Project Summary Microbial dysbiosis is known to alter physiologic homeostasis and contribute to pathogenesis. Though morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Translocated microbes may then modulate local cell signaling and gene expression. One of the most immediately vulnerable compartments following bacterial dissemination is the intestinal wall, containing many terminal processes of extrinsic primary afferent neurons (EPANs) from dorsal root ganglia (DRG). These neurons play an integral role in a major limiting factor of narcotic use, analgesic tolerance; however, the impact of gastrointestinal microbiota on the development of tolerance in these cells has not been well characterized. This F30 fellowship application seeks to investigate the interaction between the gastrointestinal microenvironment and morphine tolerance of dorsal root ganglion nociceptors. Using voltage-clamp electrophysiology, we will test whether tetrodotoxin-resistant Na+ channels are involved in morphine responses and tolerance in these cells, and if microbial manipulation via antibiotic treatment impacts these processes. Using supernatants from mouse colon tissue samples, we will assess whether the gut microenvironment can independently alter morphine tolerance in nave DRG nociceptors. Finally, to assess the translational capacity of our findings, we will attempt to recapitulate these findings using supernatants from human colon biopsy samples. In carrying out our research strategy, we hope to improve treatment outcomes by promoting maintenance of analgesic efficacy and reducing the need for clinical dosage escalations.