Project Summary/Abstract Stress is a risk factor for the development of addiction, as well as for relapse in abstinent drug users. Over recent years, it is becoming increasingly clear that dopaminergic circuitry originating in the ventral tegmental area, which is the most well studied in the context of addiction, is also critical for the behavioral response to stress suggesting an interaction between stress and addiction. Recent studies have determined that the medial habenulo-interpeduncular (MHb-IPN) axis is an essential circuit in the pathophysiology of drugs of abuse, especially nicotine. Specifically, this circuit controls nicotine intake in rodent models, while also mediating the acute aversive effects of high doses of nicotine. In addition, the IPN has been implicated as an important hub for anxiety during nicotine withdrawal. Specifically, the stress neuropeptide, CRF is released and activates CRF1 receptors in the IPN triggering anxiety suggesting that the IPN is a component of the extrahypothalamic stress network in the brain. However, how the IPN responds to stress in vivo and if/how the IPN may contribute stress-induced behaviors is unknown. Thus, the goal of this application is to test the hypothesis that the IPN is a stress-responsive brain area and a critical mediator of stress-induced behaviors that becomes dysregulated in nicotine-dependence to drive stress effects on nicotine dependence-associated behaviors. In Aim 1, we will combine GCaMP expression with fiber photometry and optogenetics to test if stress-activation of IPN GABAergic neurons is critical for stress-induced behaviors. Aim 2 will also combine GCaMP expression with fiber photometry and test the hypothesis that nicotine dysregulates the response to stress in the IPN to drive relapse. Finally, Aim 3 will test the hypothesis that stressors engage dopaminergic pathways by activating IPN neurons that project to the lateral dorsal tegmentum. It is anticipated that elucidating circuitry and neuronal mechanisms underlying stress-induced behaviors in an understudied brain region that is emerging as a critical node for nicotine dependence should yield great insights into behaviors associated with addiction and set the foundation for therapeutic strategies for new treatment.