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PubMed2002

Immunohistochemical localization of voltage-activated calcium channels in the rat oesophagus.

Voltage-activated calcium channels play an important role in the physiology of the enteric nervous system. To determine which types of voltage-activated calcium channels are present in the rat oesophagus, an immunohistochemical study was performed using specific antibodies for the alpha1 subunits of Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 and Cav1.3 (L-type) calcium channels. All myenteric cell bodies showed Cav2.2 immunoreactivity, whereas labelling for this N-type channel was absent in nerve fibres. Cav1.2 immunoreactivity was found on nerve fibres in the myenteric plexus and on fibres innervating the striated muscle of the rat oesophagus, whereas no labelling was detected on neuronal somata. Immunoreactivity against Cav1.3 was not detected in the myenteric plexus or at the level of the striated muscle. Labelling for Cav2.1 was absent at the level of the myenteric plexus, but present in the striated muscle layer at the level of the motor endplates. Comparison with recent literature data from rat small intestine reveals region-specific distribution patterns of the various subtypes of voltage-activated calcium channels within the enteric nervous system. In addition, the present immunohistochemical data corroborate our physiological data (see accompanying paper), which indicate that the Cav2.2 (N-type) channel is the predominant channel involved in the generation of the calcium-dependent action potential evoked by intrasomatic depolarizing current pulses in all rat oesophageal myenteric neurones.

Animals↗
PubMed1987

Abnormalities of peptide-containing nerve fibers in infantile hypertrophic pyloric stenosis.

The distributions of nerve cells and fibers with immunoreactivity for the peptides enkephalin, gastrin-releasing peptide, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide were examined in specimens of myenteric plexus and external muscle from the pylorus of 20 infants with hypertrophic pyloric stenosis. These were compared with peptide distributions in pyloric samples from unaffected infants and adults. In the normal pylorus the circular muscle was richly supplied with fibers reactive for enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. In pyloric stenosis, these immunoreactive fiber bundles were either missing or less than 5% of normal. In contrast, there were reactive cell bodies and nerve fibers in the myenteric plexuses of both normal and affected specimens. In the samples from cases of stenosis, swollen nerve fibers that appeared to be in the process of degeneration were frequently encountered. It is concluded that infantile hypertrophic pyloric stenosis is associated with a loss of peptide immunoreactivity in nerve fibers in the circular muscle, although the same peptides are still revealed in fibers and in nerve cell bodies in the myenteric plexus.

Enkephalin, Leucine↗
PubMed2000

Acidic fibroblast growth factor is present in the enteric nervous system of the large intestine.

Acidic fibroblast growth factor (aFGF) is a heparin binding protein that displays pleiotropic activity. The purpose of this study was to document the presence of the translated aFGF product, its mRNA, and its location in the colon. mRNA was extracted from bovine large intestine and reverse transcribed to cDNA. Nested-primer PCR was used to determine the presence of mRNA using primers homologous to the previously published bovine aFGF cDNA. Purification of translated aFGF was performed using an established HPLC protocol. Western blot analysis of the HPLC fractions was performed using two epitope-independent antibodies against aFGF. Immunohistochemistry employed these antibodies to determine the locus of aFGF expression. The nested-primer PCR product of predicted size was homologous to the published bovine aFGF mRNA sequence, as determined by DNA sequencing. Intestinal aFGF had a mass similar to bovine aFGF isolated from other tissues, and immunocrossreacted with two peptide-based, epitope-independent anti-aFGF antisera on Western blotting. Immunohistochemical analysis of large intestine using these two independent antisera localized aFGF within the myenteric plexus. These data demonstrate that aFGF is present within the myenteric plexus of the enteric nervous system.

Animals↗
PubMed1999

A correlative morphometric and clinical investigation of hypoganglionosis of the colon in children.

Hypoganglionosis of the myenteric plexus of the colon is not clearly defined and seldom investigated. Colon segments from 15 children with an extended oligoeuronal hypoganglionosis up to the proximal resection end were morphometrically studied and compared to normally innervated colon segments. The study was performed with resected specimens from 7 children with isolated hypoganglionoses, 8 children with a Hirschsprung-associated hypoganglionosis, and 12 colon segments with normal innervation. The resected colon specimens were caudo-cranial coiled. The native tissue was frozen at -80 degrees C on a cryostat carrier and cut at -20 degrees C in 15 microns-thick sections (equivalent to 4-5-micron-thick paraffin sections). The air-dried sections underwent an enzyme-histochemical procedure for an acetylcholinesterase reaction to stain the parasympathetically innervated myenteric plexus. For histological identification and morphometric measurements, ganglia and nerve cells were selectively stained using a lactic dehydrogenase reaction. The morphometric measurements were performed with an optic-electronic image analysis system that determined ganglion size, ganglion distances, nerve cell number per ganglion, and ganglion number per mm colon. The results showed that hypoganglionosis of the myenteric plexus is characterised by a 42% decrease in plexus area and a 55% decrease of the nerve cell number per mm length of colon. The number and area of myenteric ganglia showed a decrease of 59% and a doubling of the ganglion distances. The histopathological diagnosis of a hypoganglionosis of the colon was not necessarily an indication of a chronic constipation, but rather an indication of a disposition for constipation. A chronic constipation is often caused by a long hypoganglionic segment proximal to a resected short Hirschsprung segment.

Cell Count↗
PubMed1998

Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine.

Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.

Aging↗
PubMed2006

Toxicokinetics of lambda-cyhalothrin in rats.

The toxicokinetics of lambda-cyhalothrin after single 20 mg kg(-1) oral and 3 mg kg(-1) intravenous doses were studied in rats. Serial blood samples were obtained after oral and intravenous administration. Liver, brain, spinal cord, sciatic nerve, vas deferens, anococcygeus and myenteric plexus tissue samples were also collected. Plasma, liver, hypothalamus, cerebellum, medulla oblongata, frontal cortex, striatum, hippocampus, midbrain, spinal cord, vas deferens, anococcygeus, myenteric plexus and sciatic nerve concentrations of lambda-cyhalothrin were determined by HPLC. The plasma and tissue concentration-time data for lambda-cyhalothrin were found to fit a two-compartment open model. For lambda-cyhalothrin, the elimination half-life (T1/2beta) and the mean residence time from plasma were 7.55 and 8.55 h after i.v. and 10.27 and 14.43 h after oral administration. The total plasma clearance was not influenced by dose concentration or route and reached a value of 0.060l h(-1)kg(-1). After i.v. administration, the apparent volume of distribution and at steady state were 0.68 and 0.53l kg(-1), suggesting a diffusion of the pyrethroid into tissue. After oral administration, lambda-cyhalothrin was extensively but slowly absorbed (Tmax, 2.69 h). The oral bioavailability was found to be 67.37%. Significant differences in the kinetic parameters between nervous tissues and plasma was observed. The maximum concentrations in hypothalamus (Cmax, 24.12 microg g(-1)) and myenteric plexus (Cmax, 25.12 microg g(-1)) were about 1.5 times higher than in plasma (Cmax, 15.65 microg ml(-1)) and 1.3 times higher than in liver (Cmax, 18.42 microg ml(-1)). Nervous tissue accumulation of lambda-cyhalothrin was also reflected by the area under the concentration curve ratios of tissue/plasma (liver). The T1/2beta for lambda-cyhalothrin was significantly greater for the nerve tissues, including neuromuscular fibres, (range 12-26 and 15-34 h, after i.v. and oral doses) than for plasma (7.55 and 10.27 h, respectively).

Administration, Oral↗
PubMed1996

Leukemia inhibitory factor mediates cytokine-induced suppression of myenteric neurotransmitter release from rat intestine.

BACKGROUND & AIMS: Exposure of rat longitudinal muscle myenteric plexus to the proinflammatory cytokine interleukin (IL) 1 beta mimics the effects of nematode infection on enteric nerve function through a hitherto unidentified protein intermediate. It is postulated that leukemia inhibitory factor (LIF), the downstream intermediate of several IL-1-induced neuroimmune interactions, mediates IL-1 beta-induced suppression of acetylcholine release from rat jejunum. METHODS: Preparations were preloaded with [3H]choline, and [3H]-acetylcholine release was induced by either electrical field stimulation or by 50 mmol/L KCl. Cytokines and anti-LIF antibodies were added to the preincubation media or to the superfusate before stimulation. RESULTS: Human recombinant LIF had no immediate effects, but preincubation with the cytokine induced a concentration-dependent (2-100 ng/mL) and reversible suppression of acetylcholine release from rat longitudinal muscle myenteric plexus. The effects of human recombinant LIF on acetylcholine release were reversed by anti-human recombinant LIF-neutralizing antibody. Human recombinant IL-1 beta (10 ng/mL) induced a similar suppression of acetylcholine release, and the addition of anti-rat LIF antibody abolished the effects of exogenous IL-1 on acetylcholine release. CONCLUSIONS: IL-1 beta suppresses neurotransmitter release from rat myenteric plexus via the induction of LIF as a downstream intermediate.

Acetylcholine↗
PubMed1983

Immunohistochemical studies on the gastrointestinal tract using antisera to Met-enkephalin and Met-enkephalin Arg6Phe7.

Antisera specific for the C-terminus of Met-enkephalin and two of its variants isolated from adrenal medulla and brain, namely Met-enk Arg6 and Met-enk Arg6Phe7, have been used in immunohistochemical studies of the gastrointestinal tract in rat, mouse and guinea pig. Met-enk and Met-enk Arg6Phe7-like immunoreactivities were found with similar distribution in nerve cell bodies of the myenteric plexus, and in fibers that were particularly dense in the myenteric plexus and circular smooth muscle. The Met-enk Arg6 antiserum showed weak staining of nerve cells and fibers. In rat and mouse, the antiserum to Met-enk Arg6Phe7, but not those to Met-enk or Met-enk Arg6, also stained numerous endocrine-like cells of the antral mucosa. These were identified as gastrin cells by elution and re-staining experiments with C-terminal gastrin antisera. The rat and mouse gastrin cells might conceivably express the enkephalin gene, but fail to process it to yield Met-enkephalin; alternatively, the Met-enk Arg6Phe7-like immunoreactivity in gastrin cells could be due to a cross-reacting peptide that does not belong to the opioid series.

Animals↗
PubMed1990

Visceral neuropathies responsible for chronic intestinal pseudo-obstruction syndrome in pediatric practice: analysis of 26 cases.

Twenty-six children aged from 1 day to 15 years (18 boys and 8 girls) with visceral neuropathies are analyzed. Clinical symptomatology is dominated by abdominal distension, attacks of occlusion, abdominal pain, and malnutrition. Intestine bacterial overgrowth is frequent. From aspiration biopsies, surgical rectal biopsies, and, in some cases, ileal or ileocolic biopsies, histopathological studies revealed two patterns. One group had abnormalities of the myenteric plexus identified by conventional light microscopic studies, with two patterns: [myenteric plexus hyperplasia (9 patients), characterized by large ganglionic nodes, penetration into the mucosal zone, and altered argyrophilic neurons]. Clinically this pattern was observed in four patients with multiple endocrine neoplasia syndrome with risk of medullary thyroid carcinoma. The second pattern observed was characterized by glial cell hyperplasia (15 patients). Ganglion cells are present but are small and sparse, often infiltrated by collagen tissue; Schwann nerve fibers are hypertrophic. Eleven patients presented with neonatal intestinal obstruction. The second group is characterized by normal conventional light microscopic examination, but silver stains revealed important abnormalities of argyrophobic cells (one case) or argyrophilic cells (one case). In the two groups, most of the patients needed intestinal derivation and prolonged nutritional support with total parenteral nutrition.

Adolescent↗
PubMed1988

Enteric neuron numbers and sizes in Auerbach's plexus in the small and large intestine of adult and aged rats.

Neurons in the myenteric plexus of Auerbach of the jejunum, ileum, colon and rectum in 6- and 24-month-old rats have been visualised by an enzyme histochemical method in laminar preparations of the muscularis externa. Neuron somata numbers and sizes were recorded in the preparations. Significant reductions, of at least 40%, in the numbers of neurons in the ganglia of the myenteric plexus of the 24-month animals were recorded in all regions of the intestine, especially in the colon where the neuron number decreased by over 60%. These figures do not take into account changes that might have occurred in the overall length of the intestine during adult life. Measurements of neuron sizes suggest that the neuronal fall-out with age affects all categories of neuron equally and implies that all the intrinsic neuronal systems of the gut could be equally affected in old age.

Aging↗
PubMed1976

Separately developing axonal uptake of 5-hydroxytryptamine and norepinephrine in the fetal ileum of the rabbit.

Uptake of 5-hydroxytryptamine (5-HT) by adult and fetal rabbit's ileum was studied. The adult myenteric plexus accumulated tritium when incubated with tritiated 5-HT. However, in addition to labeled 5-HT, tritiated 5-hydroxyindole acetic acid and, when monoamine oxidase (MAO) was inhibited, 5-HT-o-glucuronide were found in the tissue. Two uptake processes differing in affinity could be defined. Only the high affinity process was saturable. Fetal ileum took up tritiated 5-HT but glucuronidation did not occur when MAO was inhibited. The uptake of tritiated 5-HT by the fetal ileum was due to a single, saturable, temperature sensitive (Q10 at 27-37 degress C = 2.4) process inhibited by ouabain. It was identical to the high affinity uptake found in adult tissue. This specific high affinity uptake could be found as early as the 16th day of gestation, 5-8 days before uptake of norepinephrine (NE) begins. Light and electron microscope radioautography revealed that the uptake of 5-HT was primarily into axons and a characteristic structure called the expanded process, both in the myenteric plexus. Both contained dense-cored vesicles. Axons were not labeled by tritiated NE until after 24 days and the expanded process was never labeled by tritiated NE. This study shows that uptake of 5-HT is a property of distinct system of axons in the mammalian myenteric plexus which develops prior to adrenergic axons during ontogeny.

Animals↗
PubMed1987

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum.

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.

Animals↗
PubMed2002

The guinea-pig oesophagus is a versatile in vitro preparation for pharmacological studies.

1. The isolated oesophagus of the guinea-pig is a useful preparation that can be used as an alternative to the phrenic nerve-diaphragm preparations that have been used traditionally in the determination of the actions of drugs and toxins at the neuromuscular junction. The guinea-pig isolated oesophagus can also provide information about effects at ganglionic nicotinic receptors, which are not present in the diaphragm preparations. 2. The muscularis externa of the body of the oesophagus consists exclusively of striated muscle fibres. The myenteric plexus is found between the outer longitudinally arranged layer and the inner circular layer. The muscularis mucosae contains smooth muscle fibres arranged longitudinally. 3. The vagal nerves are comprised of special vagal efferents that innervate the striated muscle fibres directly and 'parasympathetic' vagal fibres that synapse in the myenteric ganglia and, subsequently, affect the smooth muscle of the muscularis mucosae. Thus, both striated and smooth muscle responses to vagal nerve stimulation can be studied. In addition, afferent neurons run in the vagus. 4. Studies using various isolated oesophageal preparations have been reviewed. These consist of the whole oesophagus (including striated muscle, myenteric plexus and smooth muscle), the isolated mucosal layer (striated muscle and the myenteric plexus both absent) and the whole oesophagus with the vagus nerve attached. Comparative studies of the effects of drugs acting directly on the muscularis mucosae and/or indirectly via the intramural plexuses can be performed using the whole oesophagus and the isolated mucosal layer. 5. The use of the guinea-pig isolated vagus nerve-oesophagus preparation allows potency determinations for both neuromuscular and ganglion blockade of various non-depolarizing muscle relaxants to be performed simultaneously under identical conditions. Furthermore, the phenomenon of fade, a waning of tetanic tension during the stimulation period, can be studied. 6. A potential application of this preparation is the simultaneous screening of the constituents of snake venoms for activity at the neuromuscular junction and/or the ganglion. It is also suggested that new calcium channel blockers could be screened in this preparation because different voltage-sensitive calcium channels are involved in neurotransmitter release from nerve terminals at the neuromuscular junction and autonomic cholinergic neuroeffector sites.

Animals↗
PubMed1990

Increase of mammalian intestinal motility by the iminopurine caissarone isolated from the sea anemone Bunodosoma caissarum.

The effects of caissarone (C8H11N5O) on guinea-pig ileum and rat duodenum preparations are reported. Caissarone evoked, at first, a small response, as compared with that of applied acetylcholine. This effect was blocked by atropine, hexamethonium and slightly reduced by tetrodotoxin. The main caissarone effect seems to be on the myenteric plexus as it increased spontaneous activity, tone and peristalsis; these effects being counteracted by tetrodotoxin, hexamethonium and atropine. Extracellular electrophysiological recording of the action potentials of myenteric plexus neurons showed an increase in frequency in the presence of caissarone. Caissarone potentiated the longitudinal muscle tension responses elicited by transmural electric stimulation in both control conditions, and when tension was reduced by pretreatment with tetrodotoxin or adenosine triphosphate. Caissarone also potentiated the longitudinal muscle tension responses to applied acetylcholine and this effect was reduced, but not abolished, by tetrodotoxin. Caissarone may have an indirect excitatory action on nicotinic receptors at the ganglia or may act as a reversible antagonist of purine transmitters released by neurons of the myenteric plexus involved in the physiological inhibition of intestinal motility.

Acetylcholine↗
PubMed1991

Intestinal ganglioneuromatosis: mucosal and transmural types. A clinicopathologic and immunohistochemical study of six cases.

Six cases of intestinal ganglioneuromatosis (GN) included in this study reveal the occurrence of two morphologic patterns. Transmural GN was characterized by neural hyperplasia in all layers of the bowel wall with predominant involvement of the myenteric plexus. It was found in three patients affected by multiple endocrine neoplasia IIb. Mucosal GN, having predominant involvement of the mucosa without concomitant hyperplasia of the myenteric plexus, was associated with von Recklinghausen's disease, adenocarcinoma of the colon, and multiple adenomas with megacolon in one case each. Clinicopathologic correlations and review of the literature suggest that mucosal GN might represent a distinct entity with a lower morbidity rate than the transmural variant. Immunohistochemical stains reveal considerable heterogeneity. S-100 protein, neuron-specific enolase, and synapto-physin immunostaining followed the distribution of the nervous hyperplasia in the different intestinal layers as identified morphologically and allowed precise determination of the proliferating cells. Increased reactivity for vasoactive intestinal polypeptide, opioid peptides leu-enkephalin and met-enkephalin, and substance P was present in all cases with transmural involvement; mucosal GN showed normal reactivity for opioid peptides and focal increased staining for substance P (one case) and vasoactive intestinal polypeptide (two cases) in the lamina propria. Mild increased immunoreactivity for tyrosine hydroxylase was present in the myenteric plexus of four out of four cases. Histochemical determination of acetylcholinesterase, performed in one case of transmural type, demonstrated hyperplasia of parasympathetic fibers and neurons. Electron microscopic study of another case suggested the presence of several neurotransmitters. These results indicate that the physiopathology of GN is related to a complex hyperplasia of several peptidergic, cholinergic, and probably adrenergic nerve fibers instead of a selective overgrowth of one type of nerve fiber.

Acetylcholinesterase↗
PubMed1995

Cholinergic, somatostatin-immunoreactive interneurons in the guinea pig intestine: morphology, ultrastructure, connections and projections.

The shape, projection, ultrastructure and chemistry of interneurons that were initially identified by their immunoreactivity for somatostatin in the small intestine of the guinea pig were examined. Somatostatin immunoreactive nerve cell bodies and nerve fibres were located in the myenteric plexus. Simultaneous labelling for 2 antigens revealed that the somatostatin immunoreactive interneurons were also immunoreactive for choline acetyltransferase, but not for calbindin or neuropeptide Y. Cell shapes were determined by immunohistochemistry, ultrastructural analysis and intracellular dye filling. The neurons had large cell bodies (38 x 14 microns) which gave rise to long branched filamentous dendrites and a single axon. The projections of the somatostatin immunoreactive interneurons were determined by analysis of patterns of fibre loss and survival following degenerative section of myenteric nerve pathways and by analysis of individual neurons that were injected intracellularly with dye. The axons projected anally, sometimes after a short oral excursion, and were confined to the myenteric plexus. They gave rise to multiple varicose branches within myenteric ganglia: in the majority of cases the first branch was within 100 microns of the cell body. Synaptic inputs to the cells were examined by light and electron microscopy. All somatostatin immunoreactive neurons received numerous somatostatin immunoreactive inputs on the cell body and all filamentous processes. Ultrastructural investigation indicated that these constituted the majority of all inputs. It is concluded that cholinergic, somatostatin immunoreactive, interneurons have a unique soma morphology and form synaptically connected chains that run anally in the myenteric plexus.

Animals↗