Genetic Regulatory Mechanisms: insights in neurodevelopmental adaptation under alcohol stresses.

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This ongoing research on biological responses to alcohol stress during neurodevelopment focuses on behavioural alterations that are attributable to changes in gene expression and associated epigenetic mechanisms. We were among the first to consider this question at the level of the genome using expression arrays1. We identified ethanol responsive brain expressed genes from strains of mice2 and used genetical genomics to link such genes to alcohol preference3,4,5,6. The genomic technology developed in this research facilitated the extension of these studies on genome-wide gene expression to neurodevelopment. The results show that alcohol exposure in C57BL/6J mice during neurogenesis7 and synaptogenesis8 causes life-long changes in behaviours including learning/memory deficits. Also, they are related to changes in the expression of a set of candidate genes affected in relevant pathways9,10. Further we showed that, this effect on gene expression may be maintained for life by changes in DNA methylation11, microRNA12 and related epigenetic mechanisms. These results are novel, comprehensive and offer translational potential. Specifically, given that such landscapes are reversible, it is logical to consider novel experiments involving the effect of additional stress or reduced stress during rearing on molecular marks & learning deficits that forms the focus of this proposal involving hippocampus.Objective: To establish the involvement of specific epigenetic changes in response to alcohol during neurodevelopment in hippocampus. Hypothesis 1. The long lasting effect of alcohol during neurodevelopment on gene expression associated with "learning/memo deficits" is mediated by epigenetic changes (DNA methylation, miRNA, Histone modifications) in the hippocampus as suggested by our results on the whole brain.Hypothesis 2. Epigenetic changes including aberrant gene expression (hypothesis 1) could be altered by rearing conditions during early life13.Experimental Design: We will use C57BL/6J mice to generate pups exposed to ethanol via voluntary maternal consumption of 10% ethanol (T) or water only (C)7. The pups from T and C mothers will be raised under three conditions; normal housing conditions (N), enriched environment (E) or maternal separation stresses (S). The six groups of mice (TN, TE, TS, CN, CE and CS) will be followed for development and learning/memory in a Barns Maze. The mice representing six treatment groups will be sacrificed on postnatal day 70-90 (adult) and their hippocampus (involved in learning and memory) will be dissected out for studies on expression of a set on candidate genes10, miRNAs (qPCR), DNA methylation and histone modifications.Expected Results, Analysis and Interpretation: The results will quantitate the effect of neonatal ethanol and rearing environment (positive and negative) during growth and development on (i) learning/memory (ii) candidate gene expression, (iii) miRNA, (iv) DNA methylation and (v) histone modification in the hippocampus following Kleiber et al7-10 and Laufer et al 11, 12. Based on our results on total brain, we hypothesize that the hypothesis 1 will be affirmed. Further, the effect of enriched environment during development will improve learning/memory while the effect of maternal separation stress during rearing will exacerbate it. Also, such changes will follow hippocampal expression and epigenetic changes in some of the candidate genes identified in Kleiber et al11.Feasibility, Novelty and Significance: The experiments will extend our results on whole brain to hippocampus using identical methods. They will help connect learning and memory with stress induced & epigenetically regulated expression of hippocampus specific gene(s) including their postnatal reversibility.