CART Peptides Differently Regulate Firing Rates and GABAergic Synaptic Inputs of DMV Neurons Innervating the Stomach Antrum and Cecum of Adult Male Rats.

BACKGROUND/AIM: Central administration of cocaine- and amphetamine-regulated transcript peptides (CARTp) alters gastrointestinal motility and reduces food intake in rats. Since neurons in the dorsal motor nucleus of the vagus (DMV) receive GABAergic and glutamatergic inputs and innervate the smooth muscle of gastrointestinal organs, we hypothesized that CARTp acts on the DMV or presynaptic neurons. METHODS: We used 3,3'-dioctadecyloxa-carbocyanine perchlorate (DiO) retrograde tracing with electrophysiological methods to record DMV neurons innervating the stomach antrum or cecum in brainstem slices from adult rats. RESULTS: DiO application did not change the electrophysiological properties of DMV neurons. CART55-102 had no effect on the basal firing rates of neurons in either the stomach antrum-labeled group (SLG) or cecum-labeled group (CLG). When presynaptic inputs were blocked, CART55-102 further increased the firing rates of the SLG, suggesting a direct excitatory effect. Spontaneous inhibitory postsynaptic currents (sIPSCs) occurred at a higher frequency in SLG neurons than in CLG neurons. CART55-102 reduced the amplitude and the frequency of sIPSCs in SLG neurons dose-dependently, with higher doses also reducing spontaneous excitatory postsynaptic currents (sEPSCs). Higher doses of CART55-102 reduced sIPSC and sEPSC amplitudes in CLG neurons, suggesting a postsynaptic effect. In response to incremental current injections, the SLG neurons exhibited less increases in firing activity. Simultaneous applications of current injections and CART55-102 decreased the firing activity of the CLG. Therefore, stomach antrum-projecting DMV neurons possess a higher gating ability to stabilize firing activity. CONCLUSION: The mechanism by which CARTp mediates anorectic actions may be through a direct reduction in cecum-projecting DMV neuron excitability and, to a lesser extent, that of antrum-projecting DMV neurons, by acting on receptors of these neurons.

Transient receptor potential ankyrin 1 is expressed by inhibitory motoneurons of the mouse intestine.

BACKGROUND & AIMS: Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. METHODS: TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca(2+) imaging and by assays of contractile activity and transit. RESULTS: We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca(2+)](i) in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1. TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1(+/+) but not Trpa1(-/-) mice; this effect was reduced by tetrodotoxin or N(G)-nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. CONCLUSIONS: Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.

Toxoplasma gondii infection causes morphological changes in caecal myenteric neurons.

The aim of this study was to evaluate the effects of chronic infection of Toxoplasma gondii (with genotype I and genotype III strains) on the population density and morphometry of caecal myenteric neurons in rats. Fifteen, 60-day-old, male Wistar rats (Rattus norvegicus) were used. The animals were assigned into three groups: Control Group (CG), Experimental Group 1 (EG1) and Experimental Group 2 (EG2). EG1 animals received 10(5) tachyzoites of the genotype I (BTU IV) T. gondii strain orally, and the EG2 animals received 10(5) tachyzoites of the genotype III (BTU II) strain orally. Thirty days after inoculation, caecal whole-mount preparations were stained by Giemsa technique. The caecal preparations were then analysed by assessing the population density and morphometry of myenteric neurons in specific regions of the caecum: mesenteric apical (MA), antimesenteric apical (AA), antimesenteric basal (AB) and next to caecal ampulla (NA). Myenteric neurons from the AA region were more clustered in EG1 animals (P<0.05). The EG1 animals presented a 16.8% reduction in the area of the nucleus, whereas the EG2 animals showed 18.4% increase (P<0.05). There was a more marked reduction in the cytoplasm of the animals in EG1 (↓23.2%) compared to EG2 (↓6.2%). There was 35.8% neuronal atrophy in the AB region and 16.8% in the region NA of the EG1 animals (P<0.05). In conclusion, different strains of T. gondii cause morphometric changes in caecal myenteric neurons of rats. Only the genotype I strain was able to cause neuronal density changes.

Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 µg/ml). The addition of 80 µg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

Intrinsic innervation of the ileocaecal junction in the horse: preliminary study.

REASON FOR PERFORMING STUDY: In horses, morpho-functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. OBJECTIVES: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). METHODS: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP) and neurofilament NF200kDa (NF). RESULTS: Ileal FB-labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 +/- 11% and 68 +/- 4%, respectively), cholinergic (60 +/- 19% and 82 +/- 11%, respectively), NF-IR (54 +/- 9% and 78 +/- 21%, respectively), and SP-IR (about 20% in both the plexuses). CGRP-IR was expressed only by SMP descending neurons (45 +/- 21%). In both the plexuses descending neurons coexpressing nNOS- and ChAT-IR were also observed (25 +/- 11% and 61 +/- 27%, respectively). CONCLUSIONS: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. POTENTIAL RELEVANCE: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.

Inhibitory purinergic transmission in mouse caecum: role for P2Y1 receptors as prejunctional modulators of ATP release.

Using conventional microelectrode recording techniques, we investigated, in the circular muscle of the mouse caecum, the neurotransmitter(s) involved in the neurally-evoked inhibitory junction potentials (IJPs) and the existence of possible prejunctional mechanisms controlling neurotransmitter release. Electrical field stimulation with single pulses elicited IJPs, consisting only of a "fast" hyperpolarization, while using train stimuli (30-50 Hz) the initial fast hyperpolarization was followed by a slower hyperpolarization. The fast and the slow component were selectively antagonized by apamin, a blocker of calcium-activated potassium channels, and N(omega)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, respectively. Fast IJPs were antagonized also by P2 purinoceptor antagonists, suramin or 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS), P2Y purinoceptor desensitization by adenosine 5'-O-2-thiodiphosphate (ADPbetaS). 2'-Deoxy-N(6)-methyl ADP diammonium salt (MRS 2179), P2Y1 purinoceptor antagonist, at the concentration of 1 microM increased the amplitude of the fast IJP, while at the concentration of 10 microM induced a reduction. 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino (4-fluoro-3,1-phenylene) carbonylimino]] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF 157) and 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyl-oxymethyl)-propyl ester (MRS 2395), P2Y11 and P2Y12 purinoceptor antagonist, were without any effect. ATP-induced hyperpolarization was affected by apamin and by P2Y purinoceptor desensitization, but not by MRS 2179. 2-(Methylthio)ATP tetrasodium salt hydrate (2-MeSATP), P2Y1 purinoceptor agonist, at a concentration which did not cause changes in the membrane potential, reduced the amplitude of the fast IJPs. This effect was prevented by MRS 2179. Paired nerve stimulation, either using single pulses or train stimuli, did not cause any alteration of the second-evoked IJP. In conclusion, in the circular muscle of the mouse caecum, ATP is responsible for the fast IJP while nitric oxide is responsible for the slow IJP. ATP-mediated response is dependent on ADPbetaS-sensitive P2Y receptors, which are in part P2Y1, but not P2Y11 or P2Y12 receptor subtypes. In addition, the most substantial finding of this study is the functional demonstration that ATP released by nerve stimulation activates P2Y1 receptors, located prejunctionally, limiting its release by motoneurons.

Functional effects and characteristics of cecum-projecting neurons in the dorsal motor nucleus of the vagus of rats.

Preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) innervate most of the gastrointestinal tract; with the stomach and the cecum/proximal colon having a greater proportion of vagal input. Cecum-projecting neurons have been thought to be distinct from other preganglionic neurons due to their location within the DMV, but it is unknown whether these neurons innervate the cecum exclusively or what effect their activation has on cecal motor activity. Therefore, we investigated the extent of coinnervation of cecum and stomach by vagal neurons, their neurochemistry, and the effect of DMV stimulation on intracecal and intragastric volumes. Fluorescent retrograde tracers injected into the serosa of the cecum and stomach revealed that in the DMV 49+/-5% CTB-labeled cecum-projecting neurons also innervated the stomach. Immunocytochemical staining for nitric oxide (NO) synthase and tyrosine hydroxylase indicated that only 3+/-1% and 4+/-1% of cecum-projecting neurons contained these markers, respectively. In anesthetized rats gastric and cecal volumes were measured by prototypic miniaturized dual barostats that were developed for use in rodents. Microinjection of l-glutamate into the DMV increased gastric contractile activity and tone, and reduced on-going cecum contractile activity (2.6+/-0.7 contractions/2 min after injection versus 8.2+/-0.4 contractions/2 min before injection, N = 5). The barostat was able to detect decreases (-0.88+/-0.13 ml) and increases (0.25+/-0.05 ml) in cecum volume in response to carbachol and sodium nitroprusside, respectively. In summary, cecum-projecting neurons are not an entirely exclusive population within the DMV because a percentage of these also innervate the stomach. Central vagal stimulation can modulate both gastric and cecum contractile activity. Together, these data support a role of the vagus in neural reflexes involving gastric and large bowel motor function, such as the immediate phase of the gastrocolonic reflex.

Abundance and Significance of Neuroligin-1 and Neurexin II in the Enteric Nervous System of Embryonic Rats.

Aim. To investigate the abundance of neuroligin-1 and neurexin II in the enteric nervous system (ENS) of rats on different embryonic days and to explore their potential significance. Methods. The full-thickness colon specimens proximal to the ileocecal junction of rats on embryonic days 16, 18, and 20 and of newborns within 24 hours (E16, E18, E20, and Ep0) were studied, respectively. qRT-PCR was applied for detecting the expressions of neuroligin-1 and neurexin II on mRNA, and western blotting was employed for detecting their further expressions on the whole tissue. Finally, the histological appearance of neuroligin-1 and neurexin IIα was elucidated using immunohistochemical staining. Results. qRT-PCR showed that the neuroligin-1 and neurexin II mRNA expressions of groups E16, E18, E20, and Ep0 increased gradually with the growth of embryonic rats (P < 0.05). Western blotting confirmed the increasing tendency. In immunohistochemical staining, proteins neuroligin-1 and neurexin IIα positive cells concentrated mostly in the myenteric nerve plexus of the colon and their expressions depend on the embryonic time. Conclusion. Neuroligin-1 and neurexin II were both expressed in the ENS and have temporal correlation with the development of ENS, during which neuronal intestinal malformations (NIM) may occur due to their disruptions and consequent abnormal ENS development.

The effect of gut microbiome on tolerance to morphine mediated antinociception in mice.

There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1ß expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.

Mucosally-directed adrenergic nerves and sympathomimetic drugs enhance non-intimate adherence of Escherichia coli O157:H7 to porcine cecum and colon.

The sympathetic neurotransmitter norepinephrine has been found to increase mucosal adherence of enterohemorrhagic Escherichia coli O157:H7 in explants of murine cecum and porcine distal colon. In the present study, we tested the hypothesis that norepinephrine augments the initial, loose adherence of this important pathogen to the intestinal mucosa. In mucosal sheets of porcine cecum or proximal, spiral and distal colon mounted in Ussing chambers, norepinephrine (10 microM, contraluminal addition) increased mucosal adherence of wild-type E. coli O157:H7 strain 85-170; in the cecal mucosa, this effect occurred within 30-90 min after bacterial inoculation. In addition, norepinephrine transiently increased short-circuit current in cecal and colonic mucosal sheets, a measure of active anion transport. Norepinephrine was effective in promoting cecal adherence of a non-O157 E. coli strain as well as E. coli O157:H7 eae or espA mutant strains that are incapable of intimate mucosal attachment. Nerve fibers immunoreactive for the norepinephrine synthetic enzyme dopamine beta-hydroxylase appeared in close proximity to the cecal epithelium, and the norepinephrine reuptake blocker cocaine, like norepinephrine and the selective alpha2-adrenoceptor agonist UK-14,304, increased E. coli O157:H7 adherence. These results suggest that norepinephrine, acting upon the large bowel mucosa, modulates early, non-intimate adherence of E. coli O157:H7 and probably other mucosa-associated bacteria. Sympathetic nerves innervating the cecocolonic mucosa may link acute stress exposure or psychostimulant abuse with an increased microbial colonization of the intestinal surface. This in turn may alter host susceptibility to enteric infections.

Vagal preganglionic projections to the enteric nervous system characterized with Phaseolus vulgaris-leucoagglutinin.

The patterns and extent of vagal preganglionic divergence and convergence within the gastrointestinal tract of the rat were characterized with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Three weeks after tracer was iontophoretically injected into two to four sites within the dorsal motor nucleus of the vagus, wholemounts of perfused gut organs (stomach, duodenum, cecum) were prepared, counterstained with Cuprolinic blue, and processed for PHA-L using the avidin biotin complex with diaminobenzidine. Controls included animals injected with PHA-L after intracranial deafferentations. Well-positioned injections labeled an extremely dense and intricate network of varicose efferent axons throughout the gastric myenteric plexus (including that of the fundus). Individual fibers collateralized extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervated subsets of neurons within ganglia. Most enteric neurons were contacted by varicosities of more than one vagal fiber. The patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resembled the organization in the stomach in many aspects, but the projections in each organ had distinctive characteristics, and label was less dense in the intestines than in the stomach. Vagal preganglionic fibers directly innervated submucosal ganglia, although sparsely. Brainstem injections of PHA-L retrogradely labeled a few myenteric neurons in the corpus, fundus, and duodenum: These "gastrobulbar" and "duodenobulbar" neurons received reciprocal vagal preganglionic innervation. Finally, the PHA-L that spread to the nucleus of the solitary tract occasionally produced transganglionic labeling of afferent intramuscular arrays (gastric fundus). The results of this paper provide strong evidence that the traditional "command neuron" or "mother cell" hypotheses of vagal-enteric organization should be abandoned for an integrative neural network model.

Electrophysiologic identification of the location of the colonic pacemakers. A human study.

Colonic inertia, total or segmental, is a known cause of constipation, yet its etiology is poorly understood and its treatment not satisfactory. Although the colonic electric activity has been studied by many investigators, the colonic pacemakers that are assumed to generate the electric waves, are scarcely addressed and their location, to our knowledge, has not been determined. The current communication investigates the possible sites of the colonic pacemakers, aiming at a better understanding of the mechanism of colonic motility and its disorders, notably colonic inertia. The tests were performed during the repair of huge incisional hernia in 12 subjects (mean age 37.7 +/- 10.2 years, 7 women). Monopolar silver-silver chloride electrodes were applied to the terminal ileum (TI) and cecum (C), and to the ascending (AC), transverse (TC), descending (DC) and sigmoid colon (SC); they were fixed by electrode gel to the ileal and colonic serosa. The electric activity of each of the TI, C, AC, TC, DC and SC was recorded. Electric waves in the form of slow waves or pacesetter potentials and action potentials were recorded from the TI and colon. Differences in the frequency, amplitude and conduction velocity of the waves occurred between the TI and the various segments of the colon. The change in the wave variables between the TI and C occurred at the ileocecal junction, between the C and AC at the cecocolonic junction, the AC and TC at the mid third of the TC and the DC and SC at the colosigmoid junction. The colonic electric waves are suggested to be generated by at least 4 pacemakers, which are presumably located at the ileocecal junction, the cecocolonic junction, the mid third of TC and at the colosigmoid junction. The electric waves appear to be responsible for conducting the colonic motor activity. We postulate that disordered colonic pacemakers may produce segmental or total colonic inertia, a proposition that needs further investigation.

The motor efficacy of the artificial colonic pacemaker in colonic inertia patients.

We have recently demonstrated that the colon possesses at least 4 pacemakers which presumably generate electric waves and that colonic pacing evokes electric activity in colonic inertia. We tested the hypothesis that electric waves produced by artificial colonic pacing in colonic inertia patients may initiate colonic motilitiy and effect evacuation. 17 patients with constipation due to total colonic inertia were divided into 2 groups: 10 (age 45.6 ( 6.3 years, 7 women) in the study group and 7 (age 43.7 ( 5.8 years, 5 women) as controls. 7 healthy volunteers were included in the study. Colonic pacing was tested at 2 sites of pacemakers: mid-transverse colon and colosigmoid junction. A stimulating electrode was hooked to the colonic mucosa at the pacemaker site and 2-3 recording electrodes distally to it. Healthy volunteers showed resting electric waves in the form of pacesetter and action potentials which increased significantly on colonic pacing. Inertia patients exhibited no basal electric activity. Colonic pacing in the study group evoked electric waves and caused expulsion of small volume balloon. Balloon distension at pacemaker site produced mass movement of balloon, while away from pacemaker site step-wise movement. In conclusion, colonic pacing evoked electric waves in colonic inertia patients and effected balloon expulsion. We postulate that the pacemaker generates electric waves which spread along pacemaker branches that are composed of interstitial cells of Cajal and nerve fibers of the enteric nervous plexus and effect colonic mass contraction. Ex-pacemaker stimulation presumably leads to local activation of interstitial cells of Cajal and segmental step-wise contraction.

Neurokinin A mimics the slow excitatory postsynaptic current in submucous plexus neurons of the guinea-pig caecum.

Single microelectrode voltage-clamp recordings were made from submucous neurons of the guinea-pig caecum. The slow excitatory postsynaptic current was compared with the currents induced by neurokinin A and substance P. The current induced by neurokinin A (100-300 nM) was associated with a decreased membrane conductance and reversed in polarity between -90 and -100 mV. The neurokinin A current was reduced by Co2+ (1-2 mM), but was not affected by Cs+ (1-2 mM), Ba2+ (10-100 microM) or low Cl- (20-40 mM) solutions. In about 80% of the neurons, the current induced by substance P (100-300 nM) was associated with a decreased membrane conductance and did not reverse with hyperpolarization of the membrane potential up to -130 mV. The current was reduced by Co2+ (1-2 mM) and augmented by low Cl- (20-40 mM) solutions, but was not affected by Cs+ (1-2 mM) or Ba2+ (10-100 microM)-containing solutions. In about 20% of the neurons, the substance P current reversed in polarity between -100 and -120 mV. The slow excitatory postsynaptic current elicited by repetitive nerve stimulation (10-40 Hz, three to five pulses) was accompanied by a decreased membrane conductance, and reversed in polarity between -90 and -100 mV. The slow excitatory postsynaptic current was abolished by Co2+ (1-2 mM) or low Na+ (12 mM) solutions, but was not affected by Cs+ (1-2 mM), Ba2+ (10-100 microM) or low Cl- (20-40 mM) solutions. In such neurons, the neurokinin A current was reversed at approximately the same potential at which the slow excitatory postsynaptic current was reversed, while the substance P current was not reversed even by much stronger hyperpolarizations. It was concluded that the neurokinin A current was mainly due to depression of potassium conductances, while the substance P current resulted from both increased anion conductance and decreased potassium conductances. The conductance change underlying the slow excitatory postsynaptic current is similar to that caused by neurokinin A.

Slow postsynaptic potentials in neurones of submucous plexus of guinea-pig caecum and their mimicry by noradrenaline and various peptides.

Intracellular recordings of membrane potential and membrane currents were made from neurones in the submucous plexus of the guinea-pig caecum in vitro. Fast and slow excitatory postsynaptic potentials and slow inhibitory postsynaptic potentials were recorded from the majority of neurones following focal stimulation of presynaptic fibres in the plexus. The slow inhibitory postsynaptic potential was associated with an increase in membrane conductance and reversed its polarity at -90 mV; it was reversibly blocked by yohimbine. The slow excitatory postsynaptic potential and its underlying current was associated with a decrease in membrane conductance. Two kinds of voltage-dependence both of the slow excitatory postsynaptic potential and current were observed; in 80% of cells, the excitatory postsynaptic potential and current became smaller with membrane hyperpolarization and reversed polarity at -90 mV (reversing type) but in 20% of cells both the excitatory postsynaptic potential and current simply disappeared when the membrane potential reached -70 mV (non-reversing type). The effects of acetylcholine, adenosine 5'-triphosphate, bombesin, 5-hydroxytryptamine, neurotensin, noradrenaline, substance P and vasoactive intestinal polypeptide were examined. The only substance which mimicked the slow inhibitory postsynaptic potential was noradrenaline; brief applications of noradrenaline caused hyperpolarizations which had the same time-course, reversal potential and sensitivity to yohimbine as the slow inhibitory postsynaptic potential. The non-reversing type of slow excitatory postsynaptic potential was mimicked only by adenosine 5'-triphosphate. The reversing type of slow excitatory postsynaptic potential was mimicked by bombesin, neurotensin, substance P and vasoactive intestinal polypeptide. 5-Hydroxytryptamine and vasoactive intestinal polypeptide (in some neurones) caused a depolarization with an increase in membrane conductance. All three synaptic potentials were reversibly depressed by superfusion of noradrenaline but noradrenaline did not affect the potential changes evoked by brief application of exogenous acetylcholine or substance P. It is concluded that, in guinea-pig submucous plexus neurones, the slow inhibitory postsynaptic potential is mediated by noradrenaline and results from a potassium conductance increase.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurokinin-1 and neurokinin-3 receptors are expressed in vagal efferent neurons that innervate different parts of the gastro-intestinal tract.

Vagal efferent neurons innervating the digestive tract are mainly contained in the dorsal motor nucleus of the vagus. Previous studies have suggested that neurokinins and their neurokinin-1 and neurokinin-3 receptors are involved in the parasympathetic control of digestive functions. The purpose of the present study was to analyze the distribution of neurokinin-1 and neurokinin-3 receptors amongst vagal efferent neurons innervating the stomach, the duodenum, the ileum and the cecum. The immunocytochemical detection of neurokinin-1 and neurokinin-3 receptors was combined with the immunocytochemical detection of retrogradely transported cholera toxin-B subunit, previously injected in the gut wall. Neurokinin-1 and neurokinin-3 receptors were present in 19+/-7% and 8+/-3% of retrogradely labeled neurons innervating the stomach. Almost half of the labeled neurons innervating the duodenum (46+/-7%) expressed neurokinin-1 receptors but less than 0.5% contained neurokinin-3 receptors. None of the retrogradely labeled vagal efferent neurons innervating the ileum and the cecum were immunoreactive for neurokinin-1 and neurokinin-3 receptors. We conclude that neurokinin-1 and neurokinin-3 receptors are located on vagal efferent neurons which innervate the stomach and that neurokinin-1 receptors are common, whereas neurokinin-3 receptors are rare on neurons projecting to the duodenum. Additionally, the distal part of the rat small intestine is innervated by vagal efferent neurons that do not express neurokinins receptors on their membrane. This suggests that neurokinins may influence the parasympathetic control of different regions of the gastro-intestinal tract in specific ways.

Y2-receptor-mediated selective inhibition of slow, inhibitory postsynaptic potential in submucous neurones of guinea-pig caecum.

1. The subtype of neuropeptide Y receptor mediating the selective inhibition of the slow inhibitory postsynaptic potential (i.p.s.p.) of submucous neurones in guinea-pig caecum was investigated by use of conventional intracellular electrophysiological recording techniques. 2. Neuropeptide Y (NPY) (1-300 nM) was found to depress or abolish reversibly the slow i.p.s.p. evoked by focal stimulation of internodal fibre tracts. At low concentrations (1-30 nM), a reduction in the duration of the slow i.p.s.p. was often apparent before any inhibition of the amplitude of this synaptic potential. 3. These inhibitory effects of NPY were mimicked by peptide YY (PYY; 0.3-100 nM), NPY13-36 (1-300 nM) and NPY22-36 (10-100 nM); [Leu31,Pro34]NPY ([Pro34]NPY) and bovine pancreatic polypeptide (bPP) were without pre- or postsynaptic effects at concentrations of up to 300 nM. The IC50 +/- s.e. mean values for PYY, NPY, and NPY13-36 were 2.7 +/- 0.3, 7.8 +/- 2.1 and 30 +/- 4.8 nM, respectively, and were significantly different from each other. Thus, the apparent rank order of potency was PYY > NPY > NPY13-36 >> [Pro34]NPY and bPP. 4. In concentrations of up to 300 nM, NPY and its analogues had no depressant effects on the active and passive properties of the impaled neurone and did not affect the amplitude or duration of either cholinergic fast synaptic potentials or non-cholinergic, slow excitatory postsynaptic potentials (e.p.s.ps). Furthermore, none of these peptides altered the amplitude or time-course of changes in membrane potential induced by focal application of acetylcholine or noradrenaline. 5. It is, therefore, concluded that the selective inhibition of the slow i.p.s.p. is mediated by Y2-receptors,located presynaptically on noradrenergic nerve terminals.

The effect of cold storage on the adrenergic mechanisms of intestinal smooth muscle.

1. In the guinea-pig taenia caecum, fluorescent adrenergic fibres terminate in both muscle layers. The density of these fibres is greater in the taenia than in the underlying circular muscle layer. The myenteric plexus and individual ganglion cells are also densely innervated by intensely fluorescent adrenergic nerve fibres.2. After three days of cold storage, the specific fluorescence disappeared from all tissue layers of the taenia caecum and smooth muscle fibres. In contrast, cholinesterase active substances were still demonstrable in all tissue layers even after seven days of cold storage but the density of these substances was decreased.3. Cold storage (3-7 days) decreased the tissue noradrenaline content and did not modify the cholinesterase enzyme activity (4 days).4. In cold stored strips, the inhibitory response to nicotine, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) or electrical transmural stimulation was abolished and enhancement of the contractile response occurred. Cold storage also inhibited the inhibitory action of tyramine. Similar results were observed after reserpine treatment.5. In fresh taenia, the relaxation produced by nicotine, DMPP and electrical transmural stimulation was inhibited by adrenoceptor blocking agents and bretylium. In cold storage preparations, contraction produced by these stimuli was blocked by parasympathetic blocking agents and potentiated by anti-cholinesterase. These results indicate that the inhibitory response to these stimulants is mediated by stimulation of the adrenergic nerve system more than by non-adrenergic nerves; the excitatory effect is probably due to stimulation of cholinergic nerves.6. These results suggest that the adrenergic mechanisms of the taenia caecum are more labile in cold storage than the cholinergic mechanisms. Thus, the inhibitory action of cold storage on the relaxation produced by nicotine, DMPP, and transmural stimulation is probably explained by selective physical degeneration of the adrenergic nerve terminal. Also, enhancement of the contractile response to these stimulants in cold stored preparations is explained by the lack of adrenergic inhibitory mechanisms.

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea-pig taenia caeci.

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.

(3H)-adenine nucleotide and (3H)-noradrenaline release evoked by electrical field stimulation, perivascular nerve stimulation and nicotine from the taenia of the guinea-pig caecum.

1. The release of [(3)H]-noradrenaline and adenine nucleotide evoked by electrical field stimulation (20-60 V, 30 Hz), perivascular nerve stimulation (20-80 V, 60 Hz) and nicotine (10, 100 muM) was studied in the taenia of the guinea-pig caecum under various conditions.2. Electrical stimulation at high intensity (60 V) caused the release of [(3)H]-adenine nucleotide; however, the inhibitory action of electrical stimulation was proportional to [(3)H]-noradrenaline release.3. The intensity of the inhibitory effect of stimulation of the perivascular nerves was directly related to [(3)H]-noradrenaline release and not associated with the release of [(3)H]-nucleotide.4. Cold storage for more than 8 days, cooling (19 degrees C) or tetrodotoxin treatment (1 mug/ml) abolished the inhibitory responses to electrical stimulation and to nicotine. After these treatments, nicotine and electrical stimulation elicited only contractions; the release of [(3)H]-noradrenaline, but not that of [(3)H]-adenine nucleotide, was inhibited.5. The dissociation of the inhibitory effects of electrical stimulation and nicotine from [(3)H]-nucleotide release does not support the hypothesis that ATP or a related nucleotide is the humoral transmitter of the non-adrenergic inhibition in the taenia of the guinea-pig caecum.