PROJECT SUMMARY Chronic exposure to nicotine in tobacco products results in numerous health consequences (lung cancer, emphysema, hypertension, etc.) and accounts for over 6 million deaths per year. Relapse rates are high among those who attempt to quit smoking, and pharmacotherapies that seek to foster smoking cessation have limited effectiveness. Thus, there is a significant unmet need for more effective strategies to treat nicotine dependence. Nicotine exposure produces physical dependence, and the physical and/or emotional nicotine withdrawal symptoms – as compared to the rewarding effects of nicotine – are often the most important contributors to relapse. Importantly, nicotine cravings and the risk for relapse tend to peak 1 to 2 weeks after the beginning of abstinence. Unfortunately, few research studies have probed the important question of how/why relapse behavior develops. Indeed, a critical gap in knowledge exists regarding our understanding of how chronic nicotine exposure encourages nicotine seeking during abstinence. In this project, we will use a rat nicotine self- administration model to study the medial habenula (MHb) and interpeduncular nucleus (IPN), which together comprise a crucial pathway involved in nicotine-related behaviors. Nicotinic acetylcholine receptors (nAChRs), the pharmacological target of nicotine, are densely expressed in this pathway and are responsible for nicotine's psychoactive and addictive properties. In this project, we intend to identify the relevant nAChRs and brain circuits involved in nicotine seeking during abstinence. Three independent and complementary AIMs are proposed, each of which probes a specific mechanistic aspect of relapse-like behavior. In AIM 1, we will use biophysical techniques, 2-photon imaging, and fiber photometry to probe the relationship between MHb neuronal activity and nicotine seeking during abstinence. AIM 2 will use electrophysiology, 2-photon imaging, and a DREADD approach to examine the role of IPN neurons in nicotine seeking. Finally, AIM 3 will identify specific IPN nAChRs involved in nicotine seeking and determine the importance of nicotinic cholinergic receptor activity in producing nicotine craving and resultant nicotine seeking. Together, these AIMs will help us understand how cessation of nicotine intake causes the brain to generate aversive physical and emotional withdrawal responses that inevitably lead to relapse. Solving this problem could lead to new strategies or drugs to foster smoking cessation.