Although many synaptic molecules have been identified, the processes and factors that lead to the development of a mature synapse have remained largely unknown. The present proposal seeks to examine the molecular mechanisms involved in the maturation of synapses by using an attractive model system, the Drosophila neuromuscular junction. Through the analysis of mutants in which synaptic structure is disrupted, we expect to identify genes involved in its assembly and maturation. We propose to examine the differentiation of functional neuromuscular contacts using a combination of whole cell patch recordings and ultrastructural techniques. This analysis will be performed in wild type and in two mutants that alter synaptic structure. One such mutation defines the tumor suppressor gene lethal(1) discs large (dlg), whose products belong to a family of proteins present at cell junctions in both vertebrates and invertebrates, including the fly neuromuscular junction, and mammalian brain synapses. The other mutant is branch point disrupted (bpd), which was isolated in a direct screen for mutants altering synapse structure. The functional correlates of the structural defects in these mutants will be examined by electrophysiological techniques, including voltage clamp recordings of whole muscle fibers and macropatch recordings of single synaptic boutons. In addition, we will determine the contribution of the dlg locus in determining synaptic structure by targeting dlg expression to pre and postsynaptic sites, as well as to both appropriate and inappropriate motorneurons. An ongoing genetic screen, using P[Gal4]-element mutagenesis will be conducted, in a search for other mutants altering neuromuscularjunction development. This mutagenesis scheme is based on the stereotypic innervation of larval neuromuscular junctions by identified motor axons.We expect that the outcome of these studies will be relevant to neuromuscular junctions in both vertebrates and invertebrates, and to synapses in general. A better understanding of the molecular basis of the development of synapses will provide much needed insight into a host of neuromuscular and central nervous system disorders.
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