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.     

An immunohistochemical study of the distribution of enteric GABA-containing neurons in the rat and guinea-pig intestine

gamma-Aminobutyric acid (GABA) antiserum was applied to sections of rat and guinea-pig intestine which were subsequently processed to reveal any immunoreactivity using either fluorescence or peroxidase techniques. Immunopositive fibres were demonstrated in stomach, duodenum, ileum and colon of rat and guinea-pig intestine. Myenteric ganglia and nerve bundles in the circular muscle contained immunopositive nerve fibres, while the longitudinal muscle, submucosa and mucosa were only rarely innervated. In favourable sections, immunopositive fibres could be seen running from the myenteric plexus into the circular muscle, thus suggesting that the GABA-immunopositive nerves in the circular muscle originate from neurons in the myenteric plexus. In both rat and guinea-pig, immunoreactive nerve cell bodies were most numerous in the myenteric plexus of the colon. In the rat, immunopositive fibres in the circular muscle were most abundant in the ileum, whereas in the guinea-pig it was the colon circular muscle that was most richly innervated. The results demonstrate that neurons which show GABA immunoreactivity are present along the length of the gastrointestinal tract. Their distribution in both myenteric ganglia and circular muscle is heterogeneous both within and between the two species studied. It is probable that this heterogeneity reflects the diversity and specificity of function of this class of enteric neurons.     

Histochemical localization of nitric oxide-synthesizing neurons and vascular sites in the guinea-pig intestine.

Laminar preparations of fixed segments of the guinea-pig intestine were examined for nitric oxide synthase activity using reduced nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium salt as substrates. Under conditions specific for detecting nitric oxide synthase-related diaphorase activity, a subpopulation of neural elements in the myenteric plexus, deep muscular plexus and submucosa were intensely stained. Intensely stained nerve fibres were distributed throughout the meshworks of the myenteric plexus and its innervation of the circular muscle, and in the submucosa within Henle's plexus. Intensely stained nerve cells and their processes were evident in most myenteric ganglia but were rare in ganglia of Henle's plexus. Stained ganglion cells comprised types I, II and VI of the morphologically defined enteric nerve cells. Stained neural elements were increasingly prevalent within successively more caudal segments of the intestine. In addition to neuronal staining, arterioles of the submucosal vascular network displayed distinct, punctate patches of staining distributed over their surface. Perivascular nerve fibre staining was absent. These results show nitric oxide synthase activity to be present within neurons and fibres of the major enteric nerve layers and within submucosal blood vessels throughout the guinea-pig small and large intestine.     

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine.

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.     

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.     

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.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: Their projections in the guinea-pig small intestine

Changes in the distribution of 5-hydroxytryptamine-like immunoreactivity have been examined in enteric neurons at various times after microsurgical lesions of the enteric plexuses. In the myenteric plexus, varicose immunoreactive nerve fibres disappeared or were reduced in number in ganglia anal to an interruption of the myenteric plexus. Up to about 2 mm on the anal side, all varicose immunoreactive fibres disappeared from the ganglia. At about 14–16 mm below an interruption, there were about 50% of the normal number of fibres in the myenteric ganglia and at about 24 mm the innervation was normal. In the submucosa, fibres immunoreactive for 5-hydroxytryptamine were absent from an area on the anal side following interruption of the myenteric plexus. From consideration of the pattern of disappearance, it is deduced that some myenteric nerve cell bodies send immunoreactive axons in an anal direction to supply submucous ganglia. The axons run for about 8 mm in the myenteric plexus, enter the submucosa and then run for a further 4 mm approximately.Thus, varicose fibres immunoreactive for 5-hydroxytryptamine, which occur around the enteric ganglion cells of both plexuses arise from nerve cell bodies in the myenteric ganglia that send their axons in an anal direction.     

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons.

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.     

Somatostatin is present in a subpopulation of noradrenergic nerve fibres supplying the intestine

Somatostatin and dopamine β-hydroxylase have been localized in the coeliaco-mesenteric ganglia, in mesenteric nerves and in the wall of the guinea-pig small intestine. Nerve lesions were used to determine the sources of the nerves. Nerve cell bodies in the coeliaco-mesenteric ganglia with immunoreactivity for both somatostatin and dopamine β-hydroxylase project to the intestine via the mesenteric nerves. Most of their terminals are in the submucous ganglia, where they make up the full complement of noradrenergic terminals, and in the mucosa where other noradrenergic terminals, not containing somatostatin immunoreactivity, are also present. The small number of noradrenergic fibres present in the tertiary component of the myenteric plexus and in the circular muscle all show immunoreactivity for somatostatin. The noradrenergic fibres supplying the mesenteric and intestinal blood vessels and those ramifying in the myenteric ganglia do not contain somatostatin. The numerous somatostatin-immunoreactive nerves in the enteric plexuses that do not contain dopamine β-hydroxylase come from enteric nerve cell bodies.These results, considered in the context of other published work, indicate that post-ganglionic sympathetic noradrenergic neurons are chemically coded according to the target tissue they supply and suggest that neurons that were hitherto thought to be neurochemically equivalent, but which serve different functions, are in fact chemically distinct.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: their visualization and reactions to drug treatment

Immunoreactive nerve cell bodies and fibres in the intestine have been examined using three antibody preparations raised against 5-hydroxytryptamine. Cross reactivity studies indicate that the substance localized was an hydroxylated indoleamine. In the guinea-pig small intestine, nerve cell bodies were located in the myenteric plexus and varicose fibres were found in the ganglia of the myenteric and submucous plexus. The nerve cell bodies had prominent short, broad processes and a single long process. Similar nerve cells and fibres were found in the guinea-pig stomach and large intestine and areas of intestine that were examined in mice, rabbits and rats. Properties of the neurons were examined in the small intestine of the guinea-pig. The immunoreactive material was depleted by treatment with reserpine, but not by guanethidine or 6-hydroxydopamine in dose sufficient to deplete noradrenaline stores in axons in the intestine. No depletion of 5-hydroxytryptamine by the neurotoxin 5, 7-dihydroxytryptamine was observed. After depletion by reserpine, immunoreactivity of the neurons could be restored by application in vitro of 5-hydroxytryptamine, 5,7-dihydroxytryptamine or 5-hydroxytryptophan. The restoration by 5-hydroxytryptophan was prevented by the inhibitor of L-aminoacid decarboxylase, benserazide. After reserpine treatment, immunoreactivity was not restored by tryptophan. Uptake of 5, 7-dihydroxytryptamine into the nerves was antagonized by fluoxetine. The distribution of neurons with 5-hydroxytryptamine-like immunoreactivity was compared with the distribution of enteric amine-handling neurons that take up and decarboxylate L-dopa. This comparison indicated that there are two classes of aromatic amine neuron in the guinea-pig small intestine, the enteric 5-HT neurons and enteric, non-5-HT, amine handling neurons.     

Projections of substance P-containing neurons within the guinea-pig small intestine

The origins of substance P immunoreactive axons in the small intestine of the guinea-pig were investigated with an immunohistochemical technique in whole mount preparations. Nerve pathways were interrupted either in vitro or in vivo to detect the accumulation of substance P proximal to the lesion and the disappearance of immunoreactive fibres resulting from the degeneration of the severed axons. Various operations, namely, extrinsic denervation, interruption of the myenteric plexus (myotomy) or removal of the myenteric plexus with the longitudinal muscle (myectomy), were performed prior to examination of substance P-containing neurons.There are several projections of substance P-containing neurons which supply the intestine. Extrinsic neurons are the sources of two projections, one to submucosal blood vessels and one to the submucous ganglia. Intrinsic neurons located in the submucous ganglia supply the villi. Five projections arise from the myenteric plexus, a very short projection ending either within the same row of ganglia or within the adjacent rows of ganglia on both sides, a longer projection within the myenteric plexus, a very short projection to the circular muscle, a projection to the submucous ganglia where the axons surround most of submucous nerve cell bodies, and a projection to the villi.It is likely that the highly organised patterns of innervation by different substance P-containing neurons have specific roles in the intestine. Some of these neurons may act as sensory neurons, others as interneurons, and yet others as motor neurons in nerve pathways within the enteric nervous system.     

Projections of substance P, vasoactive intestinal peptide and tyrosine hydroxylase immunoreactive nerve fibres in the canine intestine, with special reference to the innervation of the circular muscle.

Antisera raised against neuron specific enolase (NSE), substance P, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) were used to reveal nerve fibres in the wall of the canine small and large intestine. The circular muscle of the colon was innervated by nerve fibre bundles that ran parallel to the muscle throughout its thickness. A plexus of fibre bundles was found against the inner (submucosal) surface of the circular muscle. Fibres with substance P, VIP and TH immunoreactivity all contributed to this innervation. The circular muscle of the small intestine was distinctly separated into outer and inner layers by a dense plexus of nerve fibres, the deep muscular plexus. The outer and inner circular muscle were innervated by substance P, VIP and TH fibres. Extrinsic denervation through the severing of nerve fibres in the mesentery caused TH fibres in the intestine to degenerate, but had no detectable effect on the fibres with substance P or VIP immunoreactivity. Myectomy (the removal of the myenteric plexus from the full circumference of the intestine over a distance of 2-3 cm), performed 7-13 days before tissue was taken, resulted in an almost complete loss of substance P fibres from the circular muscle of the colon and the outer circular muscle of the small intestine. However, many fibres persisted in the deep muscular plexus of the small intestine, and most fibres remained in its inner circular muscle. The changes in distribution of VIP fibres were almost identical, except that a small proportion of reactive fibres remained in the circular muscle of the colon and the outer circular muscle of the small intestine. It is concluded that the circular muscle layers of the small intestine and colon have dual sources of intrinsic nerve supply: the myenteric ganglia supply fibres primarily to the outer part of the muscle and the submucous ganglia supply fibres to the inner muscle. The present study further demonstrated that VIP fibres ran anally in the myenteric plexus of both the small and large intestine, whereas substance P fibres ran orally in the large intestine and both orally and anally in the small intestine. The innervation of the muscularis mucosae and mucosa by substance P and VIP fibres was not affected by myectomy or extrinsic denervation, and these structures are therefore likely to be innervated by nerve cells in the submucous ganglia.     

Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Neurofilament and glial fibrillary acid protein-related immunoreactivity in rodent enteric nervous system

Using antisera raised against neurofilaments and the glial fibrillary acidic protein (GFAP) we have examined the appearance and distribution of neurofilament- and GFAP-like immunoreactivity in the enteric nervous system of rat, mouse and guinea-pig. In whole mounts of the external circular and longitudinal muscle layers, including the myenteric plexus, a high number of neurofilament-positive perikarya were visualized both in the ganglia and in the circularly running interconnecting strands in all three species. These cells were large, usually with eccentrically placed nuclei and single, relatively thick neurofilament-positive processes. In addition, in guinea-pig myenteric plexus a small number of cells with multiple processes could be seen. Both in the longitudinal and circular interconnecting strands a large number of thin, smooth, neurofilament-positive fibres were observed. This regular network of ganglia and strands was superimposed on a sparse system of thin, usually individual neurofilament-positive fibres in the underlying circular muscle layer. Cryostat sections revealed neurofilament-positive cell bodies in the submucous plexus, whereas fibres showing neurofilament-like immunoreactivity were observed in all layers of the gut wall, with the exception of the epithelium. In whole mounts including rat and mouse myenteric plexus, a large number of cells and fibres showing GFAP-like immunoreactivity were visualized. The GFAP-positive cells were smaller and more numerous than the neurofilament-positive ones. They were present both within the ganglia and in the interconnecting strands. Several short fluorescent processes could frequently be seen emanating from the cell body. Both the strands and the ganglia contained a high number of thin, GFAP-positive fibres. Fluorescent fibres and cells were also observed in the circular muscle layer. In sections of rat and mouse small intestine, cells were observed throughout the gut wall, with the exception of the epithelium. Double labelling experiments clearly showed that neurofilament- and GFAP-positive cells represented separate cell populations. Furthermore, GFAP-positive cells and fibres outlined the neurofilament-positive perikarya. It is thus likely that the GFAP-positive cells represent enteric glial cells. The pre- and postnatal development of neurofilament- and GFAP-like immunoreactivity was studied in whole mounts from rat embryos and pups. Furthermore, the presence of neurofilament and GFAP-positive fibres was observed in whole mount preparations of rat and mouse mesenterium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Projections of chemically-specified neurons in the guinea-pig colon.

The arrangement of the enteric nerve plexuses in the colon of the guinea-pig and the distributions and projections of chemically specified neurons in this organ have been studied. Immunoreactivity for neuron specific enolase was used to examine the total population of neurons and individual subpopulations were studied using antibodies raised against calbindin, calcitonin gene-related peptide (CGRP), leu-enkephalin, gastrin releasing peptide (GRP), galanin, gamma aminobutyric acid, neurokinin A, neuropeptide Y (NPY), somatostatin, substance P, tyrosine hydroxylase and vasoactive intestinal peptide (VIP). Neuronal pathways within the colon were lesioned using myotomy and myectomy operations and extrinsic pathways running between the inferior mesenteric ganglia and the colon were also severed. Each of the antibodies revealed nerve cells and nerve fibres or only nerve fibres within the wall of the colon. VIP, galanin and GRP were in anally projecting pathways in the myenteric plexus, as they are in other species. In contrast, there are differences in the projection directions of enkephalin, substance P, NPY and somatostatin nerve fibres between regions and species. Surprisingly, somatostatin and NPY fibres have opposite projections in the small intestine and colon of the guinea-pig. The majority of nerve fibres that innervate the circular muscle, including fibres with immunoreactivity for VIP, enkephalin, substance P, NPY, galanin and GRP come from the myenteric ganglia. The mucosa is innervated by fibres from both the myenteric and submucous ganglia. The present results suggest that the guinea-pig distal colon is a suitable place in which to determine relations between structure, neurochemistry and functions of enteric neural circuits.     

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.     

Origins of synaptic inputs to calretinin immunoreactive neurons in the guinea-pig small intestine

Summary Calretinin immunoreactivity is almost completely confined to two classes of neuron in the myenteric plexus of the guinea-pig small intestine, longitudinal muscle motor neurons and ascending interneurons. Nerve cell bodies of the two classes can be readily identified by their sizes and positions in ganglia. The motor neurons, which are small Dogiel type I neurons, are about 20% and the interneurons, which are medium-sized Dogiel type I neurons, are about 5% of myenteric neurons. In the present work, we have also discovered a minor population (0.1%) of small filamentous neurons. In unoperated regions of intestine, at the light microscopic level, numerous calretinin immunoreactive nerve fibres were found in the tertiary plexus that innervates the longitudinal muscle and a medium density of varicose fibres formed pericellular endings in the myenteric ganglia. After double myotomy operations, in areas of plexus 0.5 to 1.5 mm wide which were isolated from ascending and descending inputs, calretinin-immunoreactive fibres of the tertiary plexus were unchanged, but the periceliular endings in the ganglia disappeared. Both the ascending interneurons and the longitudinal muscle motor neurons received ultrastructurally identified synapses and close axonal contacts that were calretinin-immunoreactive. These were counted in semi-serial sections from normal intestine and from regions between myotomy operations. In unoperated intestine, the proportions of calretinin-immunoreactive synapses on small, calretinin-immunoreactive, Dogiel type I nerve cells and small filamentous nerve cells were 30% and 0.1% respectively and on medium-sized Dogiel type I cells the proportion was 28%. Electron microscopy revealed an almost complete loss of immunoreactive inputs to the small Dogiel type I cells between double myotomies, but the number of unreactive inputs was the same as in normal intestine. This work demonstrates that the ascending calretinin-immunoreactive interneurons connect with one another to form ascending chains in the myenteric plexus and that they also provide about 1/3 of the inputs received by calretinin-immunoreactive longitudinal muscle motor neurons. Many of the remaining inputs to these motor neurons are local; we have deduced that these are mainly from primary sensory neurons.     

Autoradiographic study of the distribution of [3H]gamma-aminobutyrate-accumulating neural elements in guinea-pig intestine: evidence for a transmitter function of gamma-aminobutyrate

High affinity uptake, and the distribution of 3H-radiolabelled gamma-aminobutyrate (GABA), cis-3-aminocyclohexanecarboxylic acid, beta-alanine, proline, and leucine have been examined autoradiographically in laminar preparations of the myenteric plexus from the guinea-pig intestine. Following labelling with [3H]proline and [3H]leucine, which are incorporated into neurons, silver grains were concentrated over recognisable perikarya in the ganglia and meshworks of the plexus, whilst [3H]GABA labelled a smaller proportion of neurons and their processes. Specificity of labelling in the sites of [3H]GABA-uptake was established using combinations of labelled and unlabelled GABA, beta-alanine, and cis-3-aminocyclohexanecarboxylic acid, substrates for glial or neuronal high affinity GABA uptake systems. Only myenteric neurons and their processes were labelled significantly by [3H]GABA and its analogue cis-3-[3H]aminocyclohexanecarboxylic acid. Using autoradiographs of laminar preparations and paraffin sections, [3H]GABA labelling was found over nerve fibre bundles that could be traced from their ganglionic origins through the interconnecting meshworks of the myenteric plexus into the innervation of the deep muscular plexus of the circular muscle layer where GABA is evidently concerned with prejunctional modulation of transmitter release. The extensive but selective distribution of [3H]GABA high affinity uptake sites in neural elements of the guinea-pig myenteric plexus is consistent with GABA being an enteric neurotransmitter.     

Nitric oxide synthase immunoactivity and NADPH diaphorase enzyme activity in neurons of the gastrointestinal tract of the toad, Bufo marinus.

The presence of nitric oxide synthase (NOS) was demonstrated immunohistochemically, and NADPH diaphorase was demonstrated by enzyme histochemistry in neurons throughout the gastrointestinal tract of the anuran amphibian, Bufo marinus. Successive staining showed that NOS immunoreactivity and NADPH diaphorase activity occurred in precisely the same subgroup of enteric neurons. Subsequent detailed studies of the distribution of these neurons were made using NADPH diaphorase histochemistry. Numerous reactive nerve cell bodies and fibres were found in the myenteric plexus from the esophagus to the cloaca. A dense innervation of the longitudinal and circular muscle layers occurred throughout the gastrointestinal tract. The lamina muscularis mucosae was only prominent in the stomach, where it was sparsely innervated. Reactive nerve cell bodies were common in the submucosa of the large intestine, less common in the small intestine and extremely rare in the stomach and esophagus. Reactive fibres contributed to subepithelial plexuses in the esophagus, colon, rectum and cloaca. It is concluded that NOS/NADPH diaphorase is conserved amongst vertebrate classes and that NO is a likely neurotransmitter in the toad gastrointestinal tract.     

Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea-pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine beta-hydroxylase

The distribution of peptide-containing neurons in the oesophagus, stomach and small and large intestine of the rat and the guinea-pig has been studied with the indirect immunofluorescence technique ofCoons &; Co-workers (1958) using antisera to substance P, vasoactive intestinal polypeptide (VIP), enkephalin, somatostatin, gastrin and neurotensin. (The gastrin antiserum is to the C-terminal portion and consequently reacts also with cholecystokinin (CCK)-like peptides.) For comparison, the noradrenergic innervation was visualized with antiserum to dopamine β-hydroxylase. For improved visualization of peptide-containing cell bodies, a mitotic inhibitor (colchicine or vinblastine) was applied locally on the different parts of the gastro-intestinal tract of several animals.Substance P-, VIP-, enkephalin- and somatostatin-like immunoreactivity was observed in all parts of the gastro-intestinal tract studied. Gastrin/CCK had a more limited distribution, especially in the guinea-pig and neurotensin was seen only in certain regions and layers of the rat gastro-intestinal tract.Immunoreactivity to all peptides except neurotensin was observed both in cell bodies and fibres; immunoreactivity to neurotensin has so far only been seen in nerve fibres. Substance P and enkephalin immunoreactive cells were often numerous in the myenteric plexus, whereas VIP and somatostatin immunoreactive cells were preferentially located in the submucous plexus. Some VIP immunoreactive cells were observed in the lamina propria. Large numbers of especially substance P-, VIP- and enkephalin-containing fibres were often seen in the circular muscle layer and in the two ganglionic plexuses. Substance P immunoreactive fibres formed the densest network in the ganglionic plexuses, whereas VIP immunoreactive fibres constituted the most impressive network in the lamina propria and often extended into the most superficial parts of the mucosa. Enkephalin immunoreactive structures were mainly confined to the circular and longitudinal muscle layers and the myenteric plexus. Somatostatin immunoreactive fibres were mainly found in the ganglionic plexuses.Peptide-containing fibres, particularly these containing substance P and VIP were often seen along blood vessels, but never with such a density as the noradrenergic (dopamine β-hydroxylase immunoreactive) fibres. No somatostatin or neurotensin immunoreactive fibres were observed in relation to clearly identifiable blood vessels.The possible coexistence of two peptides in one neuron was studied. For this part of the study the proximal colon and five antisera, namely substance P, VIP, enkephalin. somatostatin and gastrin/CCK antisera were selected. Evidence was obtained for the occurrence of a somatostatin-like and a gastrin/ CCK-like peptide in the same neurons. This may indicate a common precursor for the two peptides in these particular neurons. Each of the substance P-, VIP- and enkephalin-like peptides. on the other hand, seem to be present in different neuronal populations, which were themselves distinct from the somatostatin-gastrin/CCK immunoreactive neurons. In addition, somatostatin immunoreactive neurons different from the gastrin/CCK immunoreactive ones seem to exist. The gastrin/CCK immunoreactive fibres around blood vessels may represent a further, separate population of fibres, since no somatostatin immunoreactive fibres were seen at this location.The findings indicate the existence of numerous subpopulations of enteric neurons, each characterized by its content of a certain peptide (or peptides). The axons of most of these neurons probably terminate in the wall of the gastro-intestinal tract, but some seem to project to other organs. In addition, some peptide-containing fibres in the gastro-intestinal wall may have an extrinsic origin. The relationship between these peptide-containing neurons and the cholinergic enteric neurons and any of the other non-cholinergic. non-adrenergic inhibitory and excitatory neurons present in the enteric nervous system is not known. It is, however, noteworthy that a somatostatin-like peptide seems to be present in noradrenergic neurons of prevertebral ganglia that project to the intestine. The possibility must be kept in mind that one or more of the peptides in the gut could be localized in neurons that contain other potential transmitters, e.g. acetylcholine.The wide variety of pharmacological actions of these neuronal peptides on smooth muscle and neurons in the gut and on its blood vessels raises the possibility that some of them may be neurotransmitters.