The long term interests of my lab have centered on establishing the respective roles of presynaptic calcium channels and postsynaptic cholinergic receptors in neuromuscular transmission. My lab has traditionally relied on use of developing Xenopus neuromuscular junction where, among other advantages, direct cellular analysis of synapse function can be assessed through voltage-clamp of pre and postsynaptic cells. During the past funding period we initiated study of Zebrafish neuromuscular junction, which is functionally similar to Xenopus, but has the additional advantage that behavioral consequences of synaptic dysfunction are directly ascertained. We have identified Zebrafish mutant lines that exhibit locomotory dysfunction resulting from defects in neuromuscular transmission. Two lines exhibit myasthenia gravis-like symptoms in the form of use-dependent fatigue, and the symptoms can be partially rescued by inhibitors of cholinesterase function. Recently, we have shown the defect in one mutant line results from a defective rapsyn gene, and we have succeeded in rescuing the behavioral defect in the mutant fish. Another line with defective synaptic receptor density shows similar progressive weakness but outgrows the behavioral phenotype with age. Both lines exhibit profound synaptic depression at the nerve-muscle junction when compared to wild type animals. We will use these two lines to establish the mechanisms through which alterations in postsynaptic receptor densityleads to the observed synaptic depression. A similar depression can be recorded at Xenopus nerve-muscle synapses where both pre and postsynaptic cells can be voltage clamped. Using this preparation we will determine whether alterations in presynaptic release or postsynaptic receptor desensitization are responsible for synaptic depression. The four specific aims that are proposed merge the collective strengths of in vitro use of Xenopus with behavioral mutants of zebrafish, in order to investigate the mechanisms underlying synaptic plasticity at the neuromuscular junction.
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