Anatomy and Neuroscience - Research Publications
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ItemEarly Development of Electrical Excitability in the Mouse Enteric Nervous System(SOC NEUROSCIENCE, 2012-08-08)Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.
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ItemA sexually dimorphic effect of cholera toxin: rapid changes in colonic motility mediated via a 5-HT3 receptor-dependent pathway in female C57Bl/6 mice(WILEY, 2016-08-01)KEY POINTS: Cholera causes more than 100,000 deaths each year as a result of severe diarrhoea, vomiting and dehydration due to the actions of cholera toxin; more females than males are affected. Cholera toxin induces hypersecretion via release of mucosal serotonin and over-activation of enteric neurons, but its effects on gastrointestinal motility are not well characterized. We found that cholera toxin rapidly and reversibly reduces colonic motility in female mice in oestrus, but not in males or females in prooestrus, an effect mediated by 5-HT in the colonic mucosa and by 5-HT3 receptors. We show that the number of mucosal enterochromaffin cells containing 5-HT changes with the oestrous cycle in mice. These findings indicate that cholera toxin's effects on motility are rapid and depend on the oestrous cycle and therefore can help us better understand differences in responses in males and female patients. ABSTRACT: Extensive studies of the mechanisms responsible for the hypersecretion produced by cholera toxin (CT) have shown that this toxin produces a massive over-activation of enteric neural secretomotor circuits. The effects of CT on gastrointestinal motility, however, have not been adequately characterized. We investigated effects of luminal CT on neurally mediated motor activity in ex vivo male and female mouse full length colon preparations. We used video recording and spatiotemporal maps of contractile activity to quantify colonic migrating motor complexes (CMMCs) and resting colonic diameter. We compared effects of CT in female colon from wild-type and mice lacking tryptophan hydroxylase (TPH1KO). We also compared CMMCs in colons of female mice in oestrus with those in prooestrus. In female (but not male) colon, CT rapidly, reversibly and concentration-dependently inhibits CMMC frequency and induces a tonic constriction. These effects were blocked by granisetron (5-HT3 antagonist) and were absent from TPH1KO females. CT effects were prominent at oestrus but absent at prooestrus. The number of EC cells containing immunohistochemically demonstrable serotonin (5-HT) was 30% greater in female mice during oestrus than during prooestrus or in males. We conclude that CT inhibits CMMCs via release of mucosal 5-HT, which activates an inhibitory pathway involving 5-HT3 receptors. This effect is sex- and oestrous cycle-dependent and is probably due to an oestrous cycle-dependent change in the number of 5-HT-containing EC cells in the colonic mucosa.
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ItemLuminal 5-HT4 receptors-A successful target for prokinetic actions(WILEY, 2019-10)The prokinetic effects of 5-HT4 receptor (5-HT4 R) agonists have been utilized clinically for almost three decades to relieve symptoms of constipation. Surprisingly, the mechanism(s) of action of these compounds is still being debated. Recent studies highlight luminal 5-HT4 Rs as an alternative and effective target for these prokinetic agents. These include the study by Shokrollahi et al (2019, Neurogastroenterol Motil, e13598) published in the current issue of Neurogastroenterology and Motility, who found that activation of mucosal 5-HT4 Rs by intraluminal prucalopride, significantly enhanced propulsive motor patterns in rabbit colon. The authors highlight the idea that development of agonists targeting luminal 5-HT4 Rs in the colonic mucosa might be more effective and safer in achieving prokinetic effects on intestinal motility. The purpose of this mini-review is to discuss the evidence for luminal 5-HT4 Rs as an emerging target for prokinetic agents in facilitating propulsive motor patterns in the colon.
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ItemOxaliplatin-induced enteric neuronal loss and intestinal dysfunction is prevented by co-treatment with BGP-15(WILEY, 2018-02)BACKGROUND AND PURPOSE: Gastrointestinal side effects of chemotherapy are an under-recognized clinical problem, leading to dose reduction, delays and cessation of treatment, presenting a constant challenge for efficient and tolerated anti-cancer treatment. We have found that oxaliplatin treatment results in intestinal dysfunction, oxidative stress and loss of enteric neurons. BGP-15 is a novel cytoprotective compound with potential HSP72 co-inducing and PARP inhibiting properties. In this study, we investigated the potential of BGP-15 to alleviate oxaliplatin-induced enteric neuropathy and intestinal dysfunction. EXPERIMENTAL APPROACH: Balb/c mice received oxaliplatin (3 mg·kg-1 ·day-1 ) with and without BGP-15 (15 mg·kg-1 ·day-1 : i.p.) tri-weekly for 14 days. Gastrointestinal transit was analysed via in vivo X-ray imaging, before and after treatment. Colons were collected to assess ex vivo motility, neuronal mitochondrial superoxide and cytochrome c levels and for immunohistochemical analysis of myenteric neurons. KEY RESULTS: Oxaliplatin-induced neuronal loss increased the proportion of neuronal NO synthase-immunoreactive neurons and increased levels of mitochondrial superoxide and cytochrome c in the myenteric plexus. These changes were correlated with an increase in PARP-2 immunoreactivity in the colonic mucosa and were attenuated by BGP-15 co-treatment. Significant delays in gastrointestinal transit, intestinal emptying and pellet formation, impaired colonic motor activity, reduced faecal water content and lack of weight gain associated with oxaliplatin treatment were restored to sham levels in mice co-treated with BGP-15. CONCLUSION AND IMPLICATIONS: Our results showed that BGP-15 ameliorated oxidative stress, increased enteric neuronal survival and alleviated oxaliplatin-induced intestinal dysfunction, suggesting that BGP-15 may relieve the gastrointestinal side effects of chemotherapy.
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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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ItemEffects of oxaliplatin on mouse myenteric neurons and colonic motility(FRONTIERS MEDIA SA, 2013)Oxaliplatin, an anti-cancer chemotherapeutic agent used for the treatment of colorectal cancer, commonly causes gastrointestinal side-effects such as constipation, diarrhoea, nausea, and vomiting. Damage to enteric neurons may underlie some of these gastrointestinal side-effects, as the enteric nervous system (ENS) controls functions of the bowel. In this study, neuronal loss and changes to the structure and immunoreactivity of myenteric neuronal nitric oxide synthase (nNOS) neurons were examined in colonic segments from mice following exposure to oxaliplatin ex vivo and following repeated intraperitoneal injections of oxaliplatin over 3 weeks in vivo, using immunohistochemistry and confocal microscopy. Significant morphological alterations and increases in the proportion of NOS-immunoreactive (IR) neurons were associated with both short-term oxaliplatin exposure and long-term oxaliplatin administration, confirming that oxaliplatin causes changes to the myenteric neurons. Long-term oxaliplatin administration induced substantial neuronal loss that was correlated with a reduction in both the frequency and propagation speed of colonic migrating motor complexes (CMMCs) in vitro. Similar changes probably produce some symptoms experienced by patients undergoing oxaliplatin treatment.
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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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ItemMultiple Neural Oscillators and Muscle Feedback Are Required for the Intestinal Fed State Motor Program(PUBLIC LIBRARY SCIENCE, 2011-05-05)After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40-60 s and separated by quiescent episodes lasting 40-200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle.
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ItemAutonomic Neuroscience: articles of interest appearing in other Frontiers journals(FRONTIERS MEDIA SA, 2012)
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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.