Project summary/Abstract Relapse defined as the resumption of alcohol drinking following a prolonged period of abstinence, represents a prevalent and significant public health concern in alcoholism. Relapse is a major impediment to treatment efforts. One promising approach for reducing high propensity for relapse in subjects with moderate to severe alcohol use disorder (AUD) is the identification of vulnerability factors in the brain that contribute to enhanced relapse to ethanol drinking; the medial prefrontal cortex (mPFC) is one of the key brain regions implicated in relapse to ethanol drinking behaviors. We have used the chronic intermittent ethanol vapor inhalation (CIE) model of ethanol dependence to demonstrate that increased ethanol self-administration during CIE in female rats predicts higher relapse to ethanol drinking triggered by ethanol contextual cues during forced abstinence compared with males. In addition, we have shown that drinking during relapse is associated with increases in endothelial cell specific angiogenesis peptide PECAM-1 in the mPFC in both sexes, indicating blood-brain barrier (BBB) instability. Treatment with an anti-angiogenic agent normalized PECAM-1 expression in both sexes and reduced relapse to ethanol drinking in female rats. To further understand the mechanisms underlying BBB instability and dysfunction, we have begun studies to isolate endothelial cells from the mPFC via endothelial cell enrichment assay. We have performed RNA sequencing to initially profile endothelial transcriptome of cells isolated from ethanol naïve adult female and male rats. Our results show that endothelial cells in females have higher expression of genes associated with angiogenesis and endothelial cell stability, whereas cells from males have higher expression of genes associated with immune responses. We have published findings from cytokine and chemokine multiplex analyses to show that the mPFC in ethanol naïve adult male rats has higher expression of pro- and anti- inflammatory cytokines and chemokines compared with females. These findings support sexual dimorphism in the endothelial transcriptome and immune responses in the mPFC under control, ethanol naïve conditions and open questions on how they are altered during ethanol dependence. In further support of BBB instability, we show that adherens junction protein cadherin5 (Cdh5, VE- cadherin) is significantly altered in the mPFC in ethanol dependent female and male rats. Specifically we show that Cdh5 is increased during acute withdrawal, significantly decreased during prolonged abstinence, and increased following relapse to ethanol drinking compared to ethanol naïve controls in both sexes. In addition to the endothelial changes in the mPFC, electrophysiological studies demonstrate altered synaptic plasticity in ethanol dependent female and male rats. Therefore, in the renewal application we hope to define the role of Cdh5 during abstinence in promoting relapse to ethanol ...