Enteric Inhibitory Neurons Research Articles

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

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

Evidence that nitric oxide participates in non-adrenergic inhibitory transmission to intestinal muscle in the guinea-pig.

The possible involvement of nitric oxide (NO) in non-adrenergic nerve-mediated relaxations of guinea-pig gastrointestinal smooth muscle was investigated using longitudinal muscle that lies between the taenia of the caecum. Relaxations induced by electrical field stimulation in this preparation are similar to those described in other preparations of guinea-pig intestinal muscle when enteric inhibitory neurons are stimulated; they were blocked by tetrodotoxin (10(-6) M) but were unaffected by guanethidine (10(-6) M). N-Nitro-L-arginine (NOLA) (5 x 10(-5) to 10(-4) M), an inhibitor of NO synthase, and haemoglobin (10(-5) M), which reduces bioavailability of NO, both reduced the amplitude of nerve-mediated relaxations to less than 50%, without affecting the ability of the muscle to relax. These results suggest that NO may play a role in inhibitory neurotransmission to this preparation.

Calcitonin gene-related peptide action on intestinal circular muscle

Isolated segments of the guinea pig small intestine were used to examine the effect of calcitonin gene-related peptide (CGRP) on the motor activity of the circular muscle. CGRP (0.3-30 nM) initiated phasic contractions of the circular muscle due to stimulation of cholinergic neurons. Peristalsis, however, was inhibited by CGRP. A further analysis of this effect showed that CGRP had no inhibitory influence on the main, cholinergic, pathway of the ascending enteric reflex (AER) contraction, whereas the hexamethonium- and atropine-resistant pathways of the AER were blocked. The inhibition of the atropine-resistant AER resulted from an action of CGRP on nerves and, since it was antagonized by apamin, might be explained by a CGRP-induced activation of enteric inhibitory neurons. The direct relaxant action of CGRP on the longitudinal muscle was not affected by apamin. These findings indicate a heterogeneity and topical selectivity in the motor actions of CGRP on the gut and suggest that this peptide, when released from nerve endings within the intestine, plays a specific role in the regulation of intestinal motility.

Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract.

Eight smooth muscle preparations from the stomach, small intestine and large intestine of the guinea-pig were used to compare apamin's actions in reducing the effectiveness of transmission from enteric inhibitory nerves and in reducing responses to inhibitory agonists alpha, beta-methylene ATP, VIP and isoprenaline. The effects of apamin on inhibitory reflexes in the ileum and colon were also evaluated. Apamin had little or no effect on responses to VIP and isoprenaline in any region, but consistently and substantially reduced responses to alpha, beta-methylene ATP. Responses to stimulation of enteric inhibitory neurons were substantially reduced by apamin in the antrum circular muscle, ileum longitudinal and circular muscle, taenia coli and distal colon longitudinal muscle, but it was ineffective in the fundus circular muscle, proximal colon longitudinal muscle and distal colon circular muscle. It caused a small reduction of the relaxation of the ileal circular muscle caused reflexly by distension, but did not modify the similar descending inhibitory reflex in the circular muscle of the colon. It is concluded that apamin can be used to distinguish two types of non-noradrenergic transmission from enteric inhibitory nerves to gastrointestinal muscle. Furthermore, neither VIP nor ATP can be the sole transmitter chemical released from enteric inhibitory neurons throughout the gastrointestinal tract.

Evidence for and significance of the projection of VIP neurons from the myenteric plexus to the taenia coli in the guinea pig

Immunohistochemical studies of the distribution of vasoactive intestinal polypeptide were made in the cecum from normal guinea pigs and from guinea pigs after lesions of the cecal wall had been made. These experiments were designed to determine the origin of the vasoactive intestinal polypeptide axons which supply the taenia of the cecum in order to ascertain whether the projections of the vasoactive intestinal polypeptide neurons were consistent with VIP being contained in enteric inhibitory neurons. Vasoactive intestinal peptide was found in nerve cell bodies of both the myenteric and submucous plexuses. After severing connections between the taenia and the myenteric plexus the vasoactive intestinal polypeptide fibers in the taenia disappeared, but there was no effect on these fibers if the taenia was separated from the submucous plexus or surrounding muscle while leaving connections with the myenteric plexus intact. Retrograde degenerative signs were seen in VIP fibers and nerve cell bodies of the myenteric plexus after removal of the taenia. In conclusion, VIP is contained in neurons that have the same projections as the enteric inhibitory neurons from the myenteric plexus to the taenia of the cecum.

γ-Aminobutyric acid stimulates intrinsic inhibitory and excitatory nerves in the guinea-pig intestine

The sites of action of γ-aminobutyric acid (GABA) were examined in preparations of the distal colon and ileum of guinea pigs. GABA caused transient relaxations of the longitudinal and circular muscle of the colon and transient constractions followed by relaxation of the muscle of the ileum. The responses of both parts of the intestine were antagonized by tetrodotoxin and by bicuculline. Nerve-free preparations of the longitudinal muscle of the ileum were not affected by GABA, even in concentrations up to 10 −4 g/ml. There was a marked tachyphylaxis of the responses to GABA. Relaxations in response to GABA were not affected by pentolinium or by a combination of phetolamine and propranolol. Contractions in response to GABA were blocked by hyoscine. Neither the ascending excitatory or descending inhibitory components of the peristaltic reflex in the distal colon were affected by bicuculline or by making the intestine tachyphylactic to GABA. The results indicate that GABA stimulates receptor mediating excitation of cholinergic excitatory and enteric inhibitory neurons whereas it has littel or no direct effect on the muscle of the ileum or distal colon.

Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal polypeptide in the guinea-pig intestine

Immunoreactivity for vasoactive intestinal polypeptide has been localized in neurons in the guinea-pig ileum, colon and stomach. In the ileum, 2.5% of the nerve cell bodies of the myenteric plexus and 45% of those of the submucous plexus showed vasoactive intestinal polypeptide-like immunoreactivity. Varicose axons containing vasoactive intestinal polypeptide ramified amongst the nerve cell bodies of both plexuses and in some cases formed rings of varicosities around non-reactive nerve cells. Axons were traced from the myenteric plexus to the circular muscle and deep muscular plexus. There were numerous positive axons running in fine strands within the circular muscle, parallel to the muscle bundles. Axons containing vasoactive intestinal polypeptide were associated with mucosal blood vessels, but few supplied the vascular network of the submucosa; some immunoreactive axons also contributed to the periglandular plexus of the mucosa. There were no changes in the distribution of axons in the ileum after extrinsic denervation. The results are discussed in relation to the possible functional roles of neurons that contain vasoactive intestinal polypeptide in the intestine: the distribution of such nerve cells in the myenteric plexus and of axons in the circular muscle and sphincters is consistent with this polypeptide being a transmitter of enteric inhibitory neurons; it is also possible that vasoactive intestinal polypeptide is the enteric vasodilator transmitter.

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.

The peristaltic reflex: an analysis of the nerve pathways and their pharmacology.

The enteric reflexes in isolated segments of the distal colon and rectum of the guinea-pig were studied by applying localized distensions and recording the consequent changes in circular muscle activity, and by recording tension changes in the circular muscle during the propulsion of a bolus in vitro. Lesions of the wall of the colon were made to locate nerve pathways involved in the reflexes and pharmacological tests were applied to investigate the natures of transmitters released and the types of receptors involved. Distension produced a transient contraction of the circular muscle on the oral side and sustained relaxation on the anal side. Both reflexes were nerve-mediated. They were elicited in segments deprived of mucosa and submucosa. Interruption of Auerbach's plexus, but not interruption of the submucosal plexus, prevented their conduction. The ascending excitatory reflex was partly blocked by hyoscine and was also partly blocked by methysergide or by making the preparation tachyphylactic to the excitatory action of 5-hydroxytryptamine. The ascending excitatory pathways apparently involve neurons releasing a 5-HT-like transmitter as well as cholinergic neurons. The descending inhibitory reflex was not antagonized by hyoscine, guanethidine, methysergide or mepyramine. It is assumed that the inhibitory neurons activated in this reflex are identical with the non-cholinergic, non-adrenergic, enteric inhibitory neurons found throughout the intestine. If both the ascending excitatory and descending inhibitory reflexes acted simultaneously on the same area of circular muscle, the inhibitory response tended to dominate. Pellets of faeces, covered by a thin layer of resin, were introduced into the oral ends of isolated segments of colon. They were propelled analwards at speeds of 0.5-1.6 mm/s. Tension records showed that the pellets were preceded by relaxation and followed by a ring of contraction. The propulsion was blocked by both hyoscine and methysergide. Descending waves of contraction were also observed in empty segments of colon. These occurred spontaneously or were initiated by stretch. They did not occur in the presence of hyoscine or tetrodotoxin. It is postulated that three factors may contribute to propulsion in the guinea-pig distal colon: ascending excitatory reflexes which evoke contractions above a bolus; descending inhibitory reflexes which cause relaxations below; and contractions which, once set up in the circular muscle, travel in an anal direction.

Excitation of intestinal muscle by atropine, tetrodotoxin, and xylocaine.

Excitation of intestinal muscle by atropine, tetrodotoxin, and xylocaine.

PERISTALSIS, SEGMENTATION, AND THE MYENTERIC REFLEX

PERISTALSIS, SEGMENTATION, AND THE MYENTERIC REFLEX