Transcriptional profiling of the anterior cingulate cortex and nucleus accumbens using a double-hit model of prenatal and adolescent alcohol exposure.

backgroundPrenatal 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.

methodsIn this study, RNA-seq was used to analyze the effects of alcohol on the anterior cingulate cortex (ACC) and nucleus accumbens (NAcc) 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. Prenatally, dams received either ethanol liquid diet (PAE: 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 given alcohol (AAE: 11 h of 10% EtOH in tap water, 1 h water, 12 h 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 were identified via DESeq2, with the resulting p values adjusted using the Benjamini-Hochberg correction (≤ 0.05).

resultsIn the ACC, males showed surprisingly few effects of ethanol, whereas adult females with a double-hit history showed 4 genes 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). In the NAcc, adolescent females showed the most robust alterations (381 genes up, 419 down) involved in signaling pathways (calcium, oxytocin, cAMP, chemokines, etc.). Both brain regions differed strongly between ages in ethanol-naïve controls. In the ACC, 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 (a similar ratio was observed in the NAcc, though there was little overlap between specific genes).

conclusionsTogether, these findings form a foundation for examining sex-specific ACC development and female-specific vulnerability to damage from the double-hit ethanol model. Examining naïve controls is an invaluable control for understanding how ethanol affects the developmental trajectory of neurodevelopmental processes.