VPAC Receptor Subtypes Tune Purinergic Neuron-to-Glia Communication in the Murine Submucosal Plexus

Abstract

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.

Structurally defined signaling in neuro‐glia units in the enteric nervous system

“…Our findings support previous reports showing that enteric neuron‐to‐glia communication can occur independent of nerve conduction (Brown et al, ; Fung et al, ; Gulbransen et al, ). It is possible that the glial responses that remain after incubation with TTX are due to activation of TTX resistant Na v channels (Padilla et al, ; Rugiero et al, ), which in turn, could make specific neurons signal to enteric glial cells in a synaptic fashion.…”

“…This mode of neuron-to-glia communication is dependent on nerve conduction and classical neurotransmitter release, and therefore is typically blocked by tetrodotoxin (TTX). However, TTX-insensitive purinergic signaling from enteric neurons to glia has also been demonstrated (Brown, McClain, Watson, Patel, & Gulbransen, 2016;Fung et al, 2017;Gulbransen et al, 2012). A neuro-glia circuit regulated by vasoactive intestinal peptide (VIP) was recently discovered in the mouse submucosal plexus (Fung et al, 2017).…”

“…However, TTX-insensitive purinergic signaling from enteric neurons to glia has also been demonstrated (Brown, McClain, Watson, Patel, & Gulbransen, 2016;Fung et al, 2017;Gulbransen et al, 2012). A neuro-glia circuit regulated by vasoactive intestinal peptide (VIP) was recently discovered in the mouse submucosal plexus (Fung et al, 2017). Here, VIP acting on vasoactive intestinal peptide receptor-1 (VPAC1) expressed by cholinergic neurons stimulates purine release to evoke Ca 2+ transients in enteric glia.…”

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System

“…Extensive murine studies with immunohistochemical analysis and various reporter mice have established the temporal development pattern of the enteric nervous system (ENS), including the onset of spontaneous and induced electrical activity in embryonic mouse gut. 4,[8][9][10][11][17][18][19][20][21][22][23][24][25] However, such knowledge about the developing human gut is lacking. In this study, we report that the onset of evoked electrical activity in the human fetal ENS appears at approximately EW16.…”

Cholinergic Submucosal Neurons Display Increased Excitability Following in Vivo Cholera Toxin Exposure in Mouse Ileum

“…The tissue was opened along the mesenteric border and pinned flat with mucosa up in a silicone elastomer-coated dish. The submucosal plexus with the mucosa intact were obtained by microdissection as previously described (Fung et al, 2017 ). The circular muscle was then removed to obtain myenteric plexus preparations.…”