PROJECT SUMMARY Combination antiretroviral therapy (cART) is the current first-line treatment for human immunodeficiency virus (HIV) infection. Unfortunately, cART is implicated in various forms of HIV-associated neurological disorders. Pain is the most common neurological complication that significantly reduces the quality of life of many people living with HIV (PLWH), and emerging evidence suggests cART critically contributes to the development of pain in PLWH. However, the mechanism by which cART promotes pain pathogenesis is still incompletely understood and rationale-based effective adjuvant therapy is not available. In this project, we aim to elucidate the pathogenic mechanism of nucleoside reverse transcriptase inhibitors (NRTIs), which are the cornerstone antiretroviral drugs in cART regimens and induce neuropathic pain. Because PLWH on cART are commonly exposed to opioids and HIV-1 gp120 protein that causes pain, we will also investigate the potential pathogenic interaction between NRTIs and opioids or gp120. Our data show that separate administration of NRTIs, morphine, or gp120 to mice causes pathological pain, and that the development of pathological pain induced by these agents shares mechanistic properties with the activation of glial cells in the spinal dorsal horn. We hypothesize that NRTIs promote pain pathogenesis in the neural circuit via glial activation and that gp120 and opioids can exacerbate this condition. We will test this hypothesis by performing: (1) single cell transcriptomic analysis to characterize the cellular neuropathogenesis induced by NRTIs in conjunction with opioids or gp120 (Aim 1), (2) whole cell patch recording to characterize the pathologic pain neural circuitry induced by NRTIs with opioids or gp120 (Aim 2), and (3) behavioral testing to characterize pathological pain induced by NRTIs with opioids or gp120 (Aim 3). Results from this research will help to define the potential detrimental impact of cART on the CNS and its interactions with HIV and opioids.