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.     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

神经激肽B受体在小鼠消化道内的定位

采用免疫组织化学ABC染色方法研究了神经激肽B受体（NK3r)在小鼠消化道的分布。MK3r样阳性的神经无胞体及神经纤维主要分布在十二指肠,空肠,回肠及结肠的粘膜下层神经丛和肌间神经丛,NK3r样阳性产物在食管,胃和直肠的神经丛中未见分布;NK3r样阳性产物大部分避限于神经细胞表面,也存在于胞和一些轴突内部,并在胞质中较细胞表面染色浅。。统计结果表明NK3r样免疫阳性神经元占肠神经系统神经元总数的0．5－1％,提示小鼠消化道内NK3r样阳性神经元可能参与消化功能的调节。     

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

Summary The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm² of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.     

Immunohistochemistry and nerve lesion experiments on the methionine-enkephalin immunopositive neurons in the small intestine of the bullfrog (Rana catesbeiana)

Summary Nerve elements in the small intestine of the bullfrog. Rana catesbeiana, were studied by immunohistochemistry with anti-methionine enkephalin antisera and by nerve lesion experiments, using laser irradiation. Methionine-enkephalin immunopositive nerve fibers occur in the myenteric plexus, circular muscle layer, submucosa, and mucosa. Immunopositive nerve cell bodies in the myenteric plexus have dendrite-like and a long axon-like processes. In the froglet (3 months after metamorphosis), these axon-like processes lead posteriorly in the nerve strand of the myenteric plexus. Some bifurcate, one branch continuing posteriorly, the other doubling back to lead anteriorly; both form terminal varicose fibers in the circular muscle layer. Nerve lesion experiments, in the adult bullfrog, resulted in accumulations of methionine-enkephalin immunoreactivity at the oral and hinder edges of the laser-irradiated necrotic area; there were sprouting and nonsprouting immunopositive stumps. It is suggested that bidirectional flow of methionine-enkephalin in the myenteric plexus is mediated via the anterior and posterior branches of the axon-like process. The difference in sprouting behavior of immunopositive nerve fiber stumps, after nerve lesion, is discussed with reference to regional differences of the axon-like process.     

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.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P

 The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10^–7 M), initially at 4°C for 1 h to allow the agonist to bind, followed by incubation at 37°C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37°C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres. Accepted: 10 March 1998     

Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine

Antibodies against choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used to determine whether neurons that have previously been identified as intrinsic primary afferent neurons in the guinea-pig small intestine have a cholinergic phenotype. Cell bodies of primary afferent neurons in the myenteric plexus were identified by their calbindin immunoreactivity and those in the submucous plexus by immunoreactivity for substance P. High proportions of both were immunoreactive for ChAT, viz. 98% of myenteric calbindin neurons and 99% of submucosal substance P neurons. ChAT immunoreactivity also occurred in all nerve cell bodies immunoreactive for calretinin and substance P in the myenteric plexus, but in only 16% of nerve cells immunoreactive for nitric oxide synthase. VAChT immunoreactivity was in the majority of calbindin-immunoreactive varicosities in the myenteric ganglia, submucous ganglia and mucosa and also in the majority of the varicosities of neurons that were immunoreactive for calretinin and somatostatin and that had been previously established as being cholinergic. We conclude that the intrinsic primary afferent neurons are cholinergic and that they may release transmitter from their sensory endings in the mucosa.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the gastrointestinal tract of rat and pig

One of the opioid precursor molecules, pre-pro-enkephalin A, contains within it, in addition to Leu-enkephalin (Leu-Enk) and Met-enkephalin (Met-Enk), Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-8), which is specific to this precursor. This study deals with the localization of Met-Enk-8-like immunoreactivity in the gastrointestinal tract of rat and pig. Immunoreactivity was identified in intramural nerve elements of rat and pig, and in gut endocrine cells of pig. Immunoreactive (IR) nerve fibers were seen mainly in the myenteric plexus of rat and in both the myenteric and submucosal plexuses of pig. Some IR fibers were dispersed throughout the lamina propria mucosae of rat. Porcine IR endocrine cells were dispersed in the epithelium from the pyloric antrum to the ileum, existing concomitantly with enterochromaffin (EC) cells. Specificity tests revealed that immunoreactivity to Met-Enk-8 antiserum was not influenced by preincubation of the antiserum with Leu-Enk and Met-Enk. This suggests the possibility that pre-pro-enkephalin A is contained in the gastroenteric nerves of rat and pig and in a population of porcine EC cells.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Comparison of opioid receptor distributions in the rat ileum

The cellular expression patterns of mu-, delta- and kappa-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular mu- and delta-ir patterns differed in ganglion neurons, mu/delta co-expression was extensive in these cells. mu/delta co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate kappa-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal mu/kappa co-localisation was not apparent, although kappa-ir fibres were often apposed against mu-ir cells. mu/kappa co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in mu and delta expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types.     

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

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.     

VIP-, substance P-, gastrin/CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout,Salmo gairdneri

Summary The presence of peptides in the gastrointestinal tract of the rainbow trout, Salmo gairdneri, was investigated immunocytochemically. VIP-like immunoreactivity was demonstrated in nerves in all layers of the stomach and the intestine, whereas substance P-like immunoreactivity was localized to endocrine cells, predominantly in the mucosa of the stomach, and to nerves mainly concentrated in the myenteric plexus throughout the gut. Endocrine cells reactive to gastrin/CCK antiserum were demonstrated in the intestinal mucosa, while no immunoreactivity was found in the stomach. Bombesin-immunoreactive and somatostatin-immunoreactive cells were localized in the stomach mucosa, and cells reactive to glucagon antiserum in the intestinal mucosa. Radioimmunoassay of stomach mucosa and muscle confirmed the presence of VIP-like and substance P-like immunoreactivity in these tissues, while gastrin/CCK-like immunoreactivity was low and bombesin-like immuno-reactivity was insignificant. In conclusion, molecules resembling the mammalian brain-gut peptides may be involved in the neuronal and hormonal control of gut function in fish.     

A neurochemical characterisation of the golden hamster myenteric plexus

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.     

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons

Summary Immunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.