Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility.

The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+ ) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.