Project Summary Neuronal development. neurons comprised coordinated understand populations proposal, premotor sensor reconstitution their the of labeled neurons interneurons are circuits arise from the activity-dependent formation and elimination of synapses during Activity-dependent synaptic wiring has been studied extensively in peripheral sensory and motor where the goal is o be excited or o excite. However, in the central nervous system, neural circuits are of neurons that both excite and inhibit common targets and so activity-dependent wiring must be during development to achieve an appropriate balance of excitation and inhibition. To better this process, it would help to simultaneously monitor the activity of excitatory and inhibitory and their synaptic connections to common targets during development. In this exploratory R21 we will develop a genetic strategy to monitor activity-dependent changes in excitatory and inhibitory synaptic connectivity in the developing zebrafish spinal cord. We will use a genetically-encoded developed in Drosophila melanogaster that uses a synaptobrevin-based GFP, YFP or CFP across synaptic partners to simultaneously label connections in different colors and monitor activity levels (Syb-XRASP) . In Aim 1 we will validate the CFP, YFP and GFP variants of Syb-XRASP at neuromuscular junction of developing zebrafish. In Aim 2, we will use Syb-XRASP to monitor the assembly connections of premotor neurons within excitatory Chx10-labeled V2a population and inhibitory En1- V1 population. Together, these aims will allow us to assess the formation of connections rom motor to muscles, from single V2a and V1 i nterneurons to all motor neurons, and from all V2a and V1 to single motor neurons. These experiments will provide an unprecedent view of how connections formed during the development of circuits comprised of excitatory and inhibitory neurons. t t f