Project Summary Benzodiazepines (BZDs) are widely prescribed drugs and exert their anxiolytic, muscle relaxant, sedative, and anticonvulsant actions by binding to GABA-A receptors (GABARs) and potentiating GABA-induced currents. Since their synthesis over 50 years ago, scientists have searched for endogenous ligands in the brain that bind to the BZD binding site and modulate GABAR activity. Recent evidence suggests a 10 kDa protein named `Diazepam binding inhibitor' (DBI) is the brain's endogenous BZD (endozepine). Depending on the brain region, DBI can potentiate or inhibit GABAR activity suggesting that the brain can modulate GABAR-mediated neuronal inhibition by controlling the levels of DBI and its cleavage products. Little, however, is known about how DBI levels are regulated, how it is processed, or how DBI exerts its positive versus negative effects on GABAR activity. Experiments proposed will address these fundamental questions, which will validate DBI's role as an endogenous BZD and provide new mechanistic insights into how endogenous BZDs regulate GABA- mediated inhibition in the brain. Decreases in GABAergic neurotransmission are linked to numerous neurological and mental health disorders such as insomnia, epilepsy, anxiety, autism, Fragile X and schizophrenia. Whether regional changes in DBI levels or DBI cleavage products contribute to human disease is unknown. Our findings will open up new areas of scientific inquiry and have the potential to reveal new targets for therapeutic drug development.