Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the presence of enterochromaffin cells

Summary The formaldehyde-induced fluorescence technique had shown 5-hydroxytryptamine-containing enteric neurons in the intestine of the teleost Platycephalus bassensis, but did not reveal such neurons in the intestine of Tetractenos glaber or Anguilla australis. Re-examination of these animals with 5-hydroxytryptamine immunohistochemistry showed immunoreactive enteric neurons in the intestine of all three teleost species. The 5-hydroxytryptamine-containing enteric neurons showed essentially the same morphology in all species examined: the somata were situated in the myenteric plexus, extending down into the circular muscle layer, but none were found in the submucosa; processes were found in the myenteric plexus, the circular muscle layer and the lamina propria. It was concluded that the neurons may innervate the muscle layers or the mucosal epithelium, but were unlikely to be interneurons. In a range of teleosts, enterochromaffin cells were found in the intestine of only those species in which the formaldehyde technique did not visualize neuronal 5-hydroxytryptamine. Available evidence suggests that, in vertebrates, 5-HT-containing enterochromaffin cells are lacking only where there is an innervation of the gut mucosa by nerve fibres containing high concentrations of 5-HT.     

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity

Summary The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytryptamine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunoreactive nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the submucosa of the posterior large intestine and in the muscularis externa of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intestine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.     

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 study of cutaneous nerves in the emu

The distribution and chemical content of cutaneous nerves in 3- to 13-day-old emu chicks (Dromaius novaehollandiae) were examined by using double-labelling immunohistochemistry. Seven different subpopulations of cutaneous nerves were identified based on their neurochemistry. No intraepidermal nerve fibres were found. However, axons were located within the dermis and were often associated with blood vessels, pennamotor muscles and feather follicles or innervated Herbst corpuscles. Both similarities and differences exist between subpopulations of cutaneous nerves in the emu and volant birds. As in volant birds, a subpopulation of cutaneous axons innervates the superficial skin layers and contains immunoreactivity to both substance P and calcitonin gene-related peptide (CGRP). This suggests that the neuropeptide content of these presumptive free nerve endings is conserved throughout the evolution of birds. In contrast, Herbst corpuscles in the emu are innervated by axons that contain immunoreactivity for CGRP or neuropeptide Y (NPY) but that lack the calbindin D-28k immunoreactivity found in fibres innervating Herbst corpuscles of volant birds. Herbst corpuscles therefore may have a different chemical content in a flightless species from that in volant birds.This work was supported by an Australian Postgraduate Award (Industry; CO9942100) and the Flinders Institute for Health and Medical Research.     

Localization and effects of orexin on fasting motility in the rat duodenum

The orexins [orexin A (OXA) and orexin B (OXB)] are novel neuropeptides that increase food intake in rodents. The aim of this study was to determine the distribution of orexin and orexin receptors (OX1R and OX2R) in the rat duodenum and examine the effects of intravenous orexin on fasting gut motility. OXA-like immunoreactivity was found in varicose nerve fibers in myenteric and submucosal ganglia, the circular muscle, the mucosa, submucosal and myenteric neurons, and numerous endocrine cells of the mucosa. OXA neurons displayed choline acetyltransferase immunoreactivity, and a subset contained vasoactive intestinal peptide. OXA-containing endocrine cells were identified as enterochromaffin (EC) cells based on the presence of 5-hydroxytryptamine immunoreactivity. OX1R was expressed by neural elements of the gut, and EC cells expressed OX2R. OXA at 100 and 500 pmol x kg(-1) x min(-1) significantly increased the myoelectric motor complex (MMC) cycle length compared with saline. Similarly, OXB increased the MMC cycle length at 100 pmol x kg(-1) x min(-1), but there was no further effect at 500 pmol x kg(-1) x min(-1). We postulate that orexins may affect the MMC through actions on enteric neurotransmission after being released from EC cells and/or enteric neurons.     

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.     

Light- and electron-microscopic immunochemical analysis of nerve fibre types innervating the taenia of the guinea-pig caecum

Summary The present work was undertaken to determine by immunocytochemical methods which of the putative enteric neurotransmitters are contained in axons supplying the guinea-pig taenia coli and what proportion of axons is accounted for by the presence of these substances. Numerous fibres displayed immunoreactivity for dynorphin (DYN), enkephalin (ENK), -aminobutyric acid (GABA), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP), but, in contrast to other gut regions, fibres showing immunoreactivity for gastrin-releasing peptide, galanin and neuropeptide Y were rare in the taenia. Fibres reactive for calbindin, calcitonin gene-related peptide, cholecystokinin, 5-hydroxytryptamine and somatostatin were also rare. Tyrosine hydroxylase-like immunoreactivity (TH-LI) was present in numerous fibres that disappeared after extrinsic denervation, a procedure that did not detectably affect any of the other major groups of fibres. Simultaneous staining of extrinsically denervated preparations revealed that SP-LI and VIP-LI were located in separate fibres, and ultrastructural studies showed these to be 58% and 33% of intrinsic fibres supplying the muscle. Immunoreactivity for the general marker, neuron-specific enolase, was located in 95–98% of axons. ENK-LI and DYN-LI were in the same axons, and similar proportions of the fibres with either SP-LI or VIP-LI, about 85%, contained immunoreactivity for ENK and DYN. All VIP-LI fibres, but no SP-LI fibres, were reactive for NOS. The results imply that the taenia of the guinea-pig caecum is innervated by two major groups of enteric neurons: (i) excitatory neurons that contain ACh, SP, other tachykinins, and, in most cases, DYN-LI and ENK-LI; and (ii) inhibitory neurons that contain NOS-LI, VIP-LI, in most cases, the two opioids and, quite probably, ATP as a transmitter. GABA-LI is contained in a smaller population of intrinsic axons. Even though the taenia represents one of the simplest tissues for examining transmission from enteric neurons to intestinal muscle, it shares some of the complexity of other regions, in that four major axon types supply the muscle and both the enteric excitatory and enteric inhibitory neurons contain multiple transmitters.     

The presence of a galanin-like peptide in the gut neuroendocrine system of Lampetra fluviatilis and Acipenser transmontanus: an immunohistochemical study

Galanin is a brain-gut neuropeptide present in the central and peripheral nervous systems of vertebrates. In the present survey, the galaninergic and the diffuse endocrine systems of the alimentary canal of the river lamprey, Lampetra fluviatilis, and the white sturgeon, Acipenser transmontanus, were studied by immunohistochemistry. The results show the presence of galanin-like immunoreactive endocrine cells in the gut of L. fluviatilis. In addition, a galanin-like immunoreactivity was detected in enteric intramural neurons of both species. It is conceivable that the galaninergic system plays in both species a role in the regulation of the gut muscle contractility and in the modulation of mucosal secretive/absorptive processes. In A. transmontanus, the presence of galanin-like immunoreactive nerve fibres associated with components of the gut associated-lymphoid tissue is possibly correlated with a control of the defensive events at this site. The presence of a galanin-like immunoreactivity in the neuroendocrine system of these two ancient fishes confirms the hypothesis on the early occurrence of this regulative molecule in the gastro-enteric system of vertebrates.     

Glucose sensing by gut endocrine cells and activation of the vagal afferent pathway is impaired in a rodent model of type 2 diabetes mellitus

Glucose in the gut lumen activates gut endocrine cells to release 5-HT, glucagon-like peptide 1/2 (GLP-1/2), and glucose-dependent insulinotropic polypeptide (GIP), which act to change gastrointestinal function and regulate postprandial plasma glucose. There is evidence that both release and action of incretin hormones is reduced in type 2 diabetes (T2D). We measured cellular activation of enteroendocrine and enterochromaffin cells, enteric neurons, and vagal afferent neurons in response to intestinal glucose in a model of type 2 diabetes mellitus, the UCD-T2DM rat. Prediabetic (PD), recent-diabetic (RD, 2 wk postonset), and 3-mo diabetic (3MD) fasted UCD-T2DM rats were given an orogastric gavage of vehicle (water, 0.5 ml /100 g body wt) or glucose (330 μmol/100 g body wt); after 6 min tissue was removed and cellular activation was determined by immunohistochemistry for phosphorylated calcium calmodulin-dependent kinase II (pCaMKII). In PD rats, pCaMKII immunoreactivity was increased in duodenal 5-HT (P < 0.001), K (P < 0.01) and L (P < 0.01) cells in response to glucose; glucose-induced activation of all three cell types was significantly reduced in RD and 3MD compared with PD rats. Immunoreactivity for GLP-1, but not GIP, was significantly reduced in RD and 3MD compared with PD rats (P < 0.01). Administration of glucose significantly increased pCaMKII in enteric and vagal afferent neurons in PD rats; glucose-induced pCaMKII immunoreactivity was attenuated in enteric and vagal afferent neurons (P < 0.01, P < 0.001, respectively) in RD and 3MD. These data suggest that glucose sensing in enteroendocrine and enterochromaffin cells and activation of neural pathways is markedly impaired in UCD-T2DM rats.     

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.     

Neuropeptide Y-like immunoreactivity in the cardiovascular nerve plexus of the elasmobranchs Raja erinacea and Raja radiata

Summary The distribution of nerves showing neuropeptide Y (NPY)-like immunoreactivity in the cardiovascular system of elasmobranchs and teleosts has been investigated. Two species of teleosts, the rainbow trout (Salmo gairdneri) and the Atlantic cod (Gadus morhua), and three species of elasmobranchs, the spiny dogfish (Squalus acanthias), the little skate (Raja erinacea) and the starry ray (Raja radiata), were used in this study. An innervation of the cardiovascular system by an NPY-like substance was found only in the two species of Raja. A rich innervation was encountered in these skates, with the highest density of fibres in the wall of the ventricle, the conus arteriosus, the coeliac artery and smaller mesenterial vessels. In the vessels, the fibres formed a plexus at the adventitio-mediol border. Few fibres were found in the walls of the dorsal aorta, the sinus venosus and the atrium, and no fibres were observed in the walls of the ventral aorta. Falck-Hillarp fluorescence histochemistry showed the presence of a rich innervation of arteries and arterioles of the gut by catecholamine-containing nerve fibres.     

Distribution of substance P in the enteric plexuses of the small intestine of the platypus,Ornithorhynchm anatinus

Summary A study was made of substance P-like-immunoreactive nerves within the small intestine of platypus, an Australian prototherian mammal. Both immunoreactive nerve cell bodies and fibres were present. No immunoreactive fibres were found in mesenteric nerves or on intramural blood vessels, suggesting that extrinsic sensory neurons containing substance P do not innervate platypus ileum. This was further supported by the result of in vitro experiments. Although applied substance P (10^-9 M-3 × 10^-8 M) caused contraction of the longitudinal muscle, neither mesenteric nerve stimulation nor application of capsaicin caused contractions.     

Glial cells revealed by GFAP immunoreactivity in fish gut

Glial fibrillary acidic protein (GFAP) is a commonly used marker to identify enteric glia in the mammalian gut. Little is however known about enteric glia in other vertebrates. The aim of the present study was to examine the distribution of GFAP immunoreactivity in adult and developing fish. In adult shorthorn sculpin (Myoxocephalus scorpius) and zebrafish (Danio rerio), GFAP immunoreactivity was seen in the myenteric plexus in all regions of the gut. Co-staining for the neuronal markers Hu C/D and acetylated tubulin showed that GFAP immunoreactivity was not associated with nerves. GFAP immunoreactivity was predominantly seen in processes with few glial cell bodies being demonstrated in adult fish. GFAP immunoreactivity was also found in the gut in larval zebrafish from 3 days post-fertilisation, i.e. at approximately the same time that differentiated enteric nerve cells first occur. Immunoreactivity was most prominent in areas with no or a low density of Hu-immunoreactive nerve cell bodies, indicating that the developing glia follows a different pattern from that of enteric neurons. The results suggest that GFAP can be used as a marker for enteric glia in fish, as in birds and mammals. The distribution of GFAP immunoreactivity implies that enteric glia are widespread in the fish gastrointestinal tract. Glia and neurons diverge early during development of the gastrointestinal tract.     

Immunocytochemical localization of histamine in flatworms

Summary Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immuno-reactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.     

Polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the endocrine and exocrine pancreas of some vertebrates: An onto- and phylogenetic study

Summary The occurrence of polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the pancreas of some species from all the eight main vertebrate groups (cyclostomes, cartilaginous fish, bony fish, amphibia, reptiles, birds, and mammals) was investigated. In addition, an ontogenetic study of these neurohormonal peptides was performed, using the rat pancreas. The distribution of these two peptides was compared with that of the structurally closely related pancreatic polypeptide.Polypeptide YY-immunoreactive cells were found to occur in the endocrine pancreas and neuropeptide Y-immunoreactivity was observed both in neurons and nerve fibres. The polypeptide YY-immunoreactive cells were limited to mammals and reptiles only. Neuropeptide Y-immunoreactive neurons and nerves were observed in reptiles, birds, and mammals only. One reptilian species (out of three) and one mammalian (out of six) failed to show any kind of immunoreactivity for the polypeptide or neuropeptide. Pancreatic polypeptide-immunoreactive cells were found in all the species examined except in the hagfish islet.In rat foetuses, polypeptide YY-immunoreactive cells and neuropeptide Y-immunoreactive nerve elements were first demonstrated at the seventeenth day of gestation, whereas pancreactic peptide-immunoreactive cells did not appear until postnatally, namely in two day-old rats. The polypeptide-containing cells, a new cell type in the endocrine pancreas, are rare. In contrast to the pancreatic peptide cells, they do not seem to have any kind of regional distribution.     

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.     

Distribution of GABA-like immunoreactive neurons in the optic lobes of Periplaneta americana

Summary Specific antisera against protein-conjugated -aminobutyric acid (GABA) were used in immunocytochemical staining procedures to study the distribution of the putative GABA-like immunoreactive neurons in the optic lobes of Periplaneta. GABA-like immunoreactive structures are evident in all three optic neuropil regions. Six different populations of GABAergic neurons, whose perikarya are grouped around the medulla, are found within the optic lobe. The number of these immunoreactive cells varies greatly and corresponds to the number of ommatidia of the eye. In the proximal part of the lamina, a coarse network of GABA-positive fibres is recognizable. These are the processes of large field tangential cells whose fibres pass through the distal surface of the medulla. A second fibre population of the lamina is made up of the processes of the centrifugal columnar neurons whose perikarya lie proximally to the medulla. The medulla contains 9 layers with GABAergic elements of variable immunoreactivity. Layers 1, 3, 5, 7 and 9 exhibit strong labelling, as a result of partial overlapping of the processes of centrifugal and centripetal columnar neurons, tangential fibres and/or lateral processes of perpendicular fibres and (possibly) processes of amacrines. A strong immunoreactivity is found in the proximal and distal layers of the lobula.     

Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Neural and glial phenotypic expression by neural crest cells in culture: effects of control and presumptive aganglionic bowel from ls/ls mice

The enteric nervous system is formed by cells that migrate to the bowel from the neural crest. Previous experiments have established that avian crest cells in vitro will colonize explants of murine bowel and there give rise to neurons. It has been proposed that phenotypic expression by the crest-derived precursors of enteric neurons and glia is critically influenced by the microenvironment these cells encounter within the gut. To test this hypothesis, quail crest cells were cocultured with explants of control or presumptive aganglionic bowel from the ls/ls mutant mouse, and the effects of the enteric tissue on five phenotypic markers of crest cell development were followed. Aganglionosis develops in the terminal region of the colon of the ls/ls mouse because viable crest-derived neural and glial precursors fail to colonize this tissue. Expression of the phenotypic markers in the cocultures was compared with that in cultures of crest alone, crest plus neural tube, and gut grown alone. The markers examined were melanogenesis and immunostaining with antisera to 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) and the monoclonal antibodies, NC-1 and GlN1. Explants of control, but not presumptive aganglionic ls/ls gut were found to increase the incidence of the expression of 5-HT and NC-1 immunoreactivities; moreover, especially near the gut, the assumption of a neuronal morphology by 5-HT-, NC-1-, and GlN1-immunoreactive cells was also increased. Coincidence of expression of 5-HT with NC-1 and GlN1 immunoreactivities was observed. The effect of the bowel was selective in that the expression of TH immunoreactivity, which is not a marker of mature enteric neurons, was reduced rather than enhanced. The effect of enteric explants on crest cell development was specific in that it was not mimicked by explants of metanephros, which inhibited expression of 5-HT immunoreactivity and the acquisition of a neuritic form by NC-1-immunoreactive cells. It is concluded that the enteric microenvironment affects the phenotypic expression of subsets of crest cells and that this action of the bowel is manifested in vitro. The inability of presumptive aganglionic gut from ls/ls mice to influence neural phenotypic expression may be due to the failure of this tissue to produce putative factor(s) required for the effect or to the inability of the crest-derived precursor cells to migrate into the abnormal enteric tissue.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.