Physiology - Research Publications
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ItemNo Preview AvailableLuminal cholera toxin alters motility in isolated guinea-pig jejunum via a pathway independent of 5-HT3 receptors(FRONTIERS MEDIA SA, 2010)Cholera toxin (CT) is well established to produce diarrhea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally. Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 or 12.5 μg/ml) in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by co-incubation with granisetron (5-HT(3) antagonist, 1 μM), which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient-induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated.
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ItemTransmission to Interneurons Is via Slow Excitatory Synaptic Potentials Mediated by P2Y1 Receptors during Descending Inhibition in Guinea-Pig Ileum(PUBLIC LIBRARY SCIENCE, 2013-02-07)BACKGROUND: The nature of synaptic transmission at functionally distinct synapses in intestinal reflex pathways has not been fully identified. In this study, we investigated whether transmission between interneurons in the descending inhibitory pathway is mediated by a purine acting at P2Y receptors to produce slow excitatory synaptic potentials (EPSPs). METHODOLOGY/PRINCIPAL FINDINGS: Myenteric neurons from guinea-pig ileum in vitro were impaled with intracellular microelectrodes. Responses to distension 15 mm oral to the recording site, in a separately perfused stimulation chamber and to electrical stimulation of local nerve trunks were recorded. A subset of neurons, previously identified as nitric oxide synthase immunoreactive descending interneurons, responded to both stimuli with slow EPSPs that were reversibly abolished by a high concentration of PPADS (30 μM, P2 receptor antagonist). When added to the central chamber of a three chambered organ bath, PPADS concentration-dependently depressed transmission through that chamber of descending inhibitory reflexes, measured as inhibitory junction potentials in the circular muscle of the anal chamber. Reflexes evoked by distension in the central chamber were unaffected. A similar depression of transmission was seen when the specific P2Y(1) receptor antagonist MRS 2179 (10 μM) was in the central chamber. Blocking either nicotinic receptors (hexamethonium 200 μM) or 5-HT(3) receptors (granisetron 1 μM) together with P2 receptors had no greater effect than blocking P2 receptors alone. CONCLUSIONS/SIGNIFICANCE: Slow EPSPs mediated by P2Y(1) receptors, play a primary role in transmission between descending interneurons of the inhibitory reflexes in the guinea-pig ileum. This is the first demonstration for a primary role of excitatory metabotropic receptors in physiological transmission at a functionally identified synapse.
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ItemNo Preview Available5-HT1A, SST1, and SST2 receptors mediate inhibitory postsynaptic potentials in the submucous plexus of the guinea pig ileum(AMER PHYSIOLOGICAL SOC, 2010-03)Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.
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ItemNo Preview AvailableNitric oxide enhances inhibitory synaptic transmission and neuronal excitability in guinea-pig submucous plexus(FRONTIERS MEDIA SA, 2010)Varicosities immunoreactive for nitric oxide synthase (NOS) make synaptic connections with submucosal neurons in the guinea-pig small intestine, but the effects of nitric oxide (NO) on these neurons are unknown. We used intracellular recording to characterize effects of sodium nitroprusside (SNP, NO donor) and nitro-l-arginine (NOLA, NOS inhibitor), on inhibitory synaptic potentials (IPSPs), slow excitatory synaptic potentials (EPSPs) and action potential firing in submucosal neurons of guinea-pig ileum in vitro. Recordings were made from neurons with the characteristic IPSPs of non-cholinergic secretomotor neurons. SNP (100 muM) markedly enhanced IPSPs evoked by single stimuli applied to intermodal strands and IPSPs evoked by trains of 2-10 pulses (30 Hz). Both noradrenergic (idazoxan-sensitive) and non-adrenergic (idazoxan-insensitive) IPSPs were affected. SNP enhanced hyperpolarizations evoked by locally applied noradrenaline or somatostatin. SNP did not affect slow EPSPs evoked by single stimuli, but depressed slow EPSPs evoked by stimulus trains. NOLA (100 muM) depressed IPSPs evoked by one to three stimulus pulses and enhanced slow EPSPs evoked by trains of two to three stimuli (30 Hz). SNP also increased the number of action potentials and the duration of firing evoked by prolonged (500 or 1000 ms) depolarizing current pulses, but NOLA had no consistent effect on action potential firing. We conclude that neurally released NO acts post-synaptically to enhance IPSPs and depress slow EPSPs, but may enhance the intrinsic excitability of these neurons. Thus, NOS neurons may locally regulate several secretomotor pathways ending on common neurons.
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Item5-HT antagonists NAN-190 and SB 269970 block α2-adrenoceptors in the guinea pig(LIPPINCOTT WILLIAMS & WILKINS, 2009-02-18)Serotonin (5-HT) plays a significant role in the regulation of intestinal secretion of water and electrolytes. The initial aim of this study was to use intracellular recording and specific antagonists to identify roles of 5-HT1A and 5-HT7 receptors of submucosal noncholinergic secretomotor neurons of guinea pig ileum, in vitro. However, it was found that the widely used 5-HT receptor antagonists NAN-190 (5-HT1A) and SB 269970 (5-HT7) both blocked alpha2-adrenoceptors, and hence depressed inhibitory synaptic potentials and hyperpolarizations evoked by noradrenaline, in these neurons. Both compounds enhanced neurally evoked contractions of the guinea pig vas deferens, an effect characteristic of blockade of alpha2-adrenoceptors. These results raise significant concerns about studies using NAN-190 and SB 269970 as specific antagonists of serotonin receptors.
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ItemDevelopment of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP Mice(WILEY, 2013-10-01)Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time.
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ItemNo Preview AvailableSerotonin and cholecystokinin mediate nutrient-induced segmentation in guinea pig small intestine(AMER PHYSIOLOGICAL SOC, 2013-04)Segmentation is an important process in nutrient mixing and absorption; however, the mechanisms underlying this motility pattern are poorly understood. Segmentation can be induced by luminal perfusion of fatty acid in guinea pig small intestine in vitro and mimicked by the serotonin (5-HT) reuptake inhibitor fluoxetine (300 nM) and by cholecystokinin (CCK). Serotonergic and CCK-related mechanisms underlying nutrient-induced segmentation were investigated using selective 5-HT and CCK receptor antagonists on isolated segments of small intestine luminally perfused with 1 mM decanoic acid. Motility patterns were analyzed using video imaging and spatiotemporal maps. Segmenting activity mediated by decanoic acid was depressed following luminal application of the 5-HT receptor antagonists granisetron (5-HT(3), 1 μM) and SB-207266 (5-HT(4), 10 nM) and the CCK receptor antagonists devazepide (CCK-1, 300 nM) and L-365260 (CCK-2, 300 nM), but these antagonists did not further depress segmentation when combined. The P2 receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonate (10 μM) had no effect on activity. Serosal application of 5-HT antagonists had little effect on segmentation in the duodenum but reduced activity in the jejunum when granisetron and SB-207266 were applied together. These results reveal that 5-HT(3) and 5-HT(4) receptors, as well as CCK-1 and CCK-2 receptors, are critical in regulating decanoic acid-induced segmentation. Computational simulation indicated that these data are consistent with decanoic acid activating two pathways in the mucosa that converge within the enteric neural circuitry, while contraction-induced release of 5-HT from the mucosa provides feedback into the neural circuit to set the time course of the overall contractile activity.
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ItemSerotonin in the gut: what does it do?(FRONTIERS RESEARCH FOUNDATION, 2012)