A central problem of neurobiology is to understand how specific connections are formed during development of the nervous system. Interactions between neurons and their targets and between presynaptic neurons competing for targets are known to be important for the establishment of proper synaptic connections, although the cellular details of these interactions are not well understood. The proposed experiments will provide a major advance in our understanding of these processes by characterizing cellular interactions between individual living motoneurons as they establish neuromuscular junctions during embryonic development. A system that we have recently developed will be used to observe living motoneurons in vivo, as their axons grow and make synaptic contacts with muscle fibers. Individual identified motoneurons in live zebrafish embryos will be labeled with fluorescent dyes and the growth of their axons will be monitored with computer enhanced video microscopy in these rapidly developing, optically clear embryos. The growth of particular motoneurons in a number of embryos will be characterized to learn if their axonal branching patterns are stereotyped and stable. These results will, for the first time, provide a detailed description of the extension and retraction of axonal branches and synaptic terminals by identified vertebrate neurons during the initial establishment of their axonal arbors. Then, the growth of motoneurons that innervate adjacent regions of muscle will be compared directly by labeling them with different colored dyes to see how individual axonal branches from different neurons interact. Finally, one or more motoneurons will be ablated with a laser and subsequent growth of the remaining motoneuron(s) will be monitored to see how competitive interactions affect the size and shape of a neuron's axonal arbor and the distribution of its synapses.
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