Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal polypeptide in the guinea-pig intestine

Immunoreactivity for vasoactive intestinal polypeptide has been localized in neurons in the guinea-pig ileum, colon and stomach. In the ileum, 2.5% of the nerve cell bodies of the myenteric plexus and 45% of those of the submucous plexus showed vasoactive intestinal polypeptide-like immunoreactivity. Varicose axons containing vasoactive intestinal polypeptide ramified amongst the nerve cell bodies of both plexuses and in some cases formed rings of varicosities around non-reactive nerve cells. Axons were traced from the myenteric plexus to the circular muscle and deep muscular plexus. There were numerous positive axons running in fine strands within the circular muscle, parallel to the muscle bundles. Axons containing vasoactive intestinal polypeptide were associated with mucosal blood vessels, but few supplied the vascular network of the submucosa; some immunoreactive axons also contributed to the periglandular plexus of the mucosa. There were no changes in the distribution of axons in the ileum after extrinsic denervation.The results are discussed in relation to the possible functional roles of neurons that contain vasoactive intestinal polypeptide in the intestine: the distribution of such nerve cells in the myenteric plexus and of axons in the circular muscle and sphincters is consistent with this polypeptide being a transmitter of enteric inhibitory neurons; it is also possible that vasoactive intestinal polypeptide is the enteric vasodilator transmitter.     

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.     

Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum

Substance P-like immunoreactivity has been localized in whole mount preparations of the isolated layers of the guinea-pig ileum. Axons containing substance P formed dense networks around the nerve cells and ran in the primary, secondary and tertiary nerve bundles of the myenteric plexus. 3.6% of the nerve cell bodies of the myenteric plexus and 11.3% of the cell bodies in the submucous plexus showed immunoreactivity for substance P. Axons ran in fine nerve bundles parallel to the longitudinal muscle, between this muscle and the myenteric plexus. Axons containing substance P also ran in small nerve trunks parallel to the circular muscle throughout its thickness and in the deep muscular plexus at the base of this muscle coat. In the submucosa, these axons ramified amongst ganglion cells of the plexus and ran in the internodal strands. In addition they formed a perivascular network around submucous arteries and contributed to the paravascular nerves following these arteries. Axons containing substance P formed a delicate plexus in the mucosa. After extrinsic denervation the nerves containing substance P that were associated with submucous arteries, and some in the submucous plexus, disappeared. The nerves in the other areas were not detectably different from normal.Comparison with the distribution of somatostatin, enkephalin and vasoactive intestinal polypeptide indicated the neurons containing substance P constitute a separate population within the enteric nervous system.     

The organisation of the enteric nervous system in the submucous and mucous layers of the small intestine of the pig studied by VIP and neurofilament protein immunohistochemistry

The arrangement of the enteric ganglia and nerve fibre plexuses was examined in the submucous and mucous layers and around Peyer's patches of the porcine small intestine to clarify their organisation. Immunohistochemistry of vasoactive intestinal peptide (VIP) and neurofilament proteins in wholemounts, chopped or paraffin sections was used to locate the neural elements. The ganglia of the internal and external submucous plexuses were situated at 2 different topographic locations, being clearly demarcated by the submucosal vascular arcades and differing in neuronal composition. The internal submucous plexus was the only contributor to the plexus surrounding the follicles of Peyer's patches as a continuous mesh of 3 ganglionated nerve subplexuses. VIP-immunoreactive fibres from this mesh innervated the dome. The mucosal plexus, which was subdivided into 4 subunits — the outer proprial, inner proprial, pericryptal and villous plexuses — contained a few solitary neuronal perikarya. Labelling for neurofilament proteins revealed Dogiel types II, IV and VI neurons. The observations reveal several new features in the enteric nervous system of the pig and clarify its nomenclature.     

Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in lethal spotted mutant mice

In order to gain insight into the process of colonization of the bowel by the neural crest-derived precursors of enteric neurons, the development of the enteric nervous system was examined in lethal spotted mutant mice, a strain in which a segment of bowel is congenitally aganglionic. In addition, nerve fibers within the ganglionic and aganglionic zones of the gut of adult mutant mice were investigated with respect to their content of acetylcholinesterase, immunoreactive substance P, vasoactive intestinal polypeptide and serotonin, and their ability to take up [³Hserotonin. In both the fetal gut of developing mutant mice and in the mature bowel of adult animals abnormalities were limited to the terminal 2 mm of colon. The enteric nervous system in the proximal alimentary tract was indistinguishable from that of control animals for all of the parameters examined. In the terminal bowel, the normal plexiform pattern of the innervation and ganglion cell bodies were replaced by a coarse reticulum of nerve fibers that stained for acetylcholineserase and were continuous with extrinsic nerves running between the colon and the pelvic plexus. These coarse nerve bundles contained greatly reduced numbers of fibers that displayed substance P- and vasoactive intestinal polypeptide-like immunoreactivity, but a serotonergic innervation was totally missing from the aganglionic bowel. During development, acetylcholineserase and uptake of [³Hserotonin appeared in neural elements in the foregut of mutant mice on the 12th day of embryonic life (E12), about the same time these markers appeared in the forgut in normal mice. By day E14, neurons expressing one or the other marker were recognizable as far distally as about 2 mm from the anus. The appearance of neurons in segments of gut grown for 2 weeks as expiants in culture was used as an assay for the presence of neuronal progenitor cells in the segments of fetal bowel at the time of explantation. Both acetyl- cholinesterase activity and uptake of [³Hserotonin developed in neuronsin vitro in expiants of proximal bowel between days E10 and E17. At all times, however, the terminal 2mm of mutant but not normal fetal gut gave rise to aneuronal cultures. In some mutant mice rare, small, ectopically-situated pelvic ganglia were found just outside aganglionic segments of fetal colon. Uptake of [³Hserotonin, normally a marker for intrinsic enteric neurites, was found in these ganglia.The experiments suppport the hypothesis that the terminal 2 mm of the gut in lethal spotted mutant mice is intrinsically abnormal and thus cannot be colonized by the precursors of enteric neurons. The defect seems to be specific in that both cells and processes of intrinsic enteric neurons, including all serotonergic and most peptidergic neurites, seem to be excluded from the abnormal region while extrinsic nerve fibers, including sympathetic and sensory axons, are able to enter the aganglionic zones. Since examination of neural progenitor cells has failed to reveal a significant proximo-distal displacement of these cells through the enteric tube during development of the murine bowel, a defect in the migration of precursor cells down the alimentary tract to the terminal gut seems unlikely to be substantially involved in the pathogenesis of aganglionosis. This conclusion is supported by the normal enteric nervous system in proximal regions of the mutant gut and the presence of enteric type neurons outside of, but at the same level as the aganglionic region.     

The origins,pathways and terminations of neurons with VIP-like immunoreactivity in the guinea-pig small intestine

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9–12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles.It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.     

Types of nerves in the enteric nervous system

The enteric nervous system is one of the three divisions of the autonomic nervous system, the others being the sympathetic and parasympathetic. In contrast to the other divisions, it can perform many functions independently of the central nervous system. It consists of ganglionated plexuses, their connections with each other, and nerve fibres which arise from the plexuses and supply the muscle, blood vessels and mucosa of the gastrointestinal tract. The enteric nervous system contains a large number of neurons, approximately 10⁷ to 10⁸. About ten or more distinct types of enteric neurons have been distinguished on electrical, pharmacological, histochemical, biochemical and ultrastructural grounds as well as on the basis of their modes of action. Both excitatory and inhibitory nerves supply the muscle and there are inhibitory and excitatory interneurons within the enteric plexuses. There are also enteric nerves which supply intestinal glands and blood vessels, but these receive less emphasis in this commentary.Correlations between groups of neurons defined on different criteria are poor and in many cases the physiological roles of the nerves are not known. The functions of noradrenergic nerves which are of extrinsic origin are reasonably well understood, but cholinergic nerves in the intestine are the only intrinsic nerves for which both the transmitter and to some extent the functions are known. In the case of non-cholinergic, non-noradrenergic enteric inhibitory nerves, the functions are understood but the transmitter is yet to be determined, both adenosine 5′-triphosphate and vasoactive intestinal polypeptide having been proposed. Other nerves have been defined pharmacologically (non-cholinergic excitatory nerves to neurons and muscle, intrinsic inhibitory inputs to neurons, and enteric, non-cholinergic vasodilator nerves) and histochemically (intrinsic amine-handling neurons and separate neurons containing peptides: substance P, somatostatin, enkephalins, vasoactive intestinal polypeptide, gastrin cholecystokinin tetrapeptide, bombesin, neurotensin and probably other peptides). Little is known of the functions of these nerves, although a number of proposals which have been made are discussed.     

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.     

How to innervate a simple gut: familiar themes and unique aspects in the formation of the insect enteric nervous system.

Like the vertebrate enteric nervous system (ENS), the insect ENS consists of interconnected ganglia and nerve plexuses that control gut motility. However, the insect ENS lies superficially on the gut musculature, and its component cells can be individually imaged and manipulated within cultured embryos. Enteric neurons and glial precursors arise via epithelial-to-mesenchymal transitions that resemble the generation of neural crest cells and sensory placodes in vertebrates; most cells then migrate extensive distances before differentiating. A balance of proneural and neurogenic genes regulates the morphogenetic programs that produce distinct structures within the insect ENS. In vivo studies have also begun to decipher the mechanisms by which enteric neurons integrate multiple guidance cues to select their pathways. Despite important differences between the ENS of vertebrates and invertebrates, common features in their programs of neurogenesis, migration, and differentiation suggest that these relatively simple preparations may provide insights into similar developmental processes in more complex systems.     

Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in the rat small intestine

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.     

The development of avian enteric nervous system: distribution of artemin immunoreactivity

Among the factors that control neural crest cell precursors within the enteric nervous system, the ligands of the glial cell line-derived neurotrophic factor family (GFL) seem to be the most influential. Artemin, a member of the GFLs, was previously described only in the oesophagus and stomach of mouse embryos. In this study, the presence and distribution of artemin is reported in duck embryos and adults. Artemin immunoreactivity was apparent in the intestinal tract at embryonic day 7 (d7), firstly in the myenteric plexus and then in the submucous plexus. Later, artemin immunoreactive nerve fibres were also seen in the longitudinal muscle plexus, the circular muscle plexus, the plexus of the muscularis mucosa and in the mucosal plexus. Furthermore, at d7, weak labeling of artemin was detected in neurons and glial cells in the oesophagus, gastric region and duodenum. Subsequently, artemin was also detected in all other intestinal segments. Moreover, during development of the gut in the domestic duck, artemin immunoreactivity decreased in neuronal cell bodies, whilst it increased in neuronal fibres and glial cells. These findings suggest an involvement of artemin in the development and biology of the gut of the domestic duck.     

Starring roles for astroglia in barrier pathologies of gut and brain

The gastrointestinal tract is a highly innervated organ and enteric neuropathy is emerging as a central feature of a wide range of gut diseases. Although most considerations of the enteric nervous system have focused on neuronal dysfunction, a large population of astrocyte-like glia populates gut muscle layers and the intestinal mucosa, and mounting new evidence points toward enteric glia as active participants in gut pathology. Similarly, in the central nervous system increasing evidence suggests that dysfunctions of astrocytes play central roles in disease mechanisms. On the basis of the premise that gut-brain disease paradigms may exist, we explore the possibility that enteric glia constitute a previously unrecognized disease target in pathologies associated with intestinal barrier dysfunction, notably inflammatory bowel disease, necrotizing enterocolitis, irritable bowel syndrome, diabetes, autoimmune disease and neurotrophic virus infection of the gut.     

Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-and neurotensin-like immunoreactive nerves in the chicken gut during development

The ontogeny and distribution of nerve cell bodies and fibres which contain vasoactive intestinal polypeptide-, substance P-, enkephalin- and neurotensin-like immunoreactivity have been studied in the chicken gastrointestinal tract, using immunocytochemistry. All four peptides were found in nerve fibres, with characteristic distribution patterns, which, in the cases of vasoactive intestinal polypeptide, substance P and methionine enkephalin were similar to those described for the mammalian gut. In addition, many of these fibres were shown to arise from intrinsic neurons, since immunoreactive nerve cell bodies for each of the peptides studied were observed. Neurotensin-immunoreactive nerves were confined to the upper part of the tract and neurotensin immunoreactive cell bodies were only observed in embryonic and newly hatched chicken gut. All four peptides were first observed at 11 days of incubation, or Hamburger-Hamilton stage 37,²⁰ in the upper part of the tract, particularly in the gizzard. Substance P and methionine enkephalin were subsequently seen in more caudal regions, while vasoactive intestinal polypeptide developed from each end of the tract. Adult patterns of immunoreactivity in nerve fibres were achieved during the first week after hatching. A striking observation was that immunoreactive neuronal cell bodies were much more abundant in the gut of young chickens and chicken embryos than in that of adult birds.     

Effect of N-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats

Intestinal ischemia impairs gastrointestinal motility. The aims of this study were to investigate the effect of intestinal ischemia on gastrointestinal transit and on the expression of enteric transmitters in the rat, and whether the glutamate N-methyl-d-aspartate receptors influence these effects. Ischemia (1 h), induced by occluding the superior mesenteric artery, was followed by 0 or 24 h of reperfusion. Normal and sham-operated rats served as controls. Serosal blood flow was measured with laser Doppler flow meter. Gastrointestinal transit was measured as time of appearance of a marker in fecal pellets. Immunohistochemistry was used to evaluate the number of neurons immunoreactive for neuronal nitric oxide synthase (NOS) or vasoactive intestinal polypeptide and the density of substance P immunoreactive fibers in the myenteric plexus. The N-methyl-d-aspartate receptors antagonist, (+)-5-methyl-10,11-dihydro-5HT-[a,b] cyclohepten-5,10-imine (MK-801) (1 mg/kg i.v.) or the NOS inhibitor, N-nitro-l-arginine (10 mg/kg i.v.) was administered prior to ischemia. Serosal blood flow was decreased by 70% during ischemia, but it was not altered in sham-operated rats. Gastrointestinal transit was significantly prolonged in ischemic/reperfused rats compared with controls. There was a significant increase in the number of vasoactive intestinal polypeptide and neuronal nitric oxide synthase immunoreactive neurons, and a marked decrease of substance P immunoreactive fibers in ischemia followed by 24 h of reperfusion animals compared with controls. These alterations were not observed in ischemia without reperfusion. A significant delay of gastrointestinal transit and increase of vasoactive intestinal polypeptide neurons were also observed in sham-operated rats. The changes in transmitter expression and gastrointestinal transit in ischemic/reperfused rats were prevented by pre-treatment with the NOS inhibitor, N-nitro-l-arginine or the N-methyl-d-aspartate receptors antagonist, MK-801. This study suggests an involvement of the glutamatergic system and its interaction with nitric oxide in intestinal ischemia/reperfusion. Ischemia/reperfusion might induce local release of glutamate that activates N-methyl-d-aspartate receptors leading to increased production of nitric oxide and adaptive changes in enteric transmitters that might contribute to gastrointestinal dysmotility.     

Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether Hand2 affects neurogenesis and/or expression of vasoactive intestinal polypeptide and choline acetyltransferase in developing enteric neurons. Gain-of-function of Hand2 in HNK-1(+) immmunoselected precursor cells resulted in increased neurogenesis. The number of neurons expressing vasoactive intestinal polypeptide increased in response to Hand2 overexpression although choline acetyltransferase was not affected. Targeted deletion of Hand2 in neural crest cells resulted in loss of all neurons expressing vasoactive intestinal polypeptide along the length of the gastrointestinal tract, patterning defects in the myenteric plexus of the stomach, and altered number and morphology of neurons expressing TH. Our data demonstrate that expression of Hand2 is sufficient and necessary for neurogenesis and expression of a subset of cell type-specific markers in the developing enteric nervous system.     

Expression of bcl-2 in enteric neurons in normal human bowel and Hirschsprung disease

OBJECTIVE: The bcl-2 protein has the functional role of blocking apoptosis, ie, programmed cell death. This protein is widely expressed in the developing central and peripheral nervous systems. The purpose of this study was to map bcl-2 expression in the human enteric nervous system, as this has not previously been done. METHODS: Rectal specimens were obtained at autopsy of 13 fetuses at 13 to 31 weeks of gestation. Normal colon was also obtained from 5 children and 2 adults, and, in addition, ganglionic and aganglionic bowel resected in 11 patients with Hirschsprung disease was examined. Specimens were fixed in formalin, embedded in paraffin, and analyzed with immunohistochemical methods, using antibodies raised against bcl-2 and neuron-specific enolase (NSE). RESULTS: The bcl-2 protein was expressed in myenteric and submucous ganglion cells in fetuses, children, and adults. Nerve fibers of the enteric plexuses that were bcl-2 immunoreactive were few compared with the number of NSE-immunoreactive nerve fibers. In aganglionic bowel no bcl-2-or NSE-immunoreactive ganglion cells were revealed. Results of NSE immunohistochemistry showed clearly stained hypertrophic nerve bundles, known to be of extrinsic origin, which were only weakly bcl-2 immunoreactive. CONCLUSION: Expression of bcl-2 in enteric ganglion cells of the myenteric and submucous plexuses is displayed in the fetus and during childhood and is also retained in adult bowel. Immunohistochemical analysis of bcl-2 provides a good marker for identification of ganglion cells in Hirschsprung disease and may also be valuable for the diagnosis of disorders characterized by hypoganglionosis or hyperganglionosis.     

Projections of peptide-containing neurons in rat small intestine

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, neuropeptide Y, somatostatin, substance P, enkephalin and calcitonin gene-related peptide were studied in the rat jejunum by immunocytochemistry and immunochemistry. Their origin was determined by the use of various procedures for extrinsic denervation (chemical sympathectomy, bilateral vagotomy or clamping of mesenterial nerves). The terminations of the different types of intramural nerve fibers were identified by examination of the loss of nerve fibers that followed local disruption of enteric nervous pathways (intestinal myectomy, transection or clamping). The majority of the peptide-containing nerve fibers in the gut wall were intramural in origin, each nerve fiber population having its own characteristic distribution and projection pattern. Nerve fibers emanating from the myenteric ganglia terminated within the myenteric ganglia and in the smooth muscle layers: those storing vasoactive intestinal peptide/neuropeptide Y, somatostatin and substance P were descending, those storing enkephalin were ascending and those containing calcitonin gene-related peptide projected in both directions. Nerve fibers emanating from the submucous ganglia terminated mainly within the submucous ganglia and in the mucosa: those storing calcitonin gene-related peptide or vasoactive intestinal peptide/neuropeptide Y were ascending and those storing substance P or somatostatin were both ascending and descending. Enkephalin nerve fibers could not be detected in the mucosa.     

Immunohistochemical studies of the enteric nervous system in tissue culture and in situ: localization of vascoactive intestinal polypeptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut

Immunofluorescence methods have been used to determine the detailed distribution of vasoactive intestinal polypeptide (VIP), substance-P and enkephalin nerve fibres in fixed cryostat sections from guinea-pig duodenum, jejunum, ileum, caecum at the site of the taenia coli, and proximal and distal colon. A novel method is used involving immunostaining of tissue culture preparations of both myenteric and submucous plexuses. These preparations allow each plexus to be studied in isolation from all axonal input for the first time, since they provide unequivocal extrinsic denervation together with severance of any intrinsic connections between the plexuses. In tissue sections the most prominent sites of VIP and substance-P immunoreactive fibres are the ganglia of the myenteric and submucous plexuses, the circular muscle layer and the longitudinal muscle of the taenia coli. In addition, VIP is prominent in the lamina propria of the submucosa except in the caecum. Enkephalin-immunopositive fibres are restricted to the ganglia of the myenteric plexus, the circular muscle layer and the longitudinal layer of the taenia coli. The culture preparations reveal that intrinsic ‘VIP neurons’ are common in the submucous plexus of the caecum and colon. They are also present, but in much lower numbers, in the myenteric plexus of the small intestine and colon but are not found in the myenteric plexus of the caecum. Intrinsic ‘substance-P neurons’ are present in the myenteric plexus from the small intestine, caecum and colon as well as in the submucous plexus of the colon; intrinsic ‘substance-P neurons’ are not found in the submucous plexus of the caecum. ‘Enkephalin neurons’ are numerous in the myenteric plexus of the small intestine, caecum and colon but are absent from the submucous plexus. Immunoreactivity is compared in the normal and denervated caecum by both the histochemical method and by radioimmunoassay of tissue extracts. In conjunction with the studies on tissue cultures, the results provide evidence for intrinsic reciprocal connections between the myenteric and submucous plexus of the caecum by neurons containing VIP and substance-P.An extensive comparison of these results with data from functional studies shows that the distribution of VIP, substance-P and enkephalin fibres in the gut is broadly in agreement with present knowledge of the action of these peptides on gut tissue, if it is assumed that they function as neurotransmitters or neuromodulators. In some instances, however, peptide-containing fibres and pathways are found which do not correlate with present knowledge obtained from functional studies. These observations provide new clues to the role of peptide neurons in gut function.     

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.