In this grant we will investigate synaptic function and plasticity in projects which depend on shared expertise of the principal investigators and a shared mouse transgenic core. Edwards will characterize transporters important in synaptic vesicle and nerve terminal function. He will identify sequences important in targeting catecholamine transporters to their appropriate organelles, characterize a novel family of vesicular glutamate and GABA transporters, characterize functions of a phosphate transporter, and examine the phenotype of mice deficient in this protein. With Copenhagen, Stryker and Malenka he will study its functions in the retina, visual cortex and hippocampus. Copenhagen will examine regulation of Ca2+ and exocytosis in rod and cone photoreceptors and in retinal ganglion cells, determining the roles of dopamine and, using homozygous mutant or genetic chimeric mice, examining roles of second messenger pathways and neurotrophin signaling. Copenhagen and Reichardt will characterize roles of neurotrophin signaling in retinal development using genetic chimeras. Stryker will use these mutant and genetically chimeric mice to determine the roles of neurotrophin signaling and second messengers in regulating plasticity during the ocular dominance critical period. Malenka will study CA3 LTP. With Copenhagen, he will examine changes in synaptic vesicle exocytosis using the membrane probe FM1-43. With Edwards, he will examine time courses of changes in cAMP and PKA activity and will determine whether proteins implicated in regulated exocytosis are phosphorylated during establishment of LTP. Using mutant mice he will determine how deficiencies in synaptic vesicle and membrane proteins affect CA3 LTP. Reichardt and Copenhagen will study the role of neurotrophin signaling pathways in modulating exocytosis at the developing Xenopus neuromuscular junction and on exocytosis in PC12 cells. Roles of neurotrophin-activated signaling pathways will be defined by mutagenesis of trk receptors and introduction of mutated molecules which act as dominant negative regulators of downstream signaling molecules. Using chimeric synaptic proteins tagged with Green Fluorescent Protein, Reichardt will determine effects of electrical activity and neurotrophins on establishment by retinal ganglion cell axons of synapses in the Xenopus optic tectum. The transgenic core will be used to generate mutant mice and genetically chimeric mice for these studies, using ES cell technologies and the lox-cre recombination system.
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