PROJECT SUMMARY Nicotine addiction is the largest cause of preventable disease and death worldwide, and current treatments are only moderately efficacious. Nicotinic acts on nicotinic acetylcholine receptors (nAChRs) in the brain. nAChRs in the ventral tegmental area (VTA) have been shown to mediate the reinforcing properties of the drug, while nAChRs in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway have been shown to mediate the aversive properties of the drug. Although studying nAChRs provides an understanding of the direct actions of nicotine, it does not elucidate the full view of all the mechanisms involved. Therefore, there is an urgent need to expand our understanding of nicotine’s effects on the brain to examine potential endogenous proteins that may mediate these processes as well. The endogenous protein, Lynx1, has been shown to directly interact with nAChRs and is co-expressed with nAChR subunits;; however, Lynx1 has yet to be examined in the context of nicotine reinforcement. Here we propose novel experiments that will characterize Lynx1 in nicotine reinforcement. To do this, Lynx1 knockout mice and their wildtype littermates will be examined for intravenous nicotine self-administration across low, moderate, and high doses of nicotine. Baseline subunit expression will also be assessed between genotypes to examine the potential for compensation in experiments. To unravel the role of Lynx1 and its colocalization with different nAChRs subunits, Lynx1 knockout mice and their wildtype littermates will be examined for c-fos, Lynx1, and nAChR subunits using RNAscope and immunohistochemistry. Finally, to examine whether Lynx1 in the VTA mediates nicotine self-administration, a separate cohort of wildtype mice will be injected in the VTA with a shRNA virus to knockdown Lynx1. Results from these studies will reveal important insights into factors underlying nicotine reinforcement. Thus, these studies may thereby elucidate a mechanism to provide a foundation for the development of efficacious treatment for nicotine dependence and lead to novel avenues for future research directions. Moreover, as a predoctoral student, these experiments will allow me to expand expertise and skills by learning multiple cutting-edge viral, molecular, surgical, and behavioral techniques. The development of these skills will promote me to achieve my career goal of becoming a leading academic scientist by making an indelible impact on the field of addiction.