Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.     

Somatostatin in human enteric nerves

Summary Somatostatin-immunoreactive nerves and endocrine cells were localized by use of immunohistochemistry in human stomach, small and large intestine. The nature of the immunoreactivity in acid extracts of separated layers of intestine was determined with separation by high pressure liquid chromatography followed by detection with radioimmunoassay; authentic somatostatin-14 was found in the external musculature, which contains nerves, and in the submucosa and mucosa, which contain both nerve fibres and endocrine cells.The distribution of somatostatin nerves in the gastric antrum, duodenum, jejunum, ileum, ascending and sigmoid colon, and rectum is described. In the intestine many positive perikarya and fine varicose fibres were seen. Mucosal fibres formed a sub-epithelial plexus and a looser network in the lamina propria; this nerve supply was less dense in the large intestine. Submucous ganglia contained positive perikarya and terminals; many terminals formed pericellular baskets, mainly around non-reactive cells. A small number of nerve fibres were associated with submucosal blood vessels. The innervation of the circular and longitudinal muscle was sparse. Positive nerve terminals were seen in the myenteric plexus, although fewer than in the submucous ganglia; positive perikarya were scarce in myenteric ganglia. Somatostatin-immunoreactive nerves were found in the muscle layers and myenteric plexus of the gastric antrum, but were not detected in the antral mucosa and all layers of the gastric body.The distribution of human enteric somatostatin nerves is compared to that in small laboratory animals, and possible roles for these nerves are discussed.     

The origin and distribution of adrenergic nerve fibres in the guinea-pig colon

Summary A detailed study of the origin and distribution of sympathetic fibres in the distal colon of the guinea-pig has been made using the fluorescent histochemical method for localizing catecholamines. The extrinsic adrenergic fibres of the colonie sympathetic nerves follow the inferior mesenteric artery and its branches to the colon. Some of the extrinsic adrenergic fibres are associated with the parasympathetic fibres of the pelvic nerves near the colon. Complete adrenergic denervation follows the removal of the inferior mesenteric ganglion or the destruction of the nerves running with the inferior mesenteric artery.No fluorescent fibres, other than those associated with blood vessels, were observed in air-dried stretch preparations of the isolated longitudinal muscle. However, a substantial number of varicose, terminal fibres, not associated with blood vessels, were observed in the circular muscle. Some varicose fibres, apart from those associated with ganglion cells, were observed in the myenteric plexus. These fibres were seen in the bundles of nerves running between the nodes of the plexus and also as single fibres which branched from the plexus to end in areas free of ganglion cells.Three plexuses of adrenergic nerve fibres have been distinguished in the submucosa: a dense plexus of terminal fibres innervating both the veins and arteries; a plexus consisting of innervated nodes of ganglion cells, connected by bundles of fluorescent and non-fluorescent nerves; and a plexus of varicose and non-varicose fibres, which is not associated with ganglion cells. Some groups of ganglion cells in the submucosa were without adrenergic innervation.A plexus of varicose fibres forms a meshwork in the lamina propria of the mucosa. The muscularis mucosae is sparsely innervated. Most of the blood vessels in the mucosa are not associated with adrenergic fibres.     

Scanning electron microscopy of the muscle coat of the guinea-pig small intestine

Summary We have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) stretch preparations of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.     

PACAP 1-38 as neurotransmitter in the porcine antrum

The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. Conclusion: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release. Work in this study was funded by the National Health and Medical Research Council (grant numbers: 114215 and 216704; Senior Research Fellowship to B.S.), a Melbourne University Research Scholarship and the Murdoch Children’s Research Institute.     

Nerve fibers in the gut and pancreas of the rat displaying neuropeptide-Y immunoreactivity

Summary Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the gut and pancreas of the rat. Immunoreactive endocrine cells could not be detected. The occurrence of NPY containing nerve-cell bodies in the submucosal and myenteric ganglia indicates an intrinsic origin of the NPY fibers. However, an additional extrinsic supply of NPY fibers is suggested by the finding that abdominal sympathectomy caused the disappearance of some NPY fibers, notably those around blood vessels. The distribution of NPY fibers in all layers of the gut wall suggests multiple functions of NPY, including a role in the regulation of intramural neuronal activities, smooth muscle tone, and local blood flow.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

An immunohistochemical study of serotonin-containing nerves in the colon of rats

The localization of the serotonin-like immunoreactive nerves of the rat colon was investigated by means of immunohistochemistry, utilizing an antibody against serotonin. In non-treated colons, serotonin-positive neuropils were consistently detected around the myenteric plexus. In pargyline-treated colons, serotonin-like fibres were demonstrated in association with either the small intramural blood vessels of the submucosa or the extramural nerve bundles. Treatment with 5-hydroxytryptophan (5-HTP) permitted the visualization of additional serotonin-immunoreactive fibres around the large extramural blood vessels. Immunoreactive nerve cell bodies were demonstrated in the myenteric plexus of colons treated with 5-HTP or colchicine. From these observations, it is suggested that the serotoninergic nerves of the rat colon comprise both intrinsic and extrinsic elements.     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.     

An immunohistochemical study of serotonin-containing nerves in the colon of rats

Summary The localization of the serotonin-like immunoreactive nerves of the rat colon was investigated by means of immunohistochemistry, utilizing an antibody against serotonin. In non-treated colons, serotonin-positive neuropils were consistently detected around the myenteric plexus. In pargyline-treated colons, serotonin-like fibres were demonstrated in association with either the small intramural blood vessels of the submucosa or the extramural nerve bundles. Treatment with 5-hydroxytryptophan (5-HTP) permitted the visualization of additional serotonin-immunoreactive fibres around the large extramural blood vessels. Immunoreactive nerve cell bodies were demonstrated in the myenteric plexus of colons treated with 5-HTP or colchicine. From these observations, it is suggested that the serotoninergic nerves of the rat colon comprise both intrinsic and extrinsic elements.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

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.     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.Abbreviations BSA Bovine serum albumin - CGRP calcitonin generelated peptide - DiA carbocyanine dye A - DiI carbocyanine dye I - dmnX dorsal motor nucleus of vagus - DMSO dimethylsulfoxide - ENK enkephalin - FITC fluorescin isothiocyanate - NADPH diaphorase nicotinamide adenine diphosphate - NPY neuropeptide Y - NTS nucleus tractus solitarii - PBS phosphate-buffered saline - VIP vasoactive intestinal peptide - WGA-HRP wheat-germ agglutinine-horseradish peroxidase     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

Evidence that myenteric neurons of the gastric corpus project to both the mucosa and the external muscle: myectomy operations on the canine stomach

Summary The distribution of nerve cell bodies and fibres in the canine stomach was investigated using antibodies to the general neuronal marker, neuron-specific enolase. Prominent ganglia containing many reactive nerve cells were found in the myenteric plexus of the gastric corpus and antrum. Nerve cells were absent from the submucosa of the corpus and were extremely rare in the antrum. Renoval of areas of longitudinal muscle and myenteric plexus from the corpus (myectomy), with 7 days allowed for axon degeneration, resulted in the loss of fibres reactive for galanin, gastrin-releasing peptide, substance P and vasoactive intestinal peptide from both the circular muscle and mucosa in the area covered by the lesion. Combined vagotomy and sympathetic denervation did not significantly affect these fibres, but did cause fibres reactive for calcitonin gene-related peptide to degenerate. It is concluded that the myenteric plexus of the gastric corpus, like the myenteric plexus of the small intestine and colon, is the source of nerve fibres innervating the circular muscle, but, in contrast to other regions of the gastrointestinal tract, myenteric ganglia, not submucous ganglia, are the major, or sole, source of the intrinsic innervation of the mucosa.     

Projections of nitric oxide synthesizing neurons in the guinea-pig colon

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.