proportion of CALR/SOM-co-reactive neurons and may be near to one tenth of the total myenteric neuronal population.     

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.     

Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture

Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.     

Spiny versus stubby: 3D reconstruction of human myenteric (type I) neurons

We have compared the three-dimensional (3D) morphology of stubby and spiny neurons derived from the human small intestine. After immunohistochemical triple staining for leu-enkephalin (ENK), vasoactive intestinal peptide (VIP) and neurofilament (NF), neurons were selected and scanned based on their immunoreactivity, whether ENK (stubby) or VIP (spiny). For the 3D reconstruction, we focused on confocal data pre-processing with intensity drop correction, non-blind deconvolution, an additional compression procedure in z-direction, and optimizing segmentation reliability. 3D Slicer software enabled a semi-automated segmentation based on an objective threshold (interrater and intrarater reliability, both 0.99). We found that most dendrites of stubby neurons emerged only from the somal circumference, whereas in spiny neurons, they also emerged from the luminal somal surface. In most neurons, the nucleus was positioned abluminally in its soma. The volumes of spiny neurons were significantly larger than those of stubby neurons (total mean of stubbies 806 ± 128 μm³, of spinies 2,316 ± 545 μm³), and spiny neurons had more dendrites (26.3 vs. 11.3). The ratios of somal versus dendritic volumes were 1:1.2 in spiny and 1:0.3 in stubby neurons. In conclusion, 3D reconstruction revealed new differences between stubby and spiny neurons and allowed estimations of volumetric data of these neuron populations.     

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).     

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.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.     

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.     

Shapes of nerve cells in the myenteric plexus of the guinea-pig small intestine revealed by the intracellular injection of dye

Summary The shapes of myenteric neurons in the guineapig small intestine were determined after injecting living neurons with the dye Lucifer yellow via a microelectrode. The cells were fixed and the distribution of Lucifer yellow rendered permanent by an immunohistochemical method. Each of 204 nerve cells was examined in whole-mount preparations of the myenteric plexus and drawn using a camera lucida at 1250 x magnification. Four cell shapes were distinguished: (1) neurons with several long processes corresponding to type II of Dogiel; (2) neurons with a single long process and lamellar dendrites corresponding to type I of Dogiel; (3) neurons with numerous filamentous dendrites; and (4) small neurons with few processes. About 15% of the neurons could not be placed into these classes or into any single class. The type II neurons (39% of the sample) had generally smooth somata and up to 7 (average 3.3) long processes, most of which ran circumferentially. Dogiel type I neurons (34% of sampled neurons) had characteristic lamellar dendrites, i.e., broad dendrites that were flattened in the plane of the plexus. The filamentous neurons (7% of the sample), had, on average, 14 fine processes up to about 50 m in length. Small neurons with smooth outlines and a few fine processes made up 5% of the neurons encountered. We conclude that myenteric neurons that have been injected with dye can be separated into morphologically distinct classes and that the different morphological classes probably correspond to different functional groupings of neurons.     

Morphology of enkephalin-immunoreactive myenteric neurons in the human gut

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259±47 m² and 487±113 m²) than those of putative sensory type II neurons (between 700±217 m² and 1,164±396 m²). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term Dogiel type I neurons for this population.     

Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

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.     

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A_2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A_2B and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A_2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A_2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A_2A and A₃) receptors by endogenous adenosine. The selective A_2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1–10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A_2A receptors. In contrast to 2-Cl-IB MECA, activation of A_2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A_2A receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A_2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.     

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.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.