Phosphatase activity in the myenteric plexus

Intense and very intense reactions were obtained for acid phosphatase, calcium activated ATP-ase (pH 9.4), magnesium activated ATP-ase (pH 7.2) and glucose-6-phosphatase in the cytoplasms of the myenteric plexus nerve cells of the small intestine of Macacus rhesus and rabbit. Nucleotidase activity was moderate or slight and unspecific alkaline phosphatase activity absent. Both ATP-ases presented an intense activity in the myenteric plexus nerve cells of human fetuses 30, 33, and 34 weeks old; 5-nucleotidase activity, slight in the 30-week-old fetuses became more intense in the 33- and 34-week-old fetuses. The satellite neuroglial cells, nerve fibers and blood capillaries presented negative alkaline phosphatase reactions and intense or very intense activities of the other phosphatases.     

Catecholamine containing nerve cells in the mammalian myenteric plexus

Summary Previous fluorescence histochemical studies have shown that extrinsic denervation causes a disappearance of adrenergic fibres from the gut wall. However, in the present work, adrenergic terminals persisted in the myenteric plexus of the guinea-pig proximal colon following interruption of paravascular nerves. Fluorescent cell bodies are found in the myenteric plexus. The fluorescence reaction of the cells does not appear after reserpine treatment and is restored by -methyl-noradrenaline.     

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.     

Mechanosensitive enteric neurons in the myenteric plexus of the mouse intestine

Background

Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.

Methodology/Principal Findings

We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca⁺⁺/high Mg⁺⁺. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.

Conclusions/Significance

We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.     

Hormonal regulation of beta-endorphin in the testis

It is well established that beta-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that beta-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, beta-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of beta-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites (Kd in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic beta-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic beta-endorphin treatment does not affect testosterone production by Leydig cells in vitro, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of beta-endorphin. Also, fetal Leydig cells represented a preferred in vitro system to study beta-endorphin release since in adult cell culture a marked degradation of the peptide was detected (greater than 50%). beta-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50-30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10-12-fold the beta-endorphin secretion. The hormone stimulation of beta-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated beta-endorphin production (150-200%), suggesting autocrine negative modulation of Leydig cell beta-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated beta-endorphin production (by 50%). Altogether these findings indicate that beta-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.     

Vascularization of the myenteric plexus in the small intestine of pigeons]

By means of light and electron microscopy vascularization of the myenteric plexus has been studied in the pigeon small intestine. Ganglia of the plexus, their cell composition, ultrastructure of neurons have been described. Links of the microcirculatory bed of the intramural ganglia are characterized, interrelations of capillaries with neurons are described, quantitative estimation of microhemovessels, surrounding the microcirculatory bed of the myenteric plexus in the intestinal wall in birds and mammalia.     

A rapid silver-free method for staining the myenteric plexus

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionein were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic "mirroring," low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.     

Failure to demonstrate serotonergic neurons in the myenteric plexus of the rat

Summary Several recent studies suggested that serotonergic neuron-like elements are present in the guinea pig ileum. The present paper reports an extensive study of the digestive tract of the rat with the use of a histofluorescence technique. Administration of the serotonin precursor, tryptophan, associated with a monoamine oxidase inhibitor, did not allow histochemical demonstration of rapidly fading, yellow fluorescent, 6-hydroxydopamine-resistant neurons; conversely such neurons were readily detected in the brain. It is concluded that serotonergic neuron-like elements cannot be detected histochemically in the rat myenteric plexus area after chemical sympathectomy.     

Neuropeptide release from isolated myenteric nerve endings derived from the guinea pig myenteric plexus.

Isolated myenteric nerve varicosities prepared from the myenteric plexus of the guinea pig ileum were investigated as a suitable model system with which to study the release of several neuropeptide-like immunoreactivities (-LI). Basal release of substance P-LI, neurokinin A-LI, Leu-enkephalin-LI and Met-enkephalin-LI was determined, and clear depolarization-induced release of the enkephalin-LI's and neurokinin A-LI was obtained using this preparation, providing further support for their roles as putative mediators in the enteric nervous system. Evoked-release of these peptides was dependent on the presence in the incubation mixture of certain antagonists to known endogenous neuronal mediators. In the absence of such antagonists, no unequivocal evidence of release was seen. Clear evoked release of Leu-enkephalin-LI occurred only in the presence of the adenosine receptor antagonist 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX), atropine and naloxone. Release of Met-enkephalin-LI occurred in the presence of either atropine or naloxone. The release of neurokinin A-LI was evident in the presence of DPSPX. These findings suggest the existence of either distinct subpopulations of nerve varicosities or distinct neuronal pools containing each peptide and that these peptides may be under differential regulation by endogenous inhibitory mediators. It is concluded that, under suitable conditions, isolated myenteric nerve varicosities provide a useful model system for the study of release, and the modulation of release, of endogenous neuropeptides.     

Aspects of opiate dependence in the myenteric plexus of the guinea-pig.

Myenteric plexus-longitudinal muscle strips prepared from tolerant/dependent guinea-pigs and continuously exposed to normorphine, display a contracture upon naloxone challenge. This phenomenon represents a sign of abstinence. Removal of the opiate by extensive washing resulted in the failure of naloxone to induce the abstinence sign, while the plexus still displayed considerable, although reduced, tolerance to morphine. Reexposure of withdrawn preparations to normorphine reinduced the ability to display the abstinence sign. Highly tolerant preparations exhibited a 30 fold increase in sensitivity to serotonin and prostaglandin E₁ when tested a few minutes after naloxone-precipitated withdrawal. Supersensitivity rapidly declined when normorphine was washed off the preparation, while reincubation of withdrawn tissues with the opiate resulted in reinduction of supersensitivity. The data confirms a close relationship between a state of tolerance and dependence (including display of the abstinence sign) and supersensitivity to putative neurotransmitters or neuromodulators, becoming evident following administration of naloxone.     

Demonstration of GABA-like immunoreactivity in myenteric plexus of frog stomach

The GABAergic innervation of the frog stomach was studied by means of an indirect immunohistochemical method. Whole mount preparations were obtained from frog stomachs after the animals had been perfused with a mixture of picric acid, glutaraldehyde and glacial acetic acid. Samples were incubated with an antiserum specific for GABA coupled to BSA with glutaraldehyde. Anti-rabbit IgG-HRP was processed by the two step method (Eckert and Ude 1983). GABA-positive varicose fibers and also nerve cell bodies were revealed within the myenteric plexus. The density of GABA-immunoreactive neurons was not higher than 4-8 cell/cm2, which is approximately 1% of the total nerve cell number in the myenteric plexus.     

Postnatal development of nitrergic neurons in the myenteric plexus of rat stomach

The effect of age on the proportion of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons was investigated in the myenteric plexus of five different gastric areas of 1-day-, 1-week-, 2-week-, 1-month- and 2-month-old rats. Protein gene product 9.5 immunocytochemistry was used as a marker for the total enteric neuron population in order to establish the percentage of gastric nitrergic neurons in relation to age. The percentage of NADPHd-positive neurons in the proximal parts of the rat stomach (34–38%) is significantly higher than in the antral part (29%). This difference persists in all the age groups investigated. No significant relative increase with age of NADPHd-positive neurons could be observed in any of the areas studied. These findings imply that the increased nitrergic response in the rat proximal stomach as seen in pharmacological studies cannot be explained by an increased relative number of nitrergic neurons. Accepted: 31 March 1999     

Demonstration of GABA-like immunoreactivity in myenteric plexus of frog stomach

Summary The GABAergic innervation of the frog stomach was studied by means of an indirect immunohistochemical method. Whole mount preparations were obtained from frog stomachs after the animals had been perfused with a mixture of picric acid, glutaraldehyde and glacial acetic acid. Samples were incubated with an antiserum specific for GABA coupled to BSA with glutaraldehyde. Anti-rabbit IgG-HRP was processed by the two step method (Eckert and Ude 1983).GABA-positive varicose fibers and also nerve cell bodies were revealed within the myenteric plexus. The density of GABA-immunoreactive neurons was not higher than 4–8 cell/cm², which is approximately 1% of the total nerve cell number in the myenteric plexus.     

Histochemical characterization of myenteric plexus in domestic fowl small intestine

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.     

Calbindin-immunopositive cells are cholinergic interneurons in the myenteric plexus of rabbit ileum

The 28-kDa calcium-binding protein (calbindin) is a widely studied neuronal marker in the enteric nervous system of numerous species. Calbindin has previously been detected in myenteric neurons of rabbit ileum in which 3% of all myenteric neurons are calbindin-immunopositive. We have studied the detailed morphology and chemical coding of calbindin-immunopositive neurons in this segment of the gut. We have found calbindin immunoreactivity in both strongly and weakly stained neurons. Of these, the strongly immunoreactive neurons belong to the Dogiel type I category. These neurons project only to other ganglia and primary strands of the plexus and their processes never run to the muscle or mucosal layers. The neurons within this group are 29.5±6.6 m in length and 14.7±3.8 m in width. The second smaller group of immunoreactive cells (27%) label faintly and have different morphological properties. They are characterized by their round medium-sized cell bodies (long axis: 24.4±5.2 m; short axis: 15.5±2.9 m) and do not exhibit immunoreactivity either in their dendrites or in their axonal processes. Double-label studies show that all calbindin-immunopositive neurons lack immunoreactivity for nitric oxide synthase, vasoactive intestinal peptide and substance P but all are immunoreactive for the synthesizing enzyme of acetylcholine, choline acetyltransferase. Thus, populations of neurons containing calbindin are cholinergic interneurons in the myenteric plexus of rabbit ileum.This study was supported by grant OTKA T 34160     

Neuronal and glial localization of GABA transporter immunoreactivity in the myenteric plexus

The neurotransmitter gamma-aminobutyric acid (GABA) is removed from the extracellular space by sodium and chloride dependent high affinity plasma membrane transporters. In the rat central nervous system, three GABA transporters, GAT1, GAT2 and GAT3, have been cloned and localized by immunohistochemistry. The purpose of this study was to examine the distribution of these transporters within the myenteric plexus of the rat gastrointestinal tract. We investigated their cellular locations using GAT1-3 specific antisera in lightly fixed segments of rat duodenum, ileum and colon. Immunohistochemistry revealed a large number of GAT2-immunoreactive structures that surrounded neurons within each ganglion of the myenteric plexus. GAT2 was colocalized in these structures with the glial cell marker p75(NTR), suggesting that the predominant high affinity GABA transporter within enteric glia is GAT2. GAT3 immunoreactivity was localized within many nerve cell bodies, and no labeling for GAT1 was detected, although it was present in retina, which was used as a control. Double labeling for calretinin and nitric oxide synthase (NOS) revealed colocalization of GAT3 with approximately 75% of calretinin-immunoreactive neurons and 15% of NOS-immunoreactive neurons. This suggests that a small proportion of inhibitory motor neurons and at least some putative intrinsic primary afferent neurons within the rat gastrointestinal tract express GAT3. Thus NOS neurons, which appear to utilize GABA as a transmitter, and calretinin-immunoreactive neurons, which do not appear to be GABAergic, both express immunoreactivity for GABA transporters.     

Fronto-cortical regulation of beta-endorphin actions in the rat

Ablation of the frontal neocortex markedly enhanced the antinociceptive and cataleptic actions of beta-endorphin injected into the lateral ventricle of rat brain. This enhanced response was not affected by simultaneous administration of cholecystokinin octapeptide (CCK-8). In sham-operated rats, however, CCK-8 suppressed the effects of beta-endorphin in a dose-related manner. Moreover, ablation of a similar amount of occipital neocortex did neither affect beta-endorphin actions nor the interactions of CCK-8.     

Physiological analysis of delta opioid receptors in the guinea pig myenteric plexus

Ilea taken from guinea pigs that had been chronically exposed to morphine exhibit a greater tolerance to morphine and normorphine than to the opioid peptides D-ala2-D-leu5-enkephalin (DADLE) or D-met2-pro5-enkephalinamide (DMPE). This differential tolerance strongly implies the existence of at least two different types of opioid receptor in the guinea pig myenteric plexus or two different mechanisms of interaction between opioids and their receptor complex. Since DADLE is considered to be the prototypic ligand for the delta receptor, the above results imply the presence of delta receptors in the guinea pig myenteric plexus and furthermore, that this subtype of opioid receptor is associated with the modulation of release of enteric acetylcholine.