In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.

The diagnosis of Hirschsprung's disease is currently based on the identification of aganglionosis and the presence of an increase in acetylcholinesterase-positive hypertrophic nerve fibres in the large bowel submucosa. However, acetylcholinesterase staining is laborious and requires a skilled technician. The aim of this study was to identify a method for diagnosing Hirschsprung's disease reliably using an immunohistochemical panel of recently proposed markers.

Sixty-nine specimens from 37 patients were evaluated. MAP2 and calretinin antibodies were shown to stain ganglia reliably in the submucosal and myenteric plexuses of normal tissue. By contrast, reduced staining of ganglia was observed in patients with Hirschsprung's disease. Staining for GLUT1 and S100 was used to evaluate the number and thickness of nerve fibres. Gain of GLUT1 and S100 expression was in contrast to the loss of calretinin and MAP2. Hypertrophic submucosal nerve fibres in Hirschsprung's disease develop a perineurium with a ring-like GLUT1 staining pattern similar in size and intensity to that observed in deeper subserosal tissue.

The diagnosis of Hirschsprung's disease using immunohistochemical panels could be as accurate as with conventional frozen section techniques. In particular, the use of a combination of markers for ganglia and hypertrophic nerve fibres highlighting a prominent perineurium in Hirschsprung's disease could be an alternative method.

Proline-rich synapse-associated protein-1 and 2 (ProSAP1/Shank2 and ProSAP2/Shank3) were originally found as synapse-associated protein 90/postsynaptic density protein-95-associated protein (SAPAP)/guanylate-kinase-associated protein (GKAP) interaction partners and also isolated from synaptic junctional protein preparations of rat brain. They are essential components of the postsynaptic density (PSD) and are specifically targeted to excitatory asymmetric type 1 synapses. Functionally, the members of the ProSAP/Shank family are one of the postsynaptic key elements since they link and attach the postsynaptic signaling apparatus, for example N-methyl-d-aspartic acid (NMDA)-receptors via direct and indirect protein interactions to the actin-based cytoskeleton. The functional significance of ProSAP1/2 for synaptic transmission and the paucity of data with respect to the molecular composition of PSDs of the peripheral nervous system (PNS) stimulated us to investigate neuromuscular junctions (NMJs), synapses of the superior cervical ganglion (SCG), and synapses in myenteric ganglia as representative synaptic junctions of the PNS. Confocal imaging revealed ProSAP1/2-immunoreactivity (-iry) in NMJs of rat and mouse sternomastoid and tibialis anterior muscles. In contrast, ProSAP1/2-iry was only negligibly found in motor endplates of striated esophageal muscle probably caused by antigen masking or a different postsynaptic molecular anatomy at these synapses. ProSAP1/2-iry was furthermore detected in cell bodies and dendrites of superior cervical ganglion neurons and myenteric neurons in esophagus and stomach. Ultrastructural analysis of ProSAP1/2 expression in myenteric ganglia demonstrated that ProSAP1 and ProSAP2 antibodies specifically labelled PSDs of myenteric neurons. Thus, scaffolding proteins ProSAP1/2 were found within the postsynaptic specializations of synapses within the PNS, indicating a similar molecular assembly of central and peripheral postsynapses.

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-beta-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and alpha-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

A mature enteric nervous system (ENS) is required to ensure a normal pattern of intestinal motility in order to regulate digestion after birth. We hypothesized that neuronal and glial components of the ENS would mature during the first postnatal days in preterm pigs that are a sensitive animal model of food intolerance and necrotizing enterocolitis (NEC). Stereological volume densities of the general neuronal population [assessed by betaIII-tubulin immunoreactivity (IR)] and subsets of neuronal (VIP-IR and nitrergic IR) and glial cells (GFAP-IR and S100-IR) were determined in the small intestine of newborn preterm piglets (93% gestation), after 3 days of receiving total parenteral nutrition (TPN) and after 3 days of TPN plus 2 days of enteral feeding with sow's colostrum or milk formula. Following TPN, VIP in the myenteric and inner submucous plexus and GFAP in the inner submucous plexus increased, while the relative volume of the total neuronal population remained constant. Introduction of enteral food induced variable degrees of food intolerance and NEC, especially after formula feeding, a diet that gave rise to a higher myenteric VIP and GFAP content in the inner submucous plexus than colostrum feeding. However, the ENS seemed unaffected by the presence of NEC-like intestinal lesions. Nevertheless, this study shows that the ENS is highly plastic during the first days after premature birth and adapts in an age- and diet-dependent manner. The observed postnatal adaptation in enteric VIP and GFAP may help to maintain intestinal homeostasis during suboptimal feeding regimens in preterm neonates.

The aim of this study was to perform an immunohistochemical characterization of two different myenteric neuron types of the pig displaying opposite axonal projections. These were type I neurons equipped with lamellar dendrites that projected mainly orally, and type VI neurons that displayed typical axonal dendrites and projected anally. Double immunostainings of longitudinal muscle/myenteric plexus wholemounts from ileal segments of four pigs were performed to visualize neurofilaments (NF) in combination with calcitonin gene-related peptide (CGRP), leu-enkephalin (ENK) and substance P (SP), respectively. Triple immunostainings of wholemounts, using antibodies against neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) as well as against VIP and galanin (GAL), were performed. We found that 78% of type I neurons immunoreacted to ENK, 21% to CGRP and 24% to SP. The NF-positive type I neurons co-reactive for one of the three above markers displayed mostly frayed outlines of both their somal contours and their broadened dendritic endings. By contrast, most of the non-coreactive type I neurons displayed rather sharply outlined somata and dendrites. No type I neuron immunoreacted to nNOS, VIP or GAL and none of the type VI NF-reactive neurons reacted to CGRP, ENK or SP. All type VI neurons investigated displayed immunoreactivity for nNOS, 92% of which were co-reactive for VIP. Co-reactivity for VIP and GAL was found in 69% of type VI neurons, 21% were positive for VIP but negative for GAL, 9% were negative for both GAL and VIP, and 1% were positive for GAL but negative for VIP. We conclude that there are two subpopulations of morphological type I neurons. One of these displays mainly oral projections and could not be further characterized in this study. The other, which may correspond to neurons innervating the longitudinal and circular muscle layers, were partly immunoreactive for ENK, CGRP and/or SP. Type VI neurons are immunoreactive for nNOS frequently co-localized with VIP and, partly, also GAL. These may be inhibitory motor neurons and are different from VIP/GAL-coreactive minineurons described earlier.

In this study, we attempted to determine the proportion of type V neurons relative to the putative whole neuron population in the two submucosal plexuses of pigs identified by their neurofilament immunoreactivity. The total neuron number was estimated in cuprolinic blue (CB)/anti-Hu protein (HU) costained wholemounts as the sum of the number of CB+/HU+, CB+/HU- and CB-/HU+ neurons. In the external submucosal plexus (ESP), HU labelled 98.6% and CB 97.3% of neurons. In the internal submucosal plexus, HU labelled 98.3%, whereas CB only marked 92.5% of neurons. Furthermore, we investigated the chemical coding of submucosal type V neurons and searched for submucosal, non-type V neurons displaying the same chemical coding as the myenteric type V neurons described earlier, i.e. the colocalization of calcitonin gene-related peptide (CGRP) and somatostatin (SOM). In order to facilitate immunohistochemical detection of neuroactive peptides, ileal segments were pretreated with colchicine prior to fixation. Type V neurons in the ESP occurred either as single cells displaying one or few prominent dendrite(s) or within aggregates displaying a dendritic tangle. In this plexus, type V neurons amounted to between 0.9 and 1.6% of all CB-stained neurons. ESP type V neurons displayed immunoreactivities for choline acetyl transferase (95.8%) and leucine-enkephalin (73.9%). All type V neurons were negative for neuronal nitric oxide synthase. Fifty-eight percent of ESP CGRP/SOM co-immunoreactive neurons displayed type V morphology, whereas 42% were non-type V neurons. Thus, the chemical coding of ESP type V neurons is in principal similar to that of the myenteric type V neurons described earlier. In the internal submucosal plexus, we found no type V neurons. In this plexus, 0.2% of all neurons counterstained with HU displayed CGRP/SOM coreactivity. As had been observed earlier concerning the myenteric type V neurons, ESP type V neurons were also closely apposed by conspicuous accumulations of boutons reactive for the same markers as the neurons themselves. Although we cannot exclude that axons of CGRP/SOM-reactive enteric, non-type V or extrinsic neurons end synaptically on type V neurons, we suggest that the main synaptic input to type V neurons originates from other type V neurons. This presents an argument for an interneuronal role of type V neurons.

In the ciliary ganglion of the chicken and quail, somatostatin (SOM) is an exclusive marker for parasympathetic postganglionic neurons innervating the choroid. A second parasympathetic pathway projecting to the choroid originates from the pterygopalatine ganglion. The aim of this study was to investigate SOM immunoreactivity in the pterygopalatine ganglion of the Japanese quail (Coturnix coturnix japonica) and on neurons within the choroid, the intrinsic choroidal neurons (ICN). We did so using immunohistochemistry and subsequent light, electron and confocal laser scanning microscopy. Pterygopalatine neurons were characterized by nNOS-immunohistochemistry or NADPH-diaphorase cytochemistry. SOM immunoreactivity was absent in the perikarya, but neurons were densely surrounded by SOM-positive nerve fibres. Electron microscopy revealed that these fibres formed contacts with and without membrane specializations on pterygopalatine neurons. In the choroid, neuronal nitric-oxide synthase (nNOS)-immunoreactive ICN were likewise closely apposed by SOM-immunoreactive nerve fibres, as revealed by confocal microscopy. There was no detectable co-localization of the markers. In the absence of tracing studies, it is open to speculation whether SOM immunoreactivity originates from preganglionic fibres of the superior salivatory nucleus, postganglionic fibres of the ciliary ganglion or fibres of the brainstem via as yet unknown pathways. SOM may regulate the production of NO in pterygopalatine neurons and ICN, respectively, and is therefore involved in neuronal circuits regulating ocular homeostasis.

Neuronal plasticity in the enteric nervous system (ENS) is probably a key step in intestinal adaptation during growth, maturation and ageing as well as in several pathophysiological situations. Studies on cultured myenteric neurons have revealed an increased vasoactive intestinal peptide (VIP) expression in neuronal nitric oxide synthase (NOS)-expressing neurons. In addition, both VIP and nitric oxide (NO) promote survival of cultured myenteric neurons. The aim of the present study was to investigate possible changes in the expression of VIP and NOS in cultured submucous neurons from adult rat large intestine. Submucous neurons were cultured as explants or as dissociated neurons for 3 and 8 days. Immunocytochemistry was used to determine the proportions of neurons containing VIP or NOS in preparations of uncultured controls (reflects the conditions in vivo) and in cultured explants of submucosa and dissociated submucous neurons. In situ hybridization was used to determine changes in the expressions of NOS and VIP mRNA. The relative number of NOS-expressing neurons increased significantly during culturing. The percentage of all neurons expressing NOS was 22% in controls, while approximately 50% of the cultured submucous neurons expressed NOS. VIP-expressing neurons constituted approximately 80% of all submucous neurons in controls as well as in cultured explants or dissociated neurons. Studies on coexistence revealed that the VIP-containing neurons were the ones that started to express NOS during culture. The induced expression of NOS in cultured adult submucous neurons indicates that nitric oxide, possibly in cooperation with VIP, is important for neuronal adaptation, maintenance and survival.

Pseudouni- or multiaxonal Dogiel type II neurons are the intrinsic primary afferent (sensory) neurons (IPANs) in the guinea pig small intestine. Our aim was to decipher the chemical code of human myenteric type II neurons and to establish their putative vertical projections, i.e., from the myenteric plexus to the submucosa/mucosa. Additionally, we tried to distinguish them chemically from uniaxonal, dendritic type V neurons displaying, at first glance, similar shapes, i.e., smoothly contoured cell bodies with several long processes. Wholemount preparations of the myenteric plexus were immunohistochemically double or triple stained for neurofilaments (NF) and one or two of the following peptides: calbindin, calretinin (CR), calcitonin gene-related peptide (CGRP), somatostatin (SOM) and substance P (SP). In each triple stained wholemount three counts were conducted: (1) NF-positive pseudouni- or multiaxonal (type II) neurons including their reactivities for the above peptides, (2) uniaxonal or NF-negative neurons displaying coreactivities for the above peptides and (3) NF-reactive type V neurons taking into account their reactivities for the above markers. Additionally, type II neurons, which had an axon leading into (disrupted) interconnecting strands towards the submucosa were counted and somal areas of types II and V neurons were measured. The majority of myenteric type II neurons displayed coreactivities for SOM/CR (89.6%), SOM/SP (86.6%) and SP/CR (81.6%), respectively. A minority of type II neurons was positive for CGRP or calbindin. A small population with type III morphology (uniaxonal, long and slender dendrites) displayed the same coreactivities as type II neurons. In contrast, not one single type V neuron was coreactive for SOM/CR, SOM/SP or SP/CR. Out of 627 type II neurons counted in six wholemounts, 84 type II neurons displayed an axon which could be followed into disrupted interconnecting strands indicating a vertical projection pattern. Somal areas of type II neurons were twice as big as those of type V neurons (904+/-210 versus 449+/-110 microm(2)). In conclusion, most human myenteric type II neurons contain SOM, SP and CR. We suggest they are the human IPANs. Type V neurons are both morphologically and chemically distinctly different from type II neurons and may represent descending interneurons. Further studies have to decipher the type-specific chemical code of type II neurons distinguishing them also from type III neurons.

The similarities between heme oxygenase-2 (HO-2) and nitric oxide synthase (nNOS) and the transient expression of nNOS during development led us to investigate whether both systems are similarly affected by changes that occur during development and by regional differences along the small intestine. By combining NADPH diaphorase histochemistry and HO-2 immunohistochemistry on whole-mount preparations and by using stereologic methods, a qualitative and quantitative description of HO-2 and nNOS expression was obtained. Examinations were carried out on the small intestine of fetal, 1-2-day and 5-6-week-old pigs. In all age groups, three enteric plexuses were distinguished. The presence of HO-2-immunoreactive (HO-2-IR) and NADPH diaphorase-positive neurons corresponded to earlier morphological and physiological reports. Nevertheless, the total number of nitrergic neurons remained constant or decreased in the enteric plexuses, whereas the total number of HO-2-IR neurons displayed an overall increase. Changing concentrations of glucocorticoids, target-derived signals, presynaptic input, and an effect of HO-2 activity on nNOS synthesis are likely to play roles in the observed developmental changes. The numerical density of HO-2-IR neurons remained relatively constant along the intestinal tract; in contrast, the nitrergic neurons were most numerous in the inner submucous and myenteric plexus in the duodenum and ileum, respectively. It is believed that the duodenal nitrergic neurons in the inner submucous plexus could be involved in the regulation of duodenal secretion processes, whereas the region-dependent density in the myenteric plexus possibly forms the morphological basis for a regionally different participation of NO in the relaxation of the small intestine.

There exists much parallelism between carbon monoxide- and nitric oxide-generating systems. Therefore, we wondered whether developmental and functional differences along the duodenum similarly affect, part of them, namely, heme oxygenase-2-(HO-2) and neural isoform of nitric oxide synthase- (nNOS) expressing neurons. By applying NADPH diaphorase histochemistry and HO-2 immunohistochemistry on whole-mount preparations and by using stereologic methods, a qualitative and quantitative description of HO-2 and nNOS expression was obtained. Examinations were carried out on the duodenum of fetal, neonatal and weaned pigs. At all ages, three enteric plexuses were readily distinguished. The presence of both enzymes fits in with other morphological and physiological reports. However, the expression of both enzymes significantly changed during development. The number of HO-2-IR neurons increased approximately 20-fold in the inner submucous and almost doubled in the myenteric plexus. In addition, the number of nNOS-expressing neurons displayed a significant decrease in the outer submucous plexus after weaning. High levels of glucocorticoids may cause the perinatally increased HO-2 expression, whereas an influence on nNOS expression is doubtful. Therefore, it seems that notwithstanding the high similarity between both systems, their expression is regulated differently in the pig duodenum.

-c-Kit-positive interstitial cells of Cajal (ICC) appear to play a key role in the normal motility function and development of intestine. Nitric oxide is considered to be the most important messenger of inhibitory nonadrenergic, noncholinergic nerves in the enteric nervous system.

The aims of this study were to examine the distribution of nitrergic innervation and ICCs in normal human bowel and to demonstrate interconnections between ICCs and nitrergic nerves and smooth muscle fibers using histochemical and immunohistochemical double-staining methods with a whole-mount preparation technique and confocal laser scanning microscopy.

Full-thickness small and large bowel specimens were obtained at autopsy from 18 children who died of nongastrointestinal diseases. A whole-mount preparation was performed for all specimens, and double staining was carried out with nicotinamide adenine dinucleotide phosphate (reduced form, NADPH)-diaphorase and c-Kit immunohistochemistry. Double immunofluorohistochemistry with neuronal nitric oxide synthase and c-Kit using confocal laser scanning microscopy was also performed in all specimens.

The whole-mount preparation facilitated 3-dimensional visualization of the meshlike network of NADPH-diaphorase-positive nerve fibers in the myenteric plexus surrounded by a reticular network of c-Kit-positive ICCs. The dense c-Kit-positive cellular network located between longitudinal and circular muscle layers and at the innermost part of circular muscle layer intermingled with the myenteric plexus. Short, fine processes of ICCs made connections with the muscle fibers and c-Kit-positive cells.

The development of double-NADPH-diaphorase histochemistry and c-Kit immunohistochemistry staining technique in a whole-mount preparation provides an easy and useful method for investigating the association between c-Kit-positive cellular network and nitrergic neuronal network in the human bowel wall. The characteristic profiles of the c-Kit-positive cellular network and nitrergic neuronal network and their relationship with the smooth muscle fibers provide a morphologic basis for investigating intestinal motility disorders.

Intrinsic choroidal neurons represent peripherally displaced autonomic nerve cells supposed to work as a local integrative network similar to the enteric nervous system, to control choroidal vasculature and stromal smooth muscle. A typical feature of such intramural neuronal networks is the innervation by primary afferent collaterals expressing peptides, e.g. CGRP. The present study was aimed at determining primary afferent contacts on nitrergic intrinsic choroidal neurons (ICN) in the duck eye. In addition, a sympathetic innervation of ICN was assessed. Choroids were immunohistochemically processed for the following markers: neuronal nitric oxide synthase (nNOS), galanin (GAL), calcitonin gene-related peptide (CGRP), and tyrosine hydroxylase (TH). For evaluation, fluorescence as well as confocal laser scanning microscopy were used. For electron microscopy, immunoperoxidase staining for CGRP in combination with NADPH-diaphorase histochemistry was applied. ICN immunoreactive for nNOS or GAL spread over the entire choroid, although they were concentrated in an equatorial zone passing obliquely from naso-cranial to temporo-caudal. About 40% of ICN showed close relationships with CGRP-immunoreactive nerve fibres, originating most likely in the trigeminal ganglion, as seen in the fluorescence and confocal laserscanning microscope. These appositions could be ultrastructurally defined as both synapses and close contacts without synaptic specialization. Some ICN endowed with CGRP-positive fibres also received TH-immunoreactive boutons. CGRP-immunoreactive profiles were also detected in close relationship to choroidal non-vascular smooth muscle cells and collagen fibres connected to them. In many instances, they were intercalated between smooth muscle cells and processes of ICN forming triads. These results suggest that ICN, similar to other intramural autonomic systems integrate signals from trigeminal primary afferent collaterals. The 'sensory' terminals of these primary afferents may be located in the anterior eye segment but also within the smooth muscle stroma of the choroid itself. Thus, ocular homeostasis may be regulated via intraocular pre-central reflexes which are probably subject to sympathetic modulation.

The submucosal plexus is important in the control of secretomotor and motor function of the intestine. Our aim was to describe the projections of submucosal neurons to the mucosa within the submucosal plexus and to the circular muscle of human colon and to determine whether submucosal neurons that projected to different layers were located at different levels of the submucosa.

A retrogradely transported fluorescent dye was applied to the mucosa, submucosa or circular muscle layer of human colon which was then maintained in organotypic culture for 5 days. The submucosa was then dissected into two preparations, one containing the inner layer of the submucosal plexus and the other containing both the intermediate and outer layers. The dissected preparations were labelled with antibodies to nitric oxide synthase (NOS) or vasoactive intestinal peptide (VIP).

Submucosal neurons projected to the mucosa, submucosa and circular muscle layers for mean distances of 3.7, 3.0 and 4.3 mm, respectively. Ninety-seven per cent of submucosal neurons labelled from the circular muscle were located in the outer or the intermediate layers, while 51% of those projecting to the mucosa were in inner layer and 49% in the intermediate/outer layers of the submucosal plexus. Eleven per cent of submucosal neurons projecting to the circular muscle were immunoreactive for NOS and 12% were immunoreactive for VIP. Forty-five per cent of those projecting within the submucosa were immunoreactive for VIP and 38% of those projecting to the mucosa were immunoreactive for VIP.

Submucosal neurons in the human colon innervate the mucosa, circular muscle and submucosa and different functional classes of neurons are located in different layers of the submucosal plexus.

Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.