Project Summary/Abstract The bed nucleus of the stria terminalis (BNST) is a heterogeneous brain region with roles in arousal, anxiety, and alcohol addiction. It contains multiple subpopulations of neurons that can be characterized by their expression of neuropeptides and spatial locations within the BNST, some of which underlie distinct behaviors relevant to alcohol use and withdrawal. Two such subpopulations express corticotropin releasing factor (CRF) and cholecystokinin (CCK). Distinct projections from BNST neurons containing each of these neuropeptides to the lateral hypothalamus (LH) are investigated in the parent grant to determine their potential differential contributions to alcohol drinking behavior and sleep/wake states. The parent grant of this supplement explores how the BNST circuits defined by neuropeptide expression affect alcohol use and potential alcohol-related sleep disturbances. Past research suggests that the expression of neuropeptides in the BNST may differ by sex. This supplement builds off the parent grant to explore sex differences in the same BNST neurons and projections being studied in the parent. Studying whether there are differences in the amount of CRF-/CCK-expressing neurons in the BNST and in their projection patterns to brain regions involved in stress, anxiety, and arousal would allow researchers to have a more nuanced understanding of this highly complex brain region and the known sex differences in excessive alcohol consumption and related behaviors. The parent grant also proposes to investigate the activity of the CRF-expressing neurons in the BNST during sleep/wake while animals have been drinking alcohol, which will provide new insights into the role of these neurons during alcohol-related sleep disturbances. Sleep disturbance is one of multiple documented effects of alcohol use disorders. Anxiety has also been reported to develop in patients with alcohol use disorder, particularly during withdrawal from alcohol. This supplement aims to further our understanding of the mechanisms underlying this anxiety by recording CRF-/CCK-BNST activity while animals are undergoing anxiety behavior testing during acute withdrawal from long-term alcohol drinking. The candidate of this supplement will be leading the projects proposed to become an independent scientist. Through rigorous training on experimental techniques and spearheading the development of custom data analysis pipelines for the data collected, she will gain invaluable skills that will serve her in a future career in science. With the guidance of her multiple mentors, she will work on expanding her network of collaborators, peers, and mentors. She will also attend conferences and seminar series to practice her science communication skills and will contribute to the training and mentoring of more junior scientists in the lab. By the end of the training period, she will be very well prepared to both enter the next stages of her career development by applyin...