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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ItemVPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus(FRONTIERS MEDIA SA, 2017-04-25)The enteric nervous system (ENS) situated within the gastrointestinal tract comprises an intricate network of neurons and glia which together regulate intestinal function. The exact neuro-glial circuitry and the signaling molecules involved are yet to be fully elucidated. Vasoactive intestinal peptide (VIP) is one of the main neurotransmitters in the gut, and is important for regulating intestinal secretion and motility. However, the role of VIP and its VPAC receptors within the enteric circuitry is not well understood. We investigated this in the submucosal plexus of mouse jejunum using calcium (Ca2+)-imaging. Local VIP application induced Ca2+-transients primarily in neurons and these were inhibited by VPAC1- and VPAC2-antagonists (PG 99-269 and PG 99-465 respectively). These VIP-evoked neural Ca2+-transients were also inhibited by tetrodotoxin (TTX), indicating that they were secondary to action potential generation. Surprisingly, VIP induced Ca2+-transients in glia in the presence of the VPAC2 antagonist. Further, selective VPAC1 receptor activation with the agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27)) predominantly evoked glial responses. However, VPAC1-immunoreactivity did not colocalize with the glial marker glial fibrillary acidic protein (GFAP). Rather, VPAC1 expression was found on cholinergic submucosal neurons and nerve fibers. This suggests that glial responses observed were secondary to neuronal activation. Trains of electrical stimuli were applied to fiber tracts to induce endogenous VIP release. Delayed glial responses were evoked when the VPAC2 antagonist was present. These findings support the presence of an intrinsic VIP/VPAC-initiated neuron-to-glia signaling pathway. VPAC1 agonist-evoked glial responses were inhibited by purinergic antagonists (PPADS and MRS2179), thus demonstrating the involvement of P2Y1 receptors. Collectively, we showed that neurally-released VIP can activate neurons expressing VPAC1 and/or VPAC2 receptors to modulate purine-release onto glia. Selective VPAC1 activation evokes a glial response, whereas VPAC2 receptors may act to inhibit this response. Thus, we identified a component of an enteric neuron-glia circuit that is fine-tuned by endogenous VIP acting through VPAC1- and VPAC2-mediated pathways.
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ItemCholera Toxin Induces Sustained Hyperexcitability in Myenteric, but Not Submucosal, AH Neurons in Guinea Pig Jejunum(FRONTIERS MEDIA SA, 2017-04-27)Background and Aims: Cholera toxin (CT)-induced hypersecretion requires activation of secretomotor pathways in the enteric nervous system (ENS). AH neurons, which have been identified as a population of intrinsic sensory neurons (ISNs), are a source of excitatory input to the secretomotor pathways. We therefore examined effects of CT in the intestinal lumen on myenteric and submucosal AH neurons. Methods: Isolated segments of guinea pig jejunum were incubated for 90 min with saline plus CT (12.5 μg/ml) or CT + neurotransmitter antagonist, or CT + tetrodotoxin (TTX) in their lumen. After washing CT away, submucosal or myenteric plexus preparations were dissected keeping circumferentially adjacent mucosa intact. Submucosal AH neurons were impaled adjacent to intact mucosa and myenteric AH neurons were impaled adjacent to, more than 5 mm from, and in the absence of intact mucosa. Neuronal excitability was monitored by injecting 500 ms current pulses through the recording electrode. Results: After CT pre-treatment, excitability of myenteric AH neurons adjacent to intact mucosa (n = 29) was greater than that of control neurons (n = 24), but submucosal AH neurons (n = 33, control n = 27) were unaffected. CT also induced excitability increases in myenteric AH neurons impaled distant from the mucosa (n = 6) or in its absence (n = 5). Coincubation with tetrodotoxin or SR142801 (NK3 receptor antagonist), but not SR140333 (NK1 antagonist) or granisetron (5-HT3 receptor antagonist) prevented the increased excitability induced by CT. Increased excitability was associated with a reduction in the characteristic AHP and an increase in the ADP of these neurons, but not a change in the hyperpolarization-activated inward current, Ih . Conclusions: CT increases excitability of myenteric, but not submucosal, AH neurons. This is neurally mediated and depends on NK3, but not 5-HT3 receptors. Therefore, CT may act to amplify the secretomotor response to CT via an increase in the activity of the afferent limb of the enteric reflex circuitry.
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ItemCalcium Sensing Receptors Mediate Local Inhibitory Reflexes Evoked by L-Phenylalanine in Guinea Pig Jejunum(FRONTIERS MEDIA SA, 2017-12-04)Amino acids applied to the mucosa evoke inhibitory reflexes in guinea-pig jejunum, but the receptors involved in sensory transduction are still unclear. One promising candidate is the extracellular calcium sensing receptor (CaSR), which is expressed by mucosal enteroendocrine cells and is preferentially activated by aromatic L-amino acids. We tested this by applying various amino acids to the mucosa and recording the resulting inhibitory junction potentials (IJPs) in nearby circular smooth muscle via intracellular recording. The CaSR is stereospecific and L-Phenylalanine evoked a significantly larger response than D-Phenylalanine when both were applied to the same site. The same pattern was seen with L- and D-Tryptophan, another aromatic amino acid. The CaSR is preferentially activated by aromatic amino acids and responses to L-Leucine and L-Lysine were significantly lower than those to L-Phenylalanine applied to the same site. Responses to L-Phenylalanine were dose-dependently suppressed by blockade of the CaSR with NPS2143, a CaSR antagonist, and mimicked by mucosal application of cinacalcet, a CaSR agonist. Responses to cinacalcet had similar pharmacology to that of responses to L-Phenylalanine, in that each requires both P2 purinoreceptors and 5-HT receptors. L-Glutamate evoked IJPs similar to those produced by L-Phenylalanine and these were depressed by blockade of P2 receptors and 5-HT3 plus 5-HT4 receptors, but NPS2143 was ineffective. The AMPA receptor antagonists DNQX (10 μM) and CNQX (10 μM) reduced IJPs evoked by L-Glutamate by 88 and 79% respectively, but neither BAY367260 (mGluR5 antagonist) nor 2APV (NMDA antagonist) affected IJPs evoked by L-Glutamate. We conclude that local inhibitory reflexes evoked by aromatic L-amino acids in guinea pig jejunum involve activation of CaSRs which triggers release of ATP and 5-HT from the mucosa. L-Glutamate also evokes inhibitory reflexes, via a pathway that does not involve CaSRs. It is likely there are multiple receptors acting as sensory transducers for different luminal amino acids.
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ItemNeurally Released GABA Acts via GABAC Receptors to Modulate Ca2+ Transients Evoked by Trains of Synaptic Inputs, but Not Responses Evoked by Single Stimuli, in Myenteric Neurons of Mouse Ileum(FRONTIERS MEDIA SA, 2018-02-13)γ-Aminobutyric Acid (GABA) and its receptors, GABAA,B,C, are expressed in several locations along the gastrointestinal tract. Nevertheless, a role for GABA in enteric synaptic transmission remains elusive. In this study, we characterized the expression and function of GABA in the myenteric plexus of the mouse ileum. About 8% of all myenteric neurons were found to be GABA-immunoreactive (GABA+) including some Calretinin+ and some neuronal nitric oxide synthase (nNOS+) neurons. We used Wnt1-Cre;R26R-GCaMP3 mice, which express a genetically encoded fluorescent calcium indicator in all enteric neurons and glia. Exogenous GABA increased the intracellular calcium concentration, [Ca2+]i of some myenteric neurons including many that did not express GABA or nNOS (the majority), some GABA+, Calretinin+ or Neurofilament-M (NFM)+ but rarely nNOS+ neurons. GABA+ terminals contacted a significantly larger proportion of the cell body surface area of Calretinin+ neurons than of nNOS+ neurons. Numbers of neurons with GABA-induced [Ca2+]i transients were reduced by GABAA,B,C and nicotinic receptor blockade. Electrical stimulation of interganglionic fiber tracts was used to examine possible effects of endogenous GABA release. [Ca2+]i transients evoked by single pulses were unaffected by specific antagonists for each of the 3 GABA receptor subtypes. [Ca2+]i transients evoked by 20 pulse trains were significantly amplified by GABAC receptor blockade. These data suggest that GABAA and GABAB receptors are not involved in synaptic transmission, but suggest a novel role for GABAC receptors in modulating slow synaptic transmission, as indicated by changes in [Ca2+]i transients, within the ENS.
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ItemCholinergic Submucosal Neurons Display Increased Excitability Following in Vivo Cholera Toxin Exposure in Mouse Ileum(FRONTIERS MEDIA SA, 2018-03-21)Cholera-induced hypersecretion causes dehydration and death if untreated. Cholera toxin (CT) partly acts via the enteric nervous system (ENS) and induces long-lasting changes to enteric neuronal excitability following initial exposure, but the specific circuitry involved remains unclear. We examined this by first incubating CT or saline (control) in mouse ileal loops in vivo for 3.5 h and then assessed neuronal excitability in vitro using Ca2+ imaging and immunolabeling for the activity-dependent markers cFos and pCREB. Mice from a C57BL6 background, including Wnt1-Cre;R26R-GCaMP3 mice which express the fluorescent Ca2+ indicator GCaMP3 in its ENS, were used. Ca2+-imaging using this mouse model is a robust, high-throughput method which allowed us to examine the activity of numerous enteric neurons simultaneously and post-hoc immunohistochemistry enabled the neurochemical identification of the active neurons. Together, this provided novel insight into the CT-affected circuitry that was previously impossible to attain at such an accelerated pace. Ussing chamber measurements of electrogenic ion secretion showed that CT-treated preparations had higher basal secretion than controls. Recordings of Ca2+ activity from the submucous plexus showed that increased numbers of neurons were spontaneously active in CT-incubated tissue (control: 4/149; CT: 32/160; Fisher's exact test, P < 0.0001) and that cholinergic neurons were more responsive to electrical (single pulse and train of 20 pulses) or nicotinic (1,1-dimethyl-4-phenylpiperazinium (DMPP; 10 μM) stimulation. Expression of the neuronal activity marker, pCREB, was also increased in the CT-treated submucous plexus neurons. c-Fos expression and spontaneous fast excitatory postsynaptic potentials (EPSPs), recorded by intracellular electrodes, were increased by CT exposure in a small subset of myenteric neurons. However, the effect of CT on the myenteric plexus is less clear as spontaneous Ca2+ activity and electrical- or nicotinic-evoked Ca2+ responses were reduced. Thus, in a model where CT exposure evokes hypersecretion, we observed sustained activation of cholinergic secretomotor neuron activity in the submucous plexus, pointing to involvement of these neurons in the overall response to CT.
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ItemBoth exogenous 5-HT and endogenous 5-HT, released by flu oxetine, enhance distension evoked propulsion in guinea-pig ileum in vitro(FRONTIERS MEDIA SA, 2014-09-19)The roles of 5-HT3 and 5-HT4 receptors in the modulation of intestinal propulsion by luminal application of 5-HT and augmentation of endogenous 5-HT effects were studied in segments of guinea-pig ileum in vitro. Persistent propulsive contractions evoked by saline distension were examined using a modified Trendelenburg method. When 5-HT (30 nM), fluoxetine (selective serotonin reuptake inhibitor; 1 nM), 2-methyl-5-HT (5-HT3 receptor agonist; 1 mM), or RS 67506 (5-HT4 receptor agonist, 1 μM) was infused into the lumen, the pressure needed to initiate persistent propulsive activity fell significantly. A specific 5-HT4 receptor antagonist, SB 207266 (10 nM in lumen), abolished the effects of 5-HT, fluoxetine, and RS 67506, but not those of 2-methyl-5-HT. Granisetron (5-HT3 receptor antagonist; 1 μM in lumen) abolished the effect of 5-HT, fluoxetine, RS 67506, and 2-methyl-5-HT. The NK3 receptor antagonist SR 142801 (100 nM in lumen) blocked the effects of 5-HT, fluoxetine, and 2-methyl-5-HT. SB 207266, granisetron, and SR 142801 had no effect by themselves. Higher concentrations of fluoxetine (100 and 300 nM) and RS 67506 (3 and 10 μM) had no effect on the distension threshold for propulsive contractions. These results indicate that luminal application of exogenous 5-HT, or increased release of endogenous mucosal 5-HT above basal levels, acts to lower the threshold for propulsive contractions in the guinea-pig ileum via activation of 5-HT3 and 5-HT4 receptors and the release of tachykinins. The results further indicate that basal release of 5-HT is insufficient to alter the threshold for propulsive motor activity.
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ItemThe relation between cesarean birth and child cognitive development(NATURE PORTFOLIO, 2017-09-13)This is the first detailed study of the relation between cesarean birth and child cognitive development. We measure differences in child cognitive performance at 4 to 9 years of age between cesarean-born and vaginally-born children (n = 3,666) participating in the Longitudinal Study of Australian Children (LSAC). LSAC is a nationally representative birth cohort surveyed biennially. Using multivariate regression, we control for a large range of confounders related to perinatal risk factors and the socio-economic advantage associated with cesarean-born children. Across several measures, we find that cesarean-born children perform significantly below vaginally-born children, by up to a tenth of a standard deviation in national numeracy test scores at age 8-9. Estimates from a low-risk sub-sample and lower-bound analysis suggest that the relation is not spuriously related to unobserved confounding. Lower rates of breastfeeding and adverse child and maternal health outcomes that are associated with cesarean birth are found to explain less than a third of the cognitive gap, which points to the importance of other mechanisms such as disturbed gut microbiota. The findings underline the need for a precautionary approach in responding to requests for a planned cesarean when there are no apparent elevated risks from vaginal birth.
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ItemNo Preview AvailableA detailed, conductance-based computer model of intrinsic sensory neurons of the gastrointestinal tract(AMER PHYSIOLOGICAL SOC, 2014-09-01)Intrinsic sensory neurons (ISNs) of the enteric nervous system respond to stimuli such as muscle tension, muscle length, distortion of the mucosa, and the chemical content in the lumen. ISNs form recurrent networks that probably drive many intestinal motor patterns and reflexes. ISNs express a large number of voltage- and calcium-gated ion channels, some of which are modified by inflammation or repeated physiological stimuli, but how interactions between different ionic currents in ISNs produce both normal and pathological behaviors in the intestine remains unclear. We constructed a model of ISNs including voltage-gated sodium and potassium channels, N-type calcium channels, big conductance calcium-dependent potassium (BK) channels, calcium-dependent nonspecific cation channels (NSCa), intermediate conductance calcium-dependent potassium (IK) channels, hyperpolarization-activated cation (Ih) channels, and internal calcium dynamics. The model was based on data from the literature and our electrophysiological studies. The model reproduced responses to short or long depolarizing current pulses and responses to long hyperpolarizing current pulses. Sensitivity analysis showed that Ih, IK, NSCa, and BK have the largest influence on the number of action potentials observed during prolonged depolarizations. The model also predicts that changes to the voltage of activation for Ih have a large influence on excitability, but changes to the time constant of activation for Ih have a minor effect. Our model identifies how interactions between different iconic currents influence the excitability of ISNs and highlights an important role for Ih in enteric neuroplasticity resulting from disease.