ABSTRACT: Prenatal alcohol exposure through maternal drinking and adolescent alcohol intake are the two most common forms of early alcohol consumption, which may produce synergistic impacts on health. Yet, few preclinical studies have used double-hit models to examine how effects manifest across sex and lifespan. In this study, RNA-seq was used to analyze the effects of ethanol on the anterior cingulate cortex (ACC) of adult and adolescent male and female Long-Evans rats (n = 5+ per group, N = 144) that were exposed to ethanol during gestation, adolescence, or both. During prenatal exposure, dams received either ethanol liquid diet (PAE group: 2.2% v/v EtOH on gestational day [G]6-8, 4.5% on G9-10, and 6.7% on G11-20) or control diets (PF pair-fed, FCL free-choice liquid diet). In adolescence, offspring were exposed to alcohol (AAE: 11h of 10% EtOH in tap water, 1h water access, 12h of water deprivation daily from prenatal [P] day 27-41) or given ad libitum water access (VEH). Brains were collected on P42 (adolescence) and P90 (adulthood). Differentially expressed genes (DEGs) were identified via DESeq2, with the resulting p values adjusted using the Benjamini-Hochberg correction (≤ 0.05). Surprisingly, there were minimal effects of any ethanol on males in adolescence, and none enduring in adulthood. In contrast, adult females with a double-hit history showed the most robust change: 4 genes were found to be downregulated (C3, Syt6, Spetex2l2, and Akap12) and 19 upregulated. These genes did not align to a specific pathway, yet more than one (i.e. C3, Akap12) were associated with immune function (e.g. complement system, glial function). It is interesting to note that these changes represent enduring alterations to baseline ACC state 48 days after the last ethanol exposure without a subsequent challenge. When examining ethanol-naïve controls, we found that between P42 and P90, both sexes were undergoing significant change. 451 DEGs were identified for males and 95 for females, yet only 36 DEGs overlapped between the sexes, demonstrating that even in the period of late adolescence (P42 onward), males and females were on different trajectories of development. Together, these findings form a foundation for examining sex-specific ACC development and female-specific vulnerability to damage from the double-hit ethanol model.
