PROJECT SUMMARY Astrocytes are evolutionarily conserved glial cells in the central nervous system (CNS) that play a crucial role in neuronal circuit development and function. Mature astrocytes exhibit remarkably ramified and complex morphology and closely associate with neuronal synapses throughout the brain. Diazepam binding inhibitor (DBI) is a protein that has been shown to be highly enriched in glial cells, including astrocytes, in mouse and human brains. We recently discovered that it is also highly expressed by Drosophila astrocytes, suggesting its crucial and evolutionarily conserved role in astrocyte function. DBI and its proteolytic neuropeptide, Octadecaneuropeptide (ODN), have been shown to bind to the benzodiazepine (BZD) binding site on GABAA receptors and modulate the strength of inhibitory signaling, acting as endogenous ligands of BZD (endozepines). As BZDs are among the most widely prescribed treatments for anxiety disorders and seizures, understanding the astrocytic endozepine signaling will provide insights into how astrocytes can regulate the proper maturation and function of neuronal circuitry, and how the brain responds to BDZs. Another proteolytic fragment of DBI, triakontatetraneuropeptide (TTN), binds to translocator protein (TSPO) receptor on the outer mitochondrial membrane and is involved in mitochondrial cholesterol transport, cytoskeletal dynamics, and Ca2+ homeostasis, all of which are critical for astrocyte morphogenesis and function. Employing a synergistic combination of molecular-genetic tools available in Drosophila and zebrafish, this proposal aims to: determine whether DBI signaling is required for normal astrocyte development (Aim 1); define cell-intrinsic function of TTN-TSPO signaling in astrocyte mitochondrial and Ca2+ dynamics (Aim 2); and investigate the role of astrocytic DBI signaling in neuronal circuit maturation in the context of the excitatory- inhibitory (E/I) balance. This work will elucidate evolutionarily conserved function of DBI signaling in astrocyte development and astrocyte-neuron interactions in vivo and could provide novel insights into the role of astrocytes in anxiety disorders and epilepsy.