PROJECT SUMMARY/ABSTRACT Abstinence from alcohol use induces a negative affective state that can lead to maladaptive responses to stress and relapse. Preclinical studies have begun to identify neurocircuits and peptide targets that regulate negative affect during abstinence. As the field has begun to develop a deeper understanding of the circuitries participating in addiction and negative affect, the next step is to understand how communication within these circuits is modulated particularly at the neuropeptide and microcircuit level. The bed nucleus of the stria terminalis (BNST) is a fundamental component of abstinence-relevant neurocircuitry as it modulates stress and alcohol-related behavior in a neuropeptide-dependent manner. To investigate peptide-specific BNST circuitry modulating negative-affect during abstinence, we will focus on afferents from the parabrachial nucleus (PBN), a brainstem region that functions as a danger signal. PBN projections to the BNST release calcitonin gene- related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP), peptides that modulate pain and fear circuits, respectively. Our studies implicate heterogenous and sexually dimorphic control within the PBNàBNST circuit as PBN projections induce heterogeneous ex vivo activity in BNST cells and female-specific anxiety-like behavior with BNSTPBN activation. Furthermore, our preliminary studies suggest a role for the PBN in alcohol-withdrawal, as inactivation is anxiolytic following alcohol exposure. This proposal will significantly build on this foundational evidence by investigating how CGRP and PACAP contribute to PBNàBNST circuit induced abstinence-induced behavior, in vivo and ex vivo activity. Accordingly, the mentored K99 phase will build on my in vivo fiber photometry recordings and provide training in ex vivo recordings to determine the role of CGRP on BNSTàPBN activity, abstinence-induced behavior (AIM1), and the contribution of PACAP on the CGRP-neuromodulation (AIM2). The independent R00 phase will investigate the role of PACAP on BNSTàPBN at the microcircuit level and alcohol-related states, with the goal to further delineate the intricacies of peptide crosstalk (AIM3). The proposed studies and related career development training plan in this MOSAIC Pathway to Independence Award collectively provide the ideal mechanism to transition the applicant to a career as an independent addiction neuroscientist. The results will significantly advance our understanding of neurocircuit mechanisms at the peptide and microcircuit level in protracted abstinence while informing the use of peptidergic pharmacotherapies in alcohol use disorders.

PROJECT NARRATIVE Alcohol use disorder is often comorbid with anxiety, depression, and stress, thereby, making it harder to maintain sobriety. To alleviate negative affective disturbances in abstinence, this project will provide insight on novel peptide targets within neural pathways that will lead to better diagnostic tools and treatment options.