The neural regulation of the mammalian esophageal motility and its implication for esophageal diseases

In contrast to the tunica muscularis of the stomach, small intestine and large intestine, the external muscle layer of the mammalian esophagus contains not only smooth muscle but also striated muscle fibers. Although the swallowing pattern generator initiates the peristaltic movement via vagal preganglionic neurons that project to the myenteric ganglia in the smooth muscle esophagus, the progressing front of contraction is organized by a local reflex circuit composed by intrinsic neurons similarly to other gastrointestinal tracts. On the other hand, the peristalsis of the striated muscle esophagus is both initiated and organized by the swallowing pattern generator via vagal motor neurons that directly innervate the muscle fibers. The presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the esophagus had been enigmatic and neglected in terms of peristaltic control for a long time. Recently, the regulatory roles of intrinsic neurons in the esophageal striated muscle have been clarified. It was reported that esophageal striated muscle receives dual innervation from both vagal motor fibers originating in the brainstem and varicose intrinsic nerve fibers originating in the myenteric plexus, which is called ‘enteric co-innervation’ of esophageal motor endplates. Moreover, a putative local neural reflex pathway that can control the motility of the striated muscle was identified in the rodent esophagus. This reflex circuit consists of primary afferent neurons and myenteric neurons, which can modulate the release of neurotransmitters from vagal motor neurons in the striated muscle esophagus. The pathogenesis of some esophageal disorders such as achalasia and gastroesophageal reflux disease might be involved in dysfunction of the neural networks including alterations of the myenteric neurons. These evidences indicate the physiological and pathological significance of intrinsic nervous system in the regulation of the esophageal motility. In addition, it is assumed that the components of intrinsic neurons might be therapeutic targets for several esophageal diseases.     

An ultrastructural analysis of the developing enteric nervous system of the guinea-pig small intestine

Summary The developing enteric nervous system of the guinea-pig has been analysed ultrastructurally. In addition, electron microscope autoradiography, following incubation with tritiated 5-hydroxytryptamine ([³H]5-HT) or tritiated norepinephrine ([³H]NE) was used to locate the developing axons of enteric serotoninergic and adrenergic neurons respectively. Observations have been correlated with previous studies of the development of the various types of enteric neuron and the onset of intestinal neuromuscular function. Prior to 25 days of gestation no neurons can be recognized morphologically. Neurons first appear at 25 days' gestation, together with a primitive neuropil in neural islands within the outer gut mesenchyme. Ganglion cell precursors are primitive at first and resemble the cells in the surrounding mesenchyme. Growth cones are abundant but there are no terminal varicosities or synapses. The circular muscle also begins to form at this time. At 32 days' gestation the longitudinal layer of smooth muscle can be discerned and, within the myenteric plexus, terminal axonal varicosities appear containing small (about 50 nm in diameter) electron-lucent synaptic vesicles. The submucosal plexus appears to be derived from neurons and neurites that reach it from the earlier-developing myenteric plexus. The submucosal plexus can be recognized at 38 days of gestation but is not well developed until day 42. Synapses on ganglion cell somata first appear in the myenteric plexus on gestational day 38 and are numerous on day 42 when the first axo-dendritic synapses can be seen. Between days 42 and 48 the developing neural tissue and growing smooth muscle cells interdigitate but after day 48, the plexus becomes ensheathed by supporting cells and connective tissue and this interdigitation is lost. Prior to day 48 most varicosities contain small electron-lucent synaptic vesicles; however, after this time a variety of terminals appears. Between days 48 and 53 of gestation evidence of degenerating neuronal processes is common, indicating that cell death may occur. Electron microscopic autoradiography with [³H]5-HT reveals labelling of axons in the neuropil of the myenteric plexus at day 32 of gestation. Some primitive appearing cell bodies, however, are also labelled and these cells seem to be entering the myenteric plexus from the surrounding mesenchyme. After 42 days of gestation [³H]5-HT labels only axons of both nerve plexuses. Often, labelled terminals are apposed to ganglion cells or dendrites. In contrast, significant labelling by [³H]NE is not found until gestational day 48. Axons are labelled by [³H]NE and these tend to be located at the interface between the myenteric plexus and the surrounding connective tissue.     

Structure of the esophagus in the adult opossum, Didelphis virginiana.

The structure of the esophagus has been studied in the adult opossum, Didelphis virginiana. A thickening of both layers of the muscularis externa occurs at the origin of the esophagus and may represent the upper esophageal sphincter; a massive expansion of the muscularis mucosae occurs in the region of the lower esophageal sphincter. The distribution of striated, mixed and smooth muscle in the muscularis externa differs in the inner and outer layers and elements of the myenteric plexus are found to occur even in the region of striated muscle; however, the ganglia of this plexus become much more prominent as smooth muscle makes its appearance. Esophageal glands are found in the lamina propria where they are confined to the 2 ends. They are especially prominent at the distal end where they are responsible for the formation of permanent transverse folds. Similar glands are found in the submucosa, scattered throughout the length of the esophagus but distally, in the region of the transverse folds, the submucous glands disappear. In both of these layers, the glands contain mucous, serous and myoepithelial cells.     

Vagal afferents innervating the gastrointestinal tract and CCKA-receptor immunoreactivity.

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA-receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA-receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C-terminal (Ab-1) nor an N-terminal (Ab-2) specific antibody produced any specific staining; in the myenteric plexus, non-vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR-immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR-immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab-1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR-immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA-receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals.     

Direct contacts between Auerbach's ganglia and smooth muscle cells in the small intestine of the rat

The present electron microscopic study demonstrated direct contacts between Auerbach's ganglia and longitudinal smooth muscle cells in the rat small intestine. The muscle cells were often observed to extend small, foot-like processes to the Auerbach's ganglia. These processes were in contact with glial cells in the ganglia without an intervening basal lamina, or were in contact with intraganglionic axon varicosities containing many synaptic vesicles. The processes in contact with glial cells may anchor the muscle cells to the ganglia during muscle contraction and those in contact with axon varicosities may function as synaptic sites between ganglion and longitudinal muscle cells.     

Distribution and projections of nerves with enkephalin-like immunoreactivity in the guinea-pig small intestine

Whole mounts of guinea-pig small intestine were used to examine the distribution of neurons with enkephalin-like immunoreactivity and the effects of microsurgical lesions on these neurons. The enkephalin neurons are intrinsic to the intestine. Cell bodies are found in the myenteric ganglia; processes are in the myenteric plexus, circular muscle (including deep muscular plexus) and submucosa, but not in the mucosa. The cell bodies have one prominent process and several short processes, the latter occasionally are seen to give rise in turn to fine, faint processes. The prominent processes provide fibres to the circular muscle and deep muscular plexus beneath and just anal (up to about 2 mm) to the cell bodies. Fibres in the submucous ganglia come from the overlying myenteric plexus. Orally-directed processes (possibly dendrites) of myenteric cell bodies provide the varicose fibres in the myenteric ganglia. These processes are 3.5-4 mm long. The enkephalin neurons represent a population of enteric neurons, with a distinct distribution and projections, which does not correspond to any of the other populations of enteric neurons that have been studied.     

Galanin nerve fibers in the rat gut: distribution, origin and projections

Galanin, a 29 amino acid peptide, was recently isolated from the porcine gut. Immunocytochemistry revealed a dense network of galanin-immunoreactive nerve fibers in the submucosa, smooth muscle layers and intramural ganglia throughout the rat gastrointestinal tract. In the smooth muscle the density of innervation was lower in the colon than in the small intestine. In the mucosa galanin-immunoreactive fibers were quite numerous in the small intestine, less numerous in the large intestine and rare in the stomach. A moderate number of galanin-immunoreactive nerve cell bodies could be detected in the myenteric ganglia throughout the digestive tract and in the submucous ganglia of the intestines. Enteric galanin-immunoreactive fibers seem to be intrinsic to the gastrointestinal tract since their distribution and frequency were unaffected after extrinsic denervation (chemical sympathectomy, vagal denervation or clamping of nerves running within the mesenterium). Myectomy at the mid-jejunal level resulted in a total loss of galanin-immunoreactive nerve fibers 5 mm anally to the lesion with a gradual return of galanin-immunoreactive fibers up to 15-20 mm further anally; there was no overt loss of fibers orally. The findings indicate that myenteric galanin-immunoreactive neurones issue long descending projections that terminate either within the myenteric ganglia or in the smooth muscle.     

Ultrastructure and synaptology of neurons immunoreactive for gamma-aminobutyric acid in the myenteric plexus of the guinea pig small intestine

Summary Immunoreactivity for gamma-aminobutyric acid is located in one morphologically-defined class of nerve cell body in the myenteric plexus of the guinea pig small intestine. These are a subgroup of the Dogiel type I nerve cells, characterized by their lamellar dendrites, about 1 m thick and flattened in the plane of the myenteric plexus, and one (or rarely two) long axonal process that extends to either the longitudinal or the circular muscle. At an ultrastructural level the dendrites were characterized by their open cytoplasm in which were scattered granular vesicles, pale mitochondria, Golgi apparatus and endoplasmic reticulum. A large proportion of the dendritic surface was in direct contact with the extra-ganglionic space. In the cell body region, which was away from the ganglion surface, the nucleus was surrounded by a thin rim of cytoplasm. The cytoplasmic features are quite distinct from those of Dogiel type II neurons but they were shared by many other non-immunoreactive neurons. Synaptic inputs, which were all non-immunoreactive, were found on the dendrites, cell bodies, axon hillocks and axons of the gamma-aminobutyric acid-immunoreactive neurons. The predominant vesicle type in the presynaptic elements was the small clear vesicle, 40–60 nm in diameter. Based on two gamma-aminobutyric acid-immunoreactive cells that were examined in serial section, about 40–50% of synapses are dendritic, 20–25% are somatic, and 30–35% are on the axon hillock or first 50–70 m of the axon. No synapses formed by immunoreactive varicosities were found on non-immunoreactive neurons or in the neuropil of the myenteric ganglia. Moreover, the lamellar dendrites or soma of gamma-aminobutyric acid neurons were never presynaptic elements forming relationships with other elements in the ganglia. It is concluded that the gamma-aminobutyric acid reactive Dogiel type I neurons are motor neurons providing inputs to the circular and longitudinal muscle layers.     

Three-dimensional visualization of the intraganglionic structures of the rat myenteric plexus: scanning electron microscopy with the connective tissue digestion method

The rat myenteric plexus was chemically microdissected and the internal structures of the ganglia were demonstrated under a scanning electron microscope. The present preparation offers a view of the three-dimensional features of ganglion cells and permits their surface structures and the size and pattern of varicosities to be observed. Numerous finger-like processes were observed on the cell bodies of the studded neurons in close relationship with the varicose axons. The intramuscular branches of the plexus were also microdissected and their running pattern was observed.     

P2X2受体在大鼠胃肠系统肌间神经节中的分布

目的:研究P2X2受体在大鼠胃肠系统肌间神经节中的分布,为研究ATP作为神经递质对胃肠道肌间神经节节细胞的作用提供形态学基础。方法:免疫组织化学。结果:从胃到结肠均有P2X2受体阳性神经元和神经纤维分布,胃、小肠和结肠分别约60％、70％和50％的肌间神经节节细胞为P2X2受体阳性。结论:胃肠道系统多数肌间神经节节细胞都能通过ATP受体特别是P2X2受体对ATP产生反应。     

The fine structure of the submucous plexus of the guinea-pig ileum. I. The ganglia, neurons, Schwann cells and neuropil

Summary A fine structural study was made of the ganglia, neurons, Schwann cells and neuropil of the submucous plexus of the guinea-pig ileum. The arrangement of the plexus as seen by light microscopy is briefly described. Submucous ganglia are small, containing an average of eight neurons per ganglion (compared with 43 in myenteric ganglia) and are connected with each other by fine nerve strands.The cell bodies of neurons and Schwann cells and a neuropil consisting of neuronal and Schwann cell processes form the ganglia. No other cell types or blood vessels are found within the ganglia. Ganglia are surrounded by a continuous basal lamina but lack a well-defined connective tissue investment. The glial investment of neurons is incomplete: many neurons lie directly beneath the basal lamina with no intervening Schwann cell processes, and the plasma membranes of adjacent neurons are often directly apposed over large areas. Other areas of apposition occur between the cell bodies and processes of neurons and Schwann cells. Desmosome-like membrane specializations may be seen between neurons and other neurons or Schwann cells. Submucous neurons could not be categorized according to size, shape, organelle content or types of processes. Processes emerging from nerve-cell bodies were placed into four broad categories on the basis of shape and microtubule content.Many bundles of closely apposed small nerve profiles lacking intervening Schwann processes are found in the neuropil in addition to a large number of vesiculated varicosities, some of which are directly apposed to the plasma membranes of nerve-cell bodies. A small proportion of vesiculated profiles form synapses with nerve cell bodies, their processes and profiles in the neuropil. From their structure, submucous neurons appear to form a more homogeneous population than myenteric neurons. Because of their incomplete investment they are more likely to be freely exposed to substances diffusing in the extraganglionic tissue than are neurons of sympathetic ganglia.     

Vagal afferents innervating the gastrointestinal tract and CCKA‐receptor immunoreactivity

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA‐receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA‐receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C‐terminal (Ab‐1) nor an N‐terminal (Ab‐2) specific antibody produced any specific staining; in the myenteric plexus, non‐vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR‐immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR‐immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab‐1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR‐immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA‐receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals. Anat Rec 266:10–20, 2002. © 2002 Wiley‐Liss, Inc.     

Effects of age on sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle of Fischer 344 rats

Loss of myenteric neurons with age is well documented, however little is known about age-related changes of the sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle. The goal of the present study, therefore, was to evaluate the influence of age on the sympathetic innervation of the myenteric plexus throughout the gastrointestinal tract. Ad libitum fed virgin male Fischer 344 rats at 3, 15–16, 24, and 27–28 months of age were sampled. Whole mounts of the stomach, small intestine, and large intestine were processed with an antibody to tyrosine hydroxylase (TH). Additionally, some specimens labeled for TH were stained for NADPH-diaphorase to selectively label the nitrergic subpopulation of neurons in the myenteric plexus. Age-related changes in the TH-positive axons occurred as early as 15–16 months and became more pronounced by 27–28 months. Changes included markedly swollen axons and terminals and a decrease in the intensity of TH staining in some of the surviving processes. Similarly, swollen NADPH-diaphorase-positive axons were found in the myenteric ganglia and secondary plexus between ganglia in the whole mounts of rats 15–28 months of age, but swollen nitrergic axons and dystrophic TH-positive axons were never present in the same ganglion or connective. Therefore, in the aged rat, deterioration of the sympathetic innervation of the myenteric plexus could be one possible mechanism for the age-related decline in gastrointestinal motor function evidenced in the elderly.     

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.     

Neuron density and distribution of NADPH-diaphorase positive neurons in the human stomach

Neuron density and distribution of the NADPH-diaphorase positive neurons were studied in the fundus, corpus and antrum of adult human stomach using cresyl violet staining and NADPH-diaphorase histochemistry. The submucous plexus contained significantly less neurons than the myenteric plexus. Submucous NADPH-d positive neurons were mostly located in ganglia close to the circular muscle layer. Myenteric NADPH-d positive neurons represented 50–60% of the neurons in all the three regions; their density, however, was significantly lower in the fundus. NADPH-d positive fibers formed a rich plexus in the innermost portion of the circular muscle layer of the corpus.     

Calcitonin gene-related peptide-alpha receptor binding sites in the gastrointestinal tract

Calcitonin gene-related peptide-alpha (CGRP alpha) is a putative neurotransmitter in the brain and in peripheral tissues. Quantitative receptor autoradiography was used to localize and quantify the distribution of specific binding sites for radiolabeled human CGRP alpha in the canine gastrointestinal tract. The canine gastrointestinal tract was chosen as a model since it is similar in both size and structure to the human gastrointestinal tract. In the stomach CGRP alpha binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of medium and small arteries, neurons in the myenteric plexus, mucosal epithelial cells and the germinal centers of lymph nodules. In the intestines, the prominent cells types expressing CGRP alpha receptors were myenteric neurons and the germinal centers of lymph nodules. Since previous studies have demonstrated that CGRP-containing sensory neurons innervate the muscularis externa in the stomach and since CGRP alpha receptors are expressed by smooth muscle cells in the muscularis externa, these results suggest that sensory neurons may directly regulate gastric motility by releasing CGRP. In correlation with previous physiological data, the present study suggests that CGRP is involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion, neuronal excitability, and the inflammatory and immune response.     

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.     

Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus.

To demonstrate the normal topography and structure of the enteric nervous system (ENS) in the human colon, the colonic wall of patients (n = 10, mean age 66.3 years), who underwent abdominal surgery unrelated to intestinal motility disorders, was submitted to wholemount immunohistochemistry. The specimens were stretched out and separated into the tunica muscularis, the outer and inner portion of the tela submucosa and the tunica mucosa. Prior to the application of the neuronal marker Protein Gene Product (PGP) 9.5, the laminar preparations were pretreated with the maceration agent KOH. The plexus myentericus was composed of prominent ganglia and interconnecting nerve fiber strands (NFS) forming a polygonal network, which was denser in the descending than in the ascending colon. Nerve cells were observed within the ganglia as well as in primary, secondary and tertiary NFS. The latter ramified into the adjacent smooth muscle layers, which contained the aganglionated plexus muscularis longitudinalis and circularis. The submucous plexus comprised three nerve networks of different topography and architecture: the delicate plexus submucosus extremus consisted of parallel orientated NFS with isolated nerve cells and small ganglia and was located at the outermost border of the tela submucosa adjacent to the circular muscle layer. The plexus submucosus externus was closely associated with the plexus submucosus extremus and composed of larger ganglia and thicker NFS. The plexus submucosus internus was situated adjacent to the lamina muscularis mucosae and formed a network with denser meshes but smaller ganglia and NFS than the plexus submucosus externus. The NFS of the aganglionated plexus muscularis mucosae followed the course of the smooth muscle cells of the lamina muscularis mucosae. The honeycomb-like network of the plexus mucosus was located within the lamina propria mucosae and divided into a subglandular and a periglandular portion. Single and accumulated nerve cells were observed within the plexus mucosus as a regular feature. The findings confirm the complex structural organisation of the ENS encountered in larger mammals, in particular the subdivision of the submucous plexus into three different compartments. PGP 9.5-immunohistochemistry applied to wholemount preparations comprehensively visualized the architecture of the intramural nerve plexus in human colonic specimens. In addition to conventional cross-sections, this technique allows a subtle assessment and classification of structural alterations of the ENS in patients with colorectal motor disorders.     

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)     

Role of intrinsic nitrergic neurones on vagally mediated striated muscle contractions in the hamster oesophagus

Oesophageal peristalsis is controlled by vagal motor neurones, and intrinsic neurones have been identified in the striated muscle oesophagus. However, the effect(s) of intrinsic neurones on vagally mediated contractions of oesophageal striated muscles has not been defined. The present study was designed to investigate the role of intrinsic neurones on vagally evoked contractions of oesophageal striated muscles, using hamster oesophageal strips maintained in an organ bath. Stimulation (30 μs, 20 V) of the vagus nerve trunk produced twitch contractions. Piperine inhibited vagally evoked contractions, while capsaicin and N ^G-nitro-L-arginine methyl ester (L-NAME) abolished the inhibitory effect of piperine. The effect of L-NAME was reversed by subsequent addition of L-arginine, but not by D-arginine. L-NAME did not have any effect on the vagally mediated contractions and presumed ³H-ACh release. NONOate, a nitric oxide donor, and dibutyryl cyclic GMP inhibited twitch contractions. Inhibition of vagally evoked contractions by piperine and NONOate was fully reversed by ODQ, an inhibitor of guanylate cyclase. Immunohistochemical staining showed immunoreactivity for nitric oxide synthase (NOS) in nerve cell bodies and fibres in the myenteric plexus and the presence of choline acetyltransferase and NOS in the motor endplates. Only a few NOS-immunoreactive portions in the myenteric plexus showed vanilloid receptor 1 (VR1) immunoreactivity. Our results suggest that there is a local neural reflex that involves capsaicin-sensitive neurones, nitrergic myenteric neurones and vagal motor neurones.