PROJECT SUMMARY: Brain functions require the assembly and maintenance of distinct synaptic subtypes into stereotyped neural circuits. However, the fundamental cellular and molecular mechanisms underlying input-specific synapse formation in neural circuits remain poorly understood. Our studies during the K99 phase revealed an important role of the adhesion GPCR Latrophilins (Lphns) in hippocampal excitatory synaptic specificity. We recently found using rescue experiments that the role of Lphn2/3 in hippocampal input-specific excitatory synapse formation requires G-protein and/or u-Arrestin coupling. In parallel studies, we found that compartmentalized postsynaptic cAMP signaling is a prominent driver of excitatory synapse formation. Efforts during the R00 phase at Vanderbilt University will be focused on examining the G-protein and u-Arrestin intracellular signaling mechanisms of Lphns and other adhesion GPCRs at synapses in the mammalian central nervous system. Furthermore, we will determine the functions of unstudied adhesion GPCRs in neural circuits, and define the extracellular ligands of orphan adhesion GPCRs in the brain. We will develop novel molecular tools including viral-mediated nanobody targeting of signaling modulators to probe the roles of compartmentalized synaptic signaling pathways in input-specific excitatory, inhibitory and neuromodulatory synapse formation in neural circuits. We will also develop novel live super-resolution STORM and confocal imaging techniques to image synapse formation, maintenance and elimination in real-time. We expect these studies to reveal fundamental principles of how neural circuits are established by diverse synapses in the mammalian central nervous system, and elucidate the functions and signaling mechanisms of the understudied adhesion GPCRs in the brain.