An understanding of molecular mechanisms underlying presynaptic function is important from many perspectives; learning, memory, motor and behavioral deficits may result from defects in molecules required for presynaptic functions; excess neurotransmitter release during epileptic seizures may result in excitotoxicity and consequent cell death in the brain; vesicular release during neural development may serve important functions during normal brain development. This proposal exploits the convenience of Drosophila as an experimental organism for incisive experiments on the mechanisms of presynaptic function. Such studies in Drosophila are validated by numerous examples of conservation of neural mechanisms across phylogeny including two examples from the PI's own past research: mutations in a neural Drosophila sodium channel gene cause temperature-sensitive paralysis in flies and mutations in a similar voltage-gated sodium channel of mammalian muscle result in periodic paralysis or a form of myotonia congenita in humans. A human potassium channel gene characterized on the basis of its homology to the Drosophila Shaker channels is mutated in patients suffering from episodic ataxia, a genetic disorder in human beings. Building on previous results from the PI's research, experiments proposed here have the potential to make a significant contribution to our understanding of mechanisms involved in presynaptic function. However, as an Assistant Professor in a major undergraduate university, teaching commitments limit the amount of effort the PI can contribute to research and this, more than anything else, places limits on the productivity of the lab. Experiments proposed in this application for a K02 award are ones that specifically require more time from the PI, not only to train laboratory personnel, but also to improve his own expertise in the proposed methods of experimentation.
The specific aims of the proposed research are to study, using electrophysiological, novel cell biological and traditional molecular genetic methods, mutations in four Drosophila genes which probably affect synaptic vesicle recycling, a little-studies process vital for presynaptic function. To understand the role of these genes in presynaptic function, the experiments will use electrophysiological and quantitative cell biological assays to study potential defects in synaptic vesicle fusion and recycling in identified mutant synapses. Morphological methods will be used to examine the disposition of synaptic vesicle membrane in mutant nerve terminals. Recombinant DNA techniques will be used to identify molecules affected by the mutations, in order to examine their phylogenetic conservation, their subcellular localization and their intracellular traffic during the cycling of synaptic vesicle membrane. If successful, these experiments will constitute the first in vivo analysis of molecular mechanisms in synaptic vesicle recycling.
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