Abstract Alcohol use during pregnancy is a common occurrence that can produce a myriad of impairments termed Fetal Alcohol Spectrum Disorder (FASD). The development of anxiety and alcohol misuse are commonly seen in individuals with prenatal alcohol exposure (PAE), two maladaptive behaviors that are considered to be driven by overlapping neural circuits, namely the corticotrophin releasing factor receptor (CRFR1) system within the medial central amygdala (CeM). In the parent grant (R01AA028566), we hypothesized that moderate PAE (mPAE) during gestational day (G) 12, an embryological period when the amygdala emerges, reduces CRF1R function through ontogeny, resulting in reduced acute alcohol and CRF1R interactions. In support of this central hypothesis, in the first year of funding of the parent grant, we showed that G12 mPAE produced sex- specific alterations in CRF1R expression and function in the CeM of adolescent wildtype offspring, a developmental period when mPAE male offspring also show heightened anxiety-like behaviors. However, given the intricate microcircuitry of the CeM and recent reports demonstrating cell-specific responses to acute ethanol within this region, the research goals of this Diversity Supplement are to establish and utilize a transgenic rat line (CRF1R-Cre-tdTomato) to directly test the effects of G12 mPAE and the resulting modifications of acute ethanol action specifically on CRFR1+ and CRFR1- neurons within the CeM across development. Specifically, Aim 1 will investigate how mPAE may alter anxiety-like behaviors and subsequent ethanol intake across development. Aim 2 will examine how mPAE affects the actions of acute ethanol on CeM GABA transmission in CRFR1+ and CRFR1- neurons across development. Aim 3 will determine the impact of mPAE on the interaction of acute ethanol and CRFR1 activation on CeM GABA transmission in CRFR1+ and CRFR1- neurons across ontogeny. Testing of these novel hypotheses will advance our understanding of neurobiological mechanisms underlying anxiety-like behaviors and ethanol intake across development and as a consequence of PAE. Importantly, the proposed use of a transgenic model in this application will supplement the proposed experiments from the parent grant that were based on using wildtype subjects which restrict our ability to dissect the microcircuitry of the CeM. The mentoring components of this Diversity Supplement include direct training on 1) breeding and establishment of a transgenic rat colony, 2) behavioral assessment of anxiety-like behaviors and ethanol intake, and 3) whole-cell patch-clamp electrophysiological techniques in transgenic rats. This training will equip Ms. Winchester with tools necessary to develop novel hypotheses and combine with additional approaches later in her graduate training and beyond. Additionally, a strong and rigorous individual development plan has been designed to assure professional and career training, including 1) emphasis on writing (both manuscr...