This 18 month project will determine whether exposure to chronic social stress, either early in life or during adulthood, causes epigenetic changes in sperm cells, which then mediate the transmission of stress vulnerability to subsequent generations. Such trans-generational transmission of stress vulnerability has recently been demonstrated by the Nestler laboratory and others, however, the molecular mechanisms responsible remain unknown. While a true epigenetic basis has been suggested, it remains unproven. To address this important and timely question, Nestler and colleagues will generate mice exposed either: 1) to chronic social stress during adulthood or 2) to two degrees of maternal separation during early life which cause life-long increases or decreases in stress susceptibility. Sperm isolated from stressed and control mice will then be screened for genome-wide changes in DNA methylation (including both 5-methycytosine and 5-hydroxymethylcytosine) and microRNAs (a class of non-coding RNAs), the two forms of epigenetic regulation most likely to be involved. Identifying stress-induced changes in DNA methylation or microRNAs in sperm will set the stage for future studies, which will directly investigate the contribution of individual epigenetic modifications in sperm to trans-generational transmission of stress vulnerability. The proposed studies will thus offer definitive proof of whether epigenetic mechanisms are involved in this highly novel phenomenon. The Nestler laboratory is ideally prepared to carry out this project: the group has led the field in epigenetic studies of stress susceptibility and resilience and has the established and demonstrated expertise to carry out all of the proposed investigations. The implications for human health are enormous, as this research has the potential to identify mechanisms by which traumatic life experiences can be passed on biologically to offspring and to develop ways of preventing such epigenetic inheritance.