CONTE CENTER SUMMARY Neuroactive steroids (NAS) offer novel directions for psychiatric therapeutics. The prototypical NAS is allopregnanolone (AlloP), which under the formulation of brexanolone, has recently been approved by FDA for treatment of women with postpartum depression; a second, orally-active NAS has had a successful Phase 3 study for postpartum depression and a Phase 2B study for men and women with major depression. While AlloP is a potent and effective enhancer of GABAA receptors (GABAARs), it is not presently clear that the antidepressant effects of NAS are mediated solely by GABAergic effects, and recent data indicate that other mechanisms including effects on cellular stress and inflammatory pathways could also be involved. Members of our center have extensive experience studying the medicinal chemistry and mechanisms underlying the effects of AlloP-like NAS. In this Conte Center proposal we will leverage novel NAS compounds to probe varied molecular, synaptic, network and behavioral effects of NAS as clues to their therapeutic mechanisms and potential. Our center proposal is driven by unique NAS analogues synthesized in our Chemistry Core and involves three complementary and intertwined projects that will pursue three specific goals. First, we will test the hypothesis that selective actions of NAS on a class of GABAARs underlies effects on hippocampal electrical activity, network function and behavior in novel mouse lines and mouse models of postpartum and major depression. Second, we will examine the role of non-GABAA ion channels in mediating hippocampal and behavioral effects of NAS focusing on novel NAS that modulate NMDA glutamate receptors and low voltage activated calcium channels. These studies will use unique photoaffinity labeling approaches to elucidate sites of actions of NAS on NMDA receptors and other targets. Other studies will examine effects of NAS analogues on cellular function, neural network function, and behavior. Third, we will test the role of intracellular targets engaged by NAS on hippocampal circuits and behavior, focusing on the roles of neuroinflammation and cellular stress pathways. Our center is uniquely positioned to traverse the exploration of antidepressant NAS from molecules to sites of action and effects on dysfunctional circuits to identify potentially novel agents and targets for psychiatric therapeutic development.