Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Appearance and some neurochemical features of nitrergic neurons in the developing quail digestive tract

Using immunocytochemistry, NADPH-diaphorase (NADPHd) histochemistry and electron microscopy, the appearance of nitrergic enteric neurons in different digestive tract regions of the embryonic, neonatal and adult quail was studied in whole mounts and sections. NADPHd was first expressed by embryonic day 4–5 in two distinct locations, namely the mesenchyme of the gizzard primordium and at the caeco-colonic junction. At embryonic day 6, nitrergic neurons had already begun to form a myenteric nerve network in the wall of the proventriculus, gizzard and proximal part of the large intestine and by embryonic day 9, a myenteric network was visualized along the entire digestive tract of the quail. At the level of the stomach, this network was confined to the area covered by the intermediate muscles. By embryonic day 12–13, the NADPHd-positive myenteric neurons in the wall of the distal parts of the blind-ending paired caeca also became organized into ganglia. From this developmental stage on, a submucous nitrergic nerve network, sandwiched between the lamina muscularis mucosae and the luminal side of the outer muscle layer, became prominent in the proventriculus and intestinal walls. In the adult quail, only a minority of the NADPHd-positive neurons stained for vasoactive intestinal polypeptide (VIP) along the intestine. VIP-immunoreactive (IR) cell bodies were frequent in the myenteric plexus but not in the submucous plexus, whereas there were considerable numbers of NADPHd-positive neurons in both these plexuses. Nitrergic fibres were also observed in the outer muscle layer, but were almost absent from the lamina muscularis mucosa and lamina propria, in contrast to the dense VIP-ergic innervation encircling the bases of the intestinal crypts.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic,neurofilament immunoreactive myenteric neuron types of human small intestine

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO₄ or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO₄ or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.     

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.     

Distribution and morphological features of nitrergic neurons in the porcine large intestine

The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.     

Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons

The submucous layers of human small and large intestines contain at least two separate neuron populations. Besides morphological features, they differ in their immunoreactivities for calretinin (CALR) and somatostatin (SOM), respectively. In this study, submucosal wholemounts of 23 patients or body donors (including all segments of small intestine and colon) were immunohistochemically quadruple stained for CALR and SOM as well as for substance P (SP) and choline acetyltransferase (ChAT). We found that all SOM-positive neurons co-stained for ChAT and the majority for SP [between 50 % in the small intestinal external submucosal plexus (ESP) and 75 % in the colonic ESP]. In contrast, a majority of CALR-neurons contained ChAT (between 77 % in the small intestinal ESP and 92 % in the large intestinal ESP) whereas less than 4 % of CALR-neurons were co-immunoreactive for SP. Another set of wholemounts was co-stained for peripherin, a marker enabling morphological analysis. Where identifiable, both SOM alone- and SOM/SP-neurons displayed a uniaxonal (supposed pseudouniaxonal) morphology. We suggest that the chemical code of SOM-immunoreactive, human submucosal neurons may be “ChAT+/SOM+/SP±”. In additional sections double stained for SOM and SP, we regularly found double-labelled nerve fibres only in the mucosa. In contrast, around submucosal arteries mostly SOM alone- fibres were found and the muscularis propria contained numerous SP-alone fibres. We conclude that the main target of submucosal SOM(/SP)-neurons may be the mucosa. Due to their morpho-chemical similarity to human myenteric type II neurons, we further suggest that one function of human submucosal SOM-neurons may be a primary afferent one.     

Cholinergic, nitrergic and peptidergic (Substance P- and CGRP-utilizing) innervation of the horse intestine. A histochemical and immunohistochemical study

The small and large intestine of adult horses were histochemically and immunohistochemically investigated in order to evidence components of the intramural nervous system. The general structural organization of the intramural nervous system was examined by using Nissl-thionin staining as well as the anti-neurofilament 200 (NF200) immunoreaction, which demonstrated the presence of neurons in the submucous as well as myenteric plexuses. The additional presence of subserosal ganglia was shown in the large intestine. Acetylcholinesterase (AChEase) activity was observed in both the submucous and myenteric plexuses. Localization of acetylcholine-utilizing neurons was also evidenced by immunohistochemical reactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). With both histochemistry and immunohistochemistry possible cholinergic nerve fibres were detected in the inner musculature. The two possible cholinergic co-mediators Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) have been investigated by an immunohistochemical approach. CGRP immunoreactivity was detected in roundish nerve cell bodies as well as in nerve fibres of the submucous plexus, whereas SP immunoreactivity was evidenced in nerve fibres of the tunica mucosa, in nerve cell bodies and fibres of the submucous plexus and in nerve fibres of the myenteric plexus. NADPH-diaphorase reactivity, which is linked to the synthesis and release of nitric oxide, was detected in nerve cell bodies and nerve fibres of both the submucous and myenteric plexuses as well as in a subserosal localization of the large intestine. The nitrergic components were confirmed by the anti-NOS (nitric oxide synthase) immunoreaction. Results are compared with those of other mammals and related to the complex intestinal horse physiology and pathophysiology.     

Two submucosal nerve plexus in human intestines

We examined the architecture of human submucosal nerve networks of gut segments derived from 12 individuals (each six from small and large intestines). Twelve undivided submucosal wholemounts were prepared and immunohistochemically stained for peripherin (nerve elements) and for α-smooth muscle actin (remnants of attached muscle bundles). We found two ganglionic nerve networks. The plexus submucosus externus was generally monolayered and located under the outermost surface of the submucosal wholemounts. Its nerve fibre strands frequently joined each other in acute or obtuse angles, the meshes of the network were relatively wide and frequently polyangular shaped. The plexus submucosus internus was generally multi-(mostly two- or three-)layered and occupied at least the inner half of the thickness of the wholemount, sometimes extending abluminally beyond the great submucosal vessels. Its meshes were irregular. The shapes of ganglia of the two plexus were generally different, those of the internal plexus were frequently grape-like whereas the neurons of external ganglia were mostly embedded in the contoures of the joining nerve fibres. Both plexus were intensely connected via coiled interconnecting strands, either with or without intercalated ganglia. For use of eponyms for two different submucosal plexus, the names of Meissner (inner) and Schabadasch (outer) are historically justified.     

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

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.     

Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description

The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.     

The extracellular matrix of Gadus morhua muscle contains types III, V, VI and IV collagens in addition to type I

Confocal microscopy and immuno‐histochemistry were used to examine collagens in the extracellular matrix of cod Gadus morhua swimming muscle. In addition to the well known presence of type I fibrous collagen, types III and VI were also found in the myocommata and the endomysium. The beaded collagen, type VI, was found in the endomysium and the network forming collagen, type IV, was found in the basement membrane. This is the first report of type V collagen in cod muscle and of types II, IV and VI in the muscle of a teleost.     

NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons

NADPH-diaphorase histochemistry was combined with demonstration of acetylcholinesterase and immunocytochemistry for calcitonin gene-related peptide to study esophageal innervation in the rat. Most of the myenteric neurons stained positively for NADPH-diaphorase, as did numerous varicose nerve fibers in the myenteric plexus, among striated muscle fibers, around arterial blood vessels, and in the muscularis mucosae. A majority of motor endplates (as demonstrated by acetylcholinesterase histochemistry or calcitonin gene-related peptide immunocytochemistry) were associated with fine varicose NADPH-diaphorase-positive nerve fibers. Analysis of brainstem nuclei, sensory vagal, spinal, and sympathetic ganglia in normal and neonatally capsaicin-treated rats, and comparison with anterogradely labeled vagal branchiomotor, preganglionic and sensory fibers led to the conclusion that NADPH-diaphorase-positive fibers on motor endplates originate in esophageal myenteric neurons. No association of NADPH-diaphorasepositive nerve fibers with motor endplates was found in other organs containing striated muscle. These results suggest extensive, presumably nitrergic, co-innervation of esophageal striated muscle fibers by enteric neurons. Thus, control of peristalsis in the esophagus of the rat may be more complex than hitherto assumed.     

Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

The aim of the study was: (1) to test the suitability of neurofilament (NF) immunohistochemistry for representing the shapes of morphologically defined neuron types in the pig ileum myenteric plexus, (2) to estimate the proportions of these neuron types as related to the whole myenteric neuron population and (3) to demonstrate the usefulness of a refined morphological classification of enteric neurons on the paradigm of calcitonin gene-related peptide (CGRP)-immunoreactive neurons. So far, immunoreactivity for this peptide was supposed to be present in the pig enteric nervous system only in type II neurons. Ileal whole mounts of two pigs were stained with the cuprolinic blue (CB) method and, thereafter, incubated with an antibody pool against NF proteins (70, 160 and 210 kDa), visualised with a fluorochrome-tagged secondary antibody. The structural representation of morphologically defined myenteric neuron types typical for pig ileum (Stach I, II, IV, V and VI) was equivalent to their silver impregnated image, as demonstrated in previous studies. Counts of CB-stained neurons revealed between 2,526 and 2,662 neurons per square centimetre in one pig and between 2,027 and 2,763 in the other. As related to these total neuron numbers, the proportions of type I neurons were 1.7% and 1.5%, of type II neurons 7.2% and 7.9%, of type IV neurons 1.9% and 2.4%, of type V neurons 1.1% and 1.5%, and of type VI neurons 1.3% each. These values are generally comparable with those estimated earlier on silver impregnated material. Double labelling for NF and CGRP indicated that CGRP-immunoreactive smooth contoured neurons with long processes could be subdivided into two distinct morphological neuron types, i.e. type II and type V. We conclude that NF immunohistochemistry is an appropriate tool for representation of morphologically defined enteric neuron types in the pig. Combination of this technique with immunohistochemistry for neuroactive substances may be useful for making both morphological and chemical classification schemes mutually more precise.     

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).     

Projections of 5-hydroxytryptamine-immunoreactive neurons in guinea-pig distal colon

The presence of 5-hydroxytryptamine in enteric neurons of the guinea-pig distal colon was demonstrated by immunohistochemistry and the projections of the neurons were determined. 5-Hydroxytryptamine-containing nerve cells were observed in the myenteric plexus but no reactive nerve cells were found in submucous ganglia. Varicose reactive nerve fibres were numerous in the ganglia of both the myenteric and submucous plexuses, but were infrequent in the longitudinal muscle, circular muscle, muscularis mucosae and mucosa. Reactivity also occurred in enterochromaffin cells. Lesion studies showed that the axons of myenteric neurons projected anally to provide innervation to the circular muscle and submucosa and to other more anally located myenteric ganglia. The results suggest that a major population of 5-hydroxytryptamine neurons in the colon is descending interneurons, most of which extend for 10 to 15 mm in the myenteric plexus and innervate both 5-hydroxytryptamine and non-5-hydroxytryptamine neurons.     

Ultrastructure and localization of substance P and met-enkephalin immunoreactivity in the human fetal gastric antrum

Summary The ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14–16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1–4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.     

Postnatal development of nitrergic neurons in the myenteric plexus of rat stomach

The effect of age on the proportion of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons was investigated in the myenteric plexus of five different gastric areas of 1-day-, 1-week-, 2-week-, 1-month- and 2-month-old rats. Protein gene product 9.5 immunocytochemistry was used as a marker for the total enteric neuron population in order to establish the percentage of gastric nitrergic neurons in relation to age. The percentage of NADPHd-positive neurons in the proximal parts of the rat stomach (34–38%) is significantly higher than in the antral part (29%). This difference persists in all the age groups investigated. No significant relative increase with age of NADPHd-positive neurons could be observed in any of the areas studied. These findings imply that the increased nitrergic response in the rat proximal stomach as seen in pharmacological studies cannot be explained by an increased relative number of nitrergic neurons. Accepted: 31 March 1999