PROJECT SUMMARY The opioid epidemic in the United States has reached critical proportions, with opioid-related overdoses contributing to more than 75% of drug overdose deaths. This crisis not only leads to significant mortality rates but also poses a significant economic burden on society. Despite the prevalence of opioid use disorders (OUD), the available treatment options remain limited, with only 3 FDA-approved medications, and there is a critical need to develop novel therapeutics. One way to develop novel treatments is by examining endogenous factors that modulate opioid responses such as galanin, a neuropeptide that is known to oppose opioid reward. Galanin signals through three G-protein coupled receptors (GPCRs) including GalR1, which preferentially forms heteromers with µ-opioid receptors (MOR) in the ventral tegmental area (VTA), a key node in the mesolimbic reward system. Recent literature indicates that galanin blocks MOR-mediated dopamine release in the VTA via GalR1-MOR heteromer interactions. Recently, our lab found that intra-VTA galanin administration attenuates morphine conditioned place preference (CPP) in male and female mice, identifying a neuroanatomical substrate for galanin action in opioid reward. However, the mechanisms underlying galanin’s ability to attenuate morphine CPP are not clear. Thus, the proposed experiments were designed with 2 key goals: 1) identify the source of endogenous galanin in the VTA that opposes opioid signaling, and 2) determine the role of the GalR1-MOR heteromer in the VTA in opioid reward. The overarching hypothesis of this proposal is that galanin from the lateral hypothalamus (LH) signals through GalR1-MOR heteromers on GABAergic projections from the rostromedial tegmental nucleus (RMTg) within the VTA to oppose morphine CPP. A combination of behavioral, molecular, pharmacological, and genetic tools will be used to test this hypothesis in male and female mice. In Aim 1, I will use retrograde viral-assisted tracing and RNAscope in situ hybridization to determine all endogenous sources of galanin to the VTA. Then, I will ablate galanin in the LH, which we know contains galaninergic projections to the VTA and modulates opioid reward, by locally infusing Cre-expressing virus into the LH of floxed galanin conditional knockout mice and quantify the density of the galanin-positive fibers in the VTA. Additionally, morphine CPP will determine the consequences of removing LH galanin inputs to the VTA on opioid reward. In Aim 2, a validated GalR1-MOR heteromer interfering peptide will be co-infused with galanin into the VTA to determine whether local heteromer signaling is necessary for galanin to attenuate morphine CPP. Conversely, a novel viral construct will be used to overexpress GalR1 selectively in MOR-containing GABAergic neurons of the RMTg. Together, these experiments will provide crucial information regarding the role of galanin in opioid reward and have the potential to identify the GalR1-MO...