PROJECT SUMMARY Abdominal pain is the most common gastrointestinal issue and is a major feature of irritable bowel syndrome and inflammatory bowel disease. Visceral hypersensitivity has emerged as a key pathophysiological mechanism for pain, but effective therapies are limited because the causal mechanisms whereby nociceptors become sensitized remain largely unresolved, are heterogeneous, sexually dimorphic, and influenced by inflammation and experiencing adverse early life events. Recent data show that enteric glia exhibit bi-directional communication with nociceptors and alter sensory transduction during acute and chronic inflammation. The overall goal of this proposal is to define glial mechanisms that sensitize visceral nerve fibers in a context and sex-specific manner. This proposal tests the central hypothesis that enteric glia promote visceral hypersensitivity through sexually dimorphic mechanisms that are influenced by acute inflammation and early life adversity. This dual hypothesis will be tested in two specific aims that utilize glial and nociceptor calcium imaging, glial chemogenetics, novel transgenic lines to perturb glial signaling mechanisms, and models of visceral pain driven by acute inflammation and early life adversity. Aim 1 will use the DNBS colitis model to test the hypothesis that enteric glia regulate visceral hypersensitivity through sexually dimorphic mechanisms during acute inflammation. Aim 1.1 will involve cellular imaging studies where GCaMPs are expressed in nociceptive nerves or glia, glial chemogenetics, in vitro measurements of gliotransmitter release, and in vivo visceromotor response recordings to test the hypothesis that glia contribute to female susceptibility in visceral nociception by exhibiting greater sensing, signal transduction, and transmitter release mechanisms than males. Aim 1.2 will use novel conditional glial gene ablation models to test the hypothesis that differences in how glia control endocannabinoids and histamine promote sex differences in susceptibility to developing pain following acute inflammation. Aim 2 will use the neonatal maternal separation stress model to test the hypothesis that early life adversity promotes male biased visceral hypersensitivity through a pathophysiological shift in gliotransmission. Aim 2.1 will use glial and nociceptor GCaMP models in cellular imaging studies, in vitro measurements of gliotransmitter release, and in vivo visceromotor response recordings to test the hypothesis that early life stress causes a shift from physiological to pathophysiological gliotransmission mechanisms in males. Aim 2.2 will use protein and RNA labeling, glial gene ablation models, cellular imaging and in vivo visceromotor recordings to test the hypothesis that increased glial endocannabinoid degradation and decreased histamine clearance contribute to male biased pain following early life adversity. The results of this study will provide novel insight into glial mechanisms that regu...