PROJECT SUMMARY All of brain function, from sensory perception to behavior, is derived from the pattern and properties of the synaptic connections among billions (in humans) of individual neurons. The long-term goal of this project is to understand molecular pathways that regulate electrical synapse formation in vivo. Electrical synapses are sites of direct communication between neurons, formed by gap junctions, that allow the passage of ions and small molecules. They contribute extensively to neural circuit formation and function, both during development as well in adulthood, acting in sensory perception, central processing, and motor output. Moreover, they are thought to be disrupted in numerous human disorders, such as autism, epilepsy, and myopia. A major gap in the field is that the molecular mechanisms controlling the formation of electrical synapses are poorly understood. This proposal utilizes the zebrafish Mauthner circuit to investigate the genetics, cell biology, and biochemistry of electrical synapse formation and function. Mauthner neurons are individually identifiable and their pre- and postsynaptic partners, synapses, and function are exquisitely visualized in a living, vertebrate embryo. Using the genetic accessibility of zebrafish, we have found that a family of membrane-associated guanylate kinase scaffolds, the “ZO-family”, are required for gap junction channel localization and electrical synapse function. These findings suggest that electrical synapses are comprised of an emerging molecular complexity, and our goal is to uncover the cellular and molecular functions of the ZO-family in building functional neuronal gap junctions. Aim1 will determine how the ZO-family proteins direct the cell biological construction of electrical synapses in vivo. Aim2 will identify the molecular interactions utilized by the ZO-family proteins in directing electrical synapse formation in vivo. Aim3 will reveal the functional impacts of ZO-family proteins on synaptic transmission and behavior. The proposed studies will provide novel insight into the mechanisms of electrical synapse formation and provide a foundation for the identification of therapeutic targets for complex neurodevelopmental disorders.