Effects of Genetics and Sex on Hippocampal Gene Expression and Adolescent Behaviors Following Neonatal Ethanol Exposure in BXD Recombinant Inbred Mice

Fetal alcohol spectrum disorders (FASD) are the leading preventable neurodevelopmental disorders in the western world. A hallmark symptom of FASD is cognitive and learning deficits that present in early childhood and continue throughout adulthood. Teratogenic effects of alcohol include increased cell death in the hippocampus, a brain region critically important in learning and memory. Genetics have been shown to have a role in the severity of alcohol’s teratogenic effect on the developing brain. Previous work in our lab identified differential vulnerability to ethanol-induced call death in the hippocampus using fourteen BXD strains and the two parental strains. The goal of the present study was to examine the effect of genetics and sex on differential gene expression changes and behavioral responses in animals exposed to postnatal ethanol. To test this, we examined multiple BXD strains that showed increased susceptibility to ethanol-induced cell death in the hippocampus, multiple BXD strains that were resistant to ethanol’s effect on hippocampal cell death, and the parental B6 and D2 strains which showed moderate levels of cell death in the hippocampus after ethanol exposure. Neonatal mice were treated on postnatal day 7 (third trimester equivalent in humans). Animals received a subcutaneous injection of either 5.0g/kg ethanol in saline solution or isovolumetric saline given in two equal doses two hours apart. Animals were sacrificed 7 hours after initial ethanol exposure. Differential gene expression was examined using the Affymetrix Microarray platform across the strains. In another subset of animals exposed to the same alcohol paradigm, we investigated the long-term effects of developmental alcohol exposure on cognition and behavior in select BXD strains and parental strains. Adolescent animals exposed to postnatal ethanol were tested across the following behavioral tests: elevated plus maze, open field, Y-maze, and T-maze. We identified gene expression changes after postnatal ethanol exposure in all BXD and parental strains with little overlap between males and females in the same strain. However, there were limited gene expression changes that showed a sex x treatment interaction. Sex-specific ethanol-induced gene expression changes were limited within each strain and these changes were not carried over across strains. Multiple genes showed a significant interaction between strain x treatment and/or strain x sex x treatment. Enrichment analysis of these genes revealed a number of significant over-represented biological categories involved in cell death and apoptosis. Genes that met our criteria and were also highly correlated with a number of apoptosis and learning and memory behaviors included Bcl2l11, Jun, Txnip, Chka, and Tgfb3. Interestingly, Tgfb3 has been previously linked to a significant QTL mediating strain-specific differences in hippocampal cell death after exposure to postnatal ethanol in BXD mice. When comparing ethanol-induced gene expression changes in high cell death strains (HCD) and low cell death strains (LCD), we observed almost double the number of differentially ethanol-induced gene expression changes in the HCD strains compared to the LCD strains. Enrichment analysis revealed some overlap in significant over-represented categories between the HCD and LCD strains, though HCD showed more cell death and apoptosis categories. Significant ethanol-induced gene expression changes in the HCD and LCD strains were always regulated in the same direction suggesting 1) more perturbed effects of ethanol-induced gene expression changes in the HCD strains compared to LCD strains and 2) limited gene expression changes that confer resistance to ethanol-induced cell death in the hippocampus in the LCD strains. In our behavioral study, our results demonstrate that the effects of developmental alcohol exposure on adolescent behavioral responses are highly dependent on strain, though the strains that showed the most behavioral alterations after exposure to postnatal alcohol were the B6 and D2 parental strains and the BXD100 and BXD48a HCD strains. In these four strains, we observed many anxiety-like and activity-related behaviors that were significantly affected by postnatal ethanol exposure and in many of these measures there were sex-specific differences within the strain. The LCD strains, BXD60 and BXD71, showed minimal effect of treatment in all behavioral tests. Interestingly, the HCD strains, BXD100 and BXD48a, were the only strains that showed significant effect of postnatal ethanol exposure in hippocampal-dependent spatial learning and memory assessment. These results suggest that there are long-term effects of developmental alcohol exposure on adolescent behavior and that these effects are highly strain specific. Overall, our study aimed to better understand genetic variation in ethanol-induced susceptibility to ethanol’s teratogenic effects. Our results accomplish this by identifying differential gene expression changes and behavioral responses in animals exposed to postnatal ethanol using the BXD RI mice and parental strains. Additionally, our study identified sex differences in both ethanol-induced gene expression changes and adolescent behaviors in mice exposed to postnatal ethanol, though sex-specific effects were highly dependent on strain. To our knowledge, this is the first study using the BXD RI strains to examine the effects of genetics and sex on 1) ethanol-induced gene expression changes during development, and 2) adolescent behaviors in mice exposed to postnatal ethanol.