PROJECT SUMMARY. Opioid use disorder (OUD) is a critical problem that contributes to the spread of HIV and may intrinsically worsen neuroHIV pathology. Although the prevalence of antiretroviral therapy (ART) has improved the lifespan and quality of life of persons infected with HIV (PWH), the viral protein HIV-Tat is still present in the central nervous system (CNS) of many PWH on ART. Further, almost half of PWH still experience cognitive deficits and worsened substance misuse outcomes. Opioids can interact with HIV and HIV-Tat to promote inflammation and exacerbate neuronal damage and dysfunction, leading to increased behavioral and cognitive deficits, via µ-opioid receptors (MOR) expressed on different cell types. Medium spiny neurons (MSNs) within the striatum are uniquely vulnerable to the combined effects of opioids and HIV/Tat, and associated with opioid and HIV/Tat deficits in motivation and reward. The spiraling inflammation between astro- and microglia is driven by astroglial CCL5-CCL2 inflammatory signaling and is critical to opioid exacerbation of HIV neuropathology. However, the role of MOR on each cell sub-type are unknown. We hypothesize that the neuroinflammatory response to combined opioid and HIV/Tat, resulting in increased neuronal damage and aberrant behavior is largely mediated by MOR activation on astroglia. Aim 1 during the K99 phase will characterize the impact of astroglial MOR on opioid and HIV-Tat-induced astroglial pathology and dysfunction in vitro. MOR astroglial-null astroglia will be used to assess neuroinflammation and glutamate buffering, and MSN survival and morphology. and assessed for opioid reward and in vivo astroglial calcium transients while freely behaving and awake. Aim 2 (K99) will delineate the loss of MOR activation on astroglia on opioid and HIV-Tat-induced astroglial pathology and dysfunction in vivo. Tat-tg mice will be crossed with MOR astroglial- null mice and assessed for reward and in vivo astroglial calcium transients while freely behaving and awake. Dysfunction will be correlated with neuroinflammation and neuropathology. The above mentored training in genetically encoded indicators and in vivo calcium imaging combined with advance career development training will prepare Dr. Nass for the independent R00 phase and be essential in her pursuit of an independent research program investigating the mechanisms of substance misuse and neurovirology-induced behavioral dysfunction. Aim 3 (R00) will determine if astroglial activation is required for opioid and HIV-Tat-induced astroglial destabilization and MSN neuropathology. Tat-tg mice with attenuated astroglial function will be assessed as described in Aims 1 and 2. The proposed studies will further our understanding of the mechanisms by which MOR signaling on specific cell sub-type mediates opioid exacerbation of HIV/Tat- induced inflammation and neuropathology within the reward circuitry and identify possible targets for therapeutic interventions.