PROJECT SUMMARY Depression is sex biased not only in its rate, but also in precipitating factors and symptoms. For example, women report a lack of social support, while men report stressful events as major contributors to depression. Understanding the mechanisms that drive these sex differences involves isolating different molecular pathways to identify the causes of divergent vulnerability and symptoms. Sex chromosomes (XX vs. XY) are a major source of sex bias within any type of cell, but this category has been difficult to discriminate from gonadal hormone effects that often co-vary with sex chromosome complement. Sex chromosome effects on disease mechanisms can now be studied in newly engineered XX and XY rats that have the same type of gonad, either with testes or with ovaries. In these Four Core Genotypes (FCG)-like rats, sex chromosome effects (XX vs. XY) will be discriminated from gonadal hormone effects that differentiate the brains of gonadal males from those of gonadal females. Here we focus on the origins of sex differences in the septohippocampal circuit, which consists of the medial septum (MS) projection to the hippocampus. The septohippocampal circuit mediates memory and its dysregulation is implicated in the cognitive deficits that characterize several disorders, including depression. We previously found that administering the stress neuropeptide, corticotropin releasing factor (CRF), into the MS impairs hippocampal-dependent object location memory in rats. However, males are more sensitive to this effect than females. Ovarian hormones do not confer resilience in females, and structural sex differences in the MS of mice are causes by sex chromosome effects (SCEs). We aim to discriminate SCEs from gonadal hormone effects that cause sex differences in the septohippocampal circuit that mediates cognitive deficits in major depression, and to understand where these factors act and what molecular mechanisms they control. Aim 1 will use the FCG-like rats to test the hypothesis that sex differences in CRF receptor regulation of MS-mediated memory are due to SCEs. The beauty of the design is that even if the hypothesis is not supported, the origin of sex difference in this stress effect on memory will be identified. To determine molecular mechanisms that can underlie sex differences in the septohippocampal circuit, Aim 2 will use single-cell RNAseq to differentiate neuronal and glial subtypes of the MS and hippocampus, identify sex differences therein, and determine whether sex differences are caused by SCEs or gonadal hormones. Cell- cell communications will be modeled within and between cell types and brain regions to retrieve cellular circuits affected by SCEs vs. gonadal hormones. Collectively, the integration of sophisticated behavioral and molecular analysis will uncover cell-specific mechanisms by which diverse sex-biasing factors influence stress regulation of memory. These results will have important implications for developing n...