D.W. Scheuermann

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.

Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats.

Abstract Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-µm-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs.

Structural organization and neuropeptide distribution in the mammalian enteric nervous system, with special attention to those components involved in mucosal reflexes.

Gastrointestinal events such as peristalsis and secretion/absorption processes are influenced by the enteric nervous system, which is capable of acting largely independently from other parts of the nervous system. Several approaches have been used to further our understanding of the underlying mechanisms of specific enteric microcircuits. Apart from pharmacological and physiological studies, the deciphering of the chemical coding of distinct morphological and functional enteric neuron classes, together with a detailed analysis of their projections by the application of immunocytochemistry, of tracing, and of denervation techniques, have substantially contributed to our knowledge. In view of existing interspecies and regional differences, it is of major importance to expand our knowledge of the enteric nervous system in mammals other than the guinea-pig, the most commonly used experimental animal in this research area. This will increase our chances of finding a valid model, from which well-founded extrapolations can be made regarding the precise function of distinct enteric neuron types regulating motility and ion transport in the human gastrointestinal tract.

Distinct distribution of CGRP-, enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural networks of the porcine small intestine

SummaryIn addition to differences between the two submucosal ganglionic neural networks, i.e., the plexus submucosus externus (Schabadasch) and the plexus submucosus internus (Meissner), with respect to the occurrence and distribution of serotonin as neurotransmitter, immunocytochemistry also revealed a distinct distribution for various neuropeptides in these two plexuses. Immunoreactivity for galanin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, neuromedin U, enkephalin, somatostatin and neuropeptide Y was found in varicose and non-varicose nerve fibres of both submucosal ganglionic plexuses, albeit with a distinct distributional pattern. The difference in neurotransmitter and/or neuromodulator content between both neural networks became even more obvious when attention was focussed on the immunoreactivity of the nerve cell bodies for these substances. Indeed, neuropeptide Y, enkephalin-and somatostatin-immunoreactive neuronal perikarya as well as serotonergic neuronal cell bodies appear solely in the plexus submucosus externus. Neuromedin U-immunoreactive perikarya, mostly coexisting with substance P, are observed in large numbers in the plexus submucosus internus, whilst they are rare in the plexus submucosus externus. Double-labelling immunostaining for substance P with CGRP and galanin revealed a different coexistence pattern for the two submucosal ganglionic plexuses. The differing chemical content of the neuronal populations supports the hypothesis that the existence of the two submucosal ganglionic plexuses, present in most large mammals including man, not only reflects a morphological difference but also points to differentiated functions.

Mucosal projections of enteric neurons in the porcine small intestine.

In the present study, a combination of immunohistochemistry and retrograde 1,1`‐didodecyl‐3,3,3`,3`‐tetramethylindocarbocyanine perchlorate (DiI) tracing was used to unravel the morphology, distribution, and neurochemical coding of submucous and myenteric neurons with axonal projections to the mucosa of the porcine small intestine. The majority of traced neurons was located in the inner submucous plexus (ISP; 78%), whereas the remaining part was distributed between the outer submucous plexus (OSP; 10%) and myenteric plexus (MP; 12%). Among these traced neurons, some distinct neuronal populations could be distinguished according to their morphologic and neurochemical properties. In the ISP, several types of traced neurons were detected: 1) morphologic type II neurons expressing choline acetyltransferase (ChAT) immunoreactivity, calcitonin gene‐related peptide (CGRP) immunoreactivity, and substance P (SP) immunoreactivity; 2) ChAT/SP‐immunoreactive (‐IR) small neurons; 3) vasoactive intestinal polypeptide (VIP) ‐IR small neurons; and 4) multidendritic ChAT/somatostatin (SOM) ‐IR neurons. The traced neuronal populations of the OSP and MP were similar to each other. In both plexuses, the following DiI‐labelled neurons were found: 1) ChAT/CGRP/(SP)‐IR type II neurons; 2) multidendritic ChAT/SP‐IR neurons; and 3) multidendritic ChAT/SOM‐IR neurons. Comparison of the present findings with previously obtained data concerning the mucosal innervation pattern of the intestine of small mammals, revealed significant species differences with respect to the morphologic and neurochemical features of the involved enteric neuronal classes. Although not identical, a closer resemblance between pig and human enteric nervous system seems to be at hand, as far as the anatomic organization and the presence of neurochemically identified neuronal subtypes within the enteric nervous system are concerned. J. Comp. Neurol. 421:429–436, 2000.

Triple Immunofluorescence Staining with Antibodies Raised in the Same Species to Study the Complex Innervation Pattern of Intrapulmonary Chemoreceptors

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.

Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine

The presence and topographical distribution of nitrergic neurons in the enteric nervous system (ENS) of the pig small intestine have been investigated by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide dinucleotide phosphate diaphorase (NADPHd) histochemistry. Both techniques yielded similar results, thus confirming that within the pig ENS the neuronal isoform of NOS corresponds to NADPHd. Intrinsic nitrergic neurons were not confined to the myenteric plexus; considerable numbers were also present in the outer submucous plexus. In the inner submucous plexus, NOS immunoreactivity or NADPHd staining was restricted to a few nerve fibres and nerve cell bodies. The nitrergic neurons displayed a wide variety in size and shape, but could all be characterized as being multidendritic uniaxonal. Nerve lesion experiments showed that the majority of the myenteric nitrergic neurons project in an anal direction. Evidence is at hand to show that a substantial proportion of these neurons contribute to the dense nitrergic innervation of the tertiary plexus and the circular smooth muscle layer. Some of the nitrergic neurons of the outer submucous plexus were equally found to send their axons towards the circular muscle layer. In some of the nitrergic enteric neurons, VIP, neuropeptide Y, galanin or protein 10 occurred colocalized, but not calbindin or serotonin. The present findings provide morphological evidence for the presence of NOS in a proportion of the enteric neurons in the small intestine of a large omnivorous mammal, i.e. the pig. The topographical features of the staining patterns of NOS and NADPHd are in accord with the results of neuropharmacological studies and argue for the existence of distinct nitrergic subpopulations acting either as interneurons or as motor neurons.

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.

Neuron-specific enolase and S-100 protein immunohistochemistry for defining the structure and topographical relationship of the different enteric nerve plexuses in the small intestine of the pig

SummaryThe morphological and topographical features of the intramural enteric nervous system in the small intestine of the pig has been studied on whole mounts by means of neuron-specific enolase (NSE) and S-100 protein immu-nohistochemistry. A clear visualization of the myenteric plexus allows the recognition of its characteristic morphology, including the thin tertiary plexus coursing within the smooth muscle layers. In the tela submucosa two ganglionated plexuses, each with its own specific characteristics, can clearly be demonstrated: (1) the plexus submucosus externus (Schabadasch) located near the inner surface of the circular muscle layer at the abluminal side of the submucosal vascular arcades, and (2) the plexus submucosus internus (Meissner) close to the outer surface of the lamina muscularis mucosae at the luminal side of the submucosal vascular arcades. Due to the possibility to trace clearly the perivascular plexuses of these vascular arcades by use of immunohistochemical techniques with antibodies to NSE and S-100 protein, the two submucosal nerve plexuses can be demonstrated with exceptional clarity. This is the first report of an investigation of the intramural nerve plexuses of the small intestine of the pig using the NSE and S-100 immunostaining methods, which is sufficiently detailed to substantiate the characteristic topography and structure of the two submucosal plexuses and their relation to the smooth muscle layers and perivascular plexuses. The level of NSE immunoreactivity for enteric neurons displays great variation, a substantial proportion of the type-II neurons appearing strongly stained. Although little is known of the specific function of these enzymes, proposals are discussed.

Morphological and neurochemical identification of enteric neurones with mucosal projections in the human small intestine.

Data on the axonal projections of enteric neurones in the human intestine are still scarce. The present study aimed to identify the morphology and neurochemical coding of enteric neurones in the human small intestine, which are involved in the innervation of the mucosa. The lipophilic neuronal tracer DiI was applied to one mucosal villus of small intestinal resection specimens. The tissue was kept in organotypic culture and subsequently processed for immunohistochemistry. Neurones labelled from the mucosa were located in all ganglionated nerve networks, including the myenteric plexus. In all plexuses, at least five neurochemical types of neurones could be observed, i.e. SOM‐IR neurones, SP‐IR neurones, SOM/SP‐IR neurones, VIP‐IR neurones and neurones lacking immunoreactivity for any of these markers. Most of the DiI‐labelled neurones were multidendritic; a minority of neurones could be identified as Dogiel type II cells, suggesting the existence of a subgroup of primary afferent neurones in the DiI‐filled cell population. The ratio of labelled multidendritic neurones (assumed to be secretomotor) to labelled Dogiel type II neurones (assumed to be primary afferent) in the myenteric plexus is higher in large mammals (pig and human) than in small mammals (guinea pig). This might point to the existence of a different topographical distribution of subsets of primary afferent neurones and/or topographically distinct intrinsic mucosal reflex circuits in large mammals, including humans.