Project Summary/Abstract Addicts often report the emergence of negative emotional states during withdrawal, including stress and anxiety, and it is thought that these states can drive future drug taking through negative reinforcement processes. Hypothalamic orexin (also known as hypocretin) neurons are unique as they mediate both reward and stress behaviors, making these cells a promising target for pharmacotherapies designed to treat the multifaceted symptomology of addiction. However, our understanding of how these neurons modulate both reward and stress behaviors in addiction is currently limited. In the current proposal, I seek to receive training in several sophisticated neuroscience techniques to test my hypothesis that orexin neurons innervate discrete subpopulations of dopamine neurons in ventral tegmental area (VTA) with projections to nucleus accumbens shell (NAcSh) and medial prefrontal cortex (mPFC), to mediate reward and stress behaviors, respectively. These experiments will yield highly novel data that will inform the development of more effective therapeutics for addiction. In addition, the training and mentorship that I receive under this award will be invaluable in assisting my transition to a career as an independent scientist. During the mentored phased of this application, I will use a combination of optogenetic and behavioral electrophysiology approaches to interrogate the orexin reward system in addiction. I will record from dopaminergic VTA neurons that project to NAcSh and receive input from LH orexin neurons, and predict that both tonic and phasic activity of these neurons will be enhanced in animals exposed to a novel cocaine self- administration paradigm (intermittent access; IntA) that promotes highly-motivated drug seeking behavior. In a second set of experiments, I will use a chemogenetic approach to downregulate activity within this circuit, which I predict will normalize drug-seeking behaviors in these animals. During the independent phase, I will use the techniques that I learned during the K99 portion to investigate the orexin stress system in addiction. I have previously shown that IntA self-administration model is associated with exacerbated anxiety-like behavior during acute withdrawal, and in these experiments, I will record from VTA neurons that innervate mPFC and receive input from DMH/PF orexin neurons. I predict that the activity of these cells will be augmented during withdrawal in these animals, and that downregulation of this circuit during withdrawal (using a chemogenetic approach) will reduce anxiety-like behavior. In my final experiment, I will use a chemogenetic stimulation approach test the hypothesis that chronic activation of the orexin stress system produces plasticity at the level of the VTA that subsequently augments activity of the orexin reward system. These data will point to an integration of orexin stress and reward systems in addiction, and will form the basis of my first R01 application.