Prenatal Ethanol Exposure and Adolescent Stress Are Associated, in Male Rats, with a Compulsive-Like Phenotype and Alterations in Microbiota Composition and Neuroimmune Gene Expression.

Prenatal ethanol exposure (PEE) alters neurobehavioral function, yet the factors associated with these effects, particularly in interaction with stress, remain poorly understood. We examined whether combined exposure to PEE and adolescent restraint stress altered compulsive-like behavior and, in the sex showing the clearest combined effects, assessed gut microbiota composition and neuroimmune-related gene expression. Male and female Wistar rats exposed to ethanol on gestational days 17-20 were subjected to restraint stress (RES) or control conditions during adolescence. Behavioral assessments included the open field, light-dark box, marble burying, and perseverance tasks. Gut microbiota composition was analyzed using 16S rRNA sequencing, and central gene expression of inflammatory and neuromodulatory markers was measured. Behavioral effects were sex-dependent. In males, the combined PEE+RES condition produced a compulsive-perseverative phenotype, reflected by increased marble burying and reduced return latency to a specific object. In females, increased marble burying was observed after PEE alone. Microbiota analyses, conducted in males only due to them exhibiting the greatest joint effects of the PEE+RES exposure, revealed no significant differences in overall beta diversity, but identified several taxon-specific reductions. These reductions, which were most pronounced under combined PEE+RES exposure, involved members of the Lactobacillaceae, Muribaculaceae, Christensenellaceae, and Lachnospiraceae families, as well as the genera Butyricicoccus and Intestinimonas. PEE and RES were associated with region- and marker-specific alterations in Tnf and Oxtr gene expression. The findings suggest the involvement of the microbiota-gut-brain axis in the effects of PEE, with RES potentially exacerbating this vulnerability.