This is a study to investigate the molecular basis for both basal synaptic transmission and synaptic plasticity, including long-term potentiation, at CA3-CA1 synapses in the hippocampus. The approach is to use genetically engineered mice with mutations and/or deletions of specific synaptic vesicle proteins. The effects of such mutations will be investigated on normal synaptic transmission, short-term synaptic plasticity, including paired pulse facilitation, post-tetanic potentiation, and high frequency depression, and long-term synaptic plasticity, including early and late phases of long-term potentiation. The following aims will be addressed: 1. What is the role of the rab3 family of low molecular weight GTP binding proteins in synaptic transmission and the late phase of LTP? Earlier studies on rab3A knockouts will be augmented by studying rab3B and rab3C knockouts, either singly or in combinations. Effects of the rab3 deletions on synaptic transmission will be studied using quantal analysis of pairs of unitary recordings between a single presynaptic CA3 neuron and a single postsynaptic CA1 neuron in hippocampal slices. Effects of the rab3 deletions on synaptic vesicle cycling kinetics at single presynaptic boutons will be examined in CA3-CA1 dissociated cell cultures. The role of rab3 proteins in the cAMP-dependent late phase of LTP will be explored. 2. What is the role of the synaptophysin family of integral membrane synaptic vesicle proteins in release and plasticity? Previous studies by the applicants demonstrated that synaptophysin-synaptogyrin double knockout mice had defects in both short term forms of plasticity and LTP. Using quantal analysis of synaptic transmission at CA3-CA1 synapses in hippocampal slices, the possibility that these changes are due to an increase in the probability of transmitter release will be investigated. Possible effects on vesicle cycling kinetics will be examined in dissociated hippocampal CA3-CA1 cultures. The role of a third family member, synaptoporin, will be studied in mice made deficient in this protein, either alone or in combination with deletions of other synaptophysin family members. 3. The importance of specific functional domains of the rab3 proteins and synaptophysin family members will be addressed by expressing mutant forms of these proteins in the background of the knockout mice. For example, the role of GTP hydrolysis by rab3A will be studied by expressing a rab3A gene containing a point mutation that renders the rab3A protein defective in GTP hydrolysis, and thus constitutively active, in the rab3A deficient mice. The role of tyrosine phosphorylation in the function of synaptophysin and/or synaptoporin will be tested by expression of mutant proteins lacking the phosphotyrosine residues. 4. Mutants showing interesting phenotypes in synaptic plasticity will be further studied using region specific knockouts, inducible knockouts, and/or inducible transgene expression. Effects of such regional specific knockout or region-specific expression of proteins will be studied at the behavioral level.
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