The gastrointestinal (GI) tract is the largest environmental interface of the mammalian body, which faces the challenge of maintaining tolerance to dietary and microbial antigens while protecting against pathogen invasion. In addition to hosting a large collection of immune cells, the GI tract contains diverse and numerous populations of glial cells and neurons, both intrinsic and extrinsic. Enteric neurons are responsible for controlling various physiological functions but can be targeted during enteric infections, resulting in functional gastrointestinal disorders after pathogen clearance. While some mechanisms underlying neuronal damage have been uncovered, the host and microbial factors that trigger enteric neuron loss and regeneration remain unclear. Over the past three years, we have uncovered new mechanisms of enteric neuronal cell death following infection or microbiota depletion and described the role of the gut microbiota and intestinal immune cells in regulating enteric neuron maintenance and function. In new preliminary data presented in the application, we provide evidence that enteric glial cells (EGC) experience a similar loss as enteric neurons following infection and antibiotic treatment. Additionally, our new data strongly suggest that EGC de-differentiation may play a role in neuronal recovery observed after microbiota transplantation. Finally, candidate metabolites involved in dysbiosis- associated neuronal death and neuronal recovery were identified. Based on these observations, we hypothesize that dysbiosis triggered by infection or antibiotic treatment generates ligands that trigger inflammasome- associated neuronal loss. Upon injury, glia-to-neuron dedifferentiation mediates neuronal recovery in a microbiota-dependent manner. The three complementary aims will delve deeper into these observations to reveal novel cellular and molecular mechanisms of microbiota-dependent neuronal death and regeneration, using novel imaging, state-of-the-art single-nuclei transcriptomics and chromatic accessibility, metabolomics, murine genetic fate-mapping, gain- and loss-of-function, and gnotobiotic approaches. In Aim 1, we will define the bacterial species and metabolites associated with specific glia and neuronal cell death after infection or antibiotic treatment. In Aim 2, we will assess the cellular dynamics of EGC and neuronal recovery after microbiota transplantation, followed by fate-mapping studies to define the possible role of glial cell de-differentiation in this process. Finally, Aim 3 will use complementary gnotobiotic and metabolomic approaches to identify bacterial signals and host pathways that induce gliogenesis and neurogenesis in different contexts. Overall, this proposal seeks to uncover novel mechanisms of microbiota-dependent neuronal death and regeneration with implications for the treatment of functional gastrointestinal disorders.