PROJECT SUMMARY/ABSTRACT Synapses are the fundamental signaling components of the nervous system and mediate trans-neuronal information transfer. Brain functions require the precise, stereotyped establishment of diverse synaptic connections into circuits during development, followed by the refinement and maintenance of these circuits throughout life. Information processing by synaptic circuits underlies the brain's ability to generate behavioral responses, and circuit alterations are hallmarks of neurological disorders. Despite the importance of synaptic circuit formation for understanding brain functions and neurological diseases, the fundamental cellular and molecular framework mediating these processes in the mammalian brain remain unknown. Emerging evidence suggests that trans-synaptic signaling by networks of cell adhesion molecules directs critical aspects of synaptic circuit assembly and function. Our previous studies found that the adhesion G-protein-coupled receptor (aGPCR) Latrophilins (Lphn) mediate the synaptic wiring specificity of the hippocampal CA1 region. aGPCRs exhibit the unusual dual function of trans-cellular adhesion and control of intracellular GPCR signal transduction cascades. Studies of aGPCRs provide an opportunity into understanding the mechanisms of circuit assembly and function. aGPCRs are the second-largest GPCR class containing 33 members in humans that are poorly understood. For example, while a significant fraction of FDA-approved drugs target well-studied GPCRs, no effective therapeutics exist presently for aGPCRs although they have been linked to a range of diseases, including cancer, ADHD, and autism. Many aGPCRs are highly expressed in the brain, but their biological functions and signal transduction mechanisms remain unclear. More generally, lack of mechanistic insights into brain circuit assembly is precluding a deeper understanding of behavioral neuroscience and the basis of neurological disorders. Our studies will interrogate the biological principles of mammalian central nervous system circuit assembly using a multidisciplinary combination of approaches. First, we will determine the biological functions and signaling mechanisms of the aGPCRs. These studies will provide insights into an understudied class of GPCR and reveal novel principles of mammalian central nervous system development. We will subsequently employ genome engineering combined with live imaging approaches to monitor the dynamics of synaptic connections forming in mammalian circuits down to the nanoscale level. Our studies will use these approaches to visualize neural circuit assembly in both aGPCR mouse models and human neurons derived from patients with neurological disorders. Furthermore, using a combination of molecular and genetic approaches, we will determine the molecular codes operating in distinct synaptic subtypes during in vivo synaptogenesis. Collectively, these studies will overcome several current obstacles in the field and i...