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ItemThe role of submucosal neurons in physiological and pathophysiological intestinal secretion( 2016)The enteric nervous system (ENS) has two major types of secretomotor neurons, which contain either acetylcholine (i.e. are cholinergic) or vasoactive intestinal peptide (VIP). These are well conserved across species, and are key mediators of physiological and pathophysiological intestinal secretion. Notably, VIP is a potent secretogogue implicated in cholera toxin (CT)-evoked hypersecretion – the most widely-studied model of bacterial toxin-induced diarrhoea. As diarrhoeal disease remains a significant health problem worldwide, a better understanding of these secretomotor pathways and mediators in physiological and pathophysiological secretion is crucial for advancing therapeutic treatments. While acetylcholine is an established enteric neurotransmitter, VIP has long been considered a putative transmitter. VIP acts via VPAC1 and VPAC2 receptors, but their physiological role within the enteric circuitry remains unclear. Despite previous efforts to better characterize the role of VIP, a number of discrepancies arose from these studies. These issues were mainly due to the lack of selective pharmacological tools, as well as technical limitations. Furthermore, although the secretory effects of CT have been studied extensively, the degree of enteric neural involvement, and the relative contribution of cholinergic and VIP secretomotor neurons remains debatable. Thus, the aim of my PhD studies was to address these longstanding questions which have impeded our understanding of the control of intestinal secretion by employing specific agonists and antagonists for VPAC receptors and more advanced experimental techniques that are now available. In Chapter 3, I localized VPAC1 receptors to a subset of cholinergic secretomotor neurons and cholinergic excitatory longitudinal muscle motor neurons. Further, I demonstrated that VIP acts via VPAC1 receptors on cholinergic secretomotor neurons to stimulate secretion and longitudinal muscle contraction. This was done using a combination of molecular, immunohistochemical and functional studies, as well as selective antagonists. There was no evidence of VPAC2 receptor involvement. In Chapter 4, I identified a novel role of VIP and VPAC receptors in modulating neuro-glial communication in the mouse submucosal plexus. This was achieved by performing Ca2+ imaging on transgenic Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent Ca2+ indicator in their ENS (enteric neurons and glia). VIP application to submucosal ganglia exclusively evoked Ca2+ responses in neurons, but surprisingly, using specific agonists and antagonists for VPAC1 and 2 revealed a role for VIP as a transmitter in signaling from neurons to glia. Activating VPAC1 receptors initiates neuron-to-glia signaling by stimulating purine release. Whereas, activating VPAC2 receptors suppresses this signaling pathway. Thus, VIP may have a dual role through its activation of VPAC1 and 2 receptors in fine-tuning purinergic neuron-glia communication. In Chapter 5, I used an in vivo mouse ileal loop model of CT-incubation, followed by a series of in vitro analysis to investigate the relative contributions of cholinergic and VIP secretomotor neurons to CT-evoked hypersecretion. Following the incubation, I found an increased fluid accumulation in CT-treated loops which corresponded to an increase in basal secretion measured using Ussing chambers. This hypersecretory effect did not appear to have a significant neuronal component. However, I found that CT induced a sustained increase in the excitability of cholinergic submucosal neurons by utilizing Wnt1-Cre;R26R-GCaMP3 mice to examine Ca2+ activity within the enteric circuitry. Increased spontaneous activity and enhanced responses to neural stimulation was observed in cholinergic submucosal neurons following CT-exposure. Further, CT was found to activate most submucosal neurons with the increased expression of an activity-dependent marker pCREB. I also found that the myenteric plexus did not significantly contribute to the sustained hypersecretion. Collectively, I showed that CT induced sustained hypersecretion by a direct effect on the mucosa, but also partly by increasing the excitability of cholinergic submucosal neurons.