Abdominal pain in irritable bowel syndrome and inflammatory bowel disease is thought to be driven by processes that sensitize sensory nerves innervating the gut. How sensory nerves become sensitized is not clear, but their terminals in the gut are surrounded by enteric glia. Here, we tested the hypothesis that intercellular enteric glia-to-nociceptor signaling contributes to visceral hypersensitivity during inflammation. In vivo and in vitro models of acute inflammation were used in combination with protein and RNA labeling, and cellular assays of activity and mediator release. Mechanisms of interaction between glia and nociceptors were studied using Trpv1^{Cre;GCaMP5gtdT};GFAP-hM3Dq mice, in which glial activity is controlled by chemogenetics while simultaneously recording nociceptor activity using calcium imaging. Mice lacking glial connexin-43 were used in combination with visceromotor reflex recordings to disrupt glial intercellular signaling and study its impact on visceral sensitivity. Acute colitis induces a transient increase in proinflammatory cytokines including IL-1β, which is produced in part by glia and facilitates glial connexin-43 function. Provoking glial activity under these conditions changes the normal benign influence of glia on nociceptors to one where glia have a sensitizing effect on gut-innervating nociceptors. The mechanisms responsible for glial-driven visceral hypersensitivity involve an upregulation of glial COX-2 and an increase in stimulated glial PGE2 release which acts on nociceptor EP4 receptors. In vivo recordings show that colonic IL-1β shifts normal innocuous stimuli toward a noxious range through mechanisms that require glial connexin-43. Enteric glia sensitize gut nociceptors during inflammation. Cell-specific therapies targeting the glial mechanisms identified here could benefit treatments for visceral pain.

Whole colon samples were collected from control and DNBS treated mice at 6 hours, 48 hours, and 3 weeks. Tissue was processed for multiplex immunoassay as previously described (50). Colonic samples were homogenized in a Tris-buffered saline Tween buffer solution to extract protein. After, homogenates were centrifuged at 10,000g for 10 minutes at 4°C. Cytokine/chemokine levels were evaluated using bead-based multiplex immunoassays (Eve Technologies, Calgary, AB, Canada).