During the past decade many candidate molecules have been identified that may play roles in regulating and triggering synaptic transmission. However, there have been limited opportunities to test the role of these proteins in the synapse because there are few tractable preparations where it is possible to integrate the necessary cellular and molecular approaches for the study protein function in the synapse. Using a system of synapses which form between spherical somata of Helisoma neurons, we are now in a unique position to utilize both cellular and molecular approaches in studying the synapse. We will distinguish between two roles for rab3 in the synapse; that it 1) controls vesicle docking with the plasma membrane; or 2) is a critical component of the vesicle fusion apparatus during calcium-dependent exocytosis. Molecular perturbation of rab3: immediate effects on synaptic transmission. Helisoma neurons form synaptic connections directly between apposed neuronal somata in culture. This permits the microinjection of macromolecules directly into the presynaptic terminal while monitoring the immediate effects on synaptic transmission. Using specific domain peptides, antibodies and chromophore-assisted laser inactivation (CALI), we will determine how perturbations of rab3 affect synaptic transmission. Molecular manipulation of rab3 expression: The requirement for rab3 in synapse function. We have recently demonstrated that we can effectively use antisense oligonucleotides to selectively prevent protein expression in Helisoma. Using this approach, we will ask whether the expression of rab3 is necessary for synaptic transmission. Expression of mutant rab3: Functional consequences for synaptic transmission. We have recently demonstrated that we can express proteins in Helisoma presynaptic neurons using a pNEX expression vector. We will use this expression system in presynaptic neurons to express mutant forms of rab3 that are impaired in their ability to bind GTP and will determine the consequences for synaptic transmission. We have known for decades that calcium, acting through a series of molecular events, stimulates neurotransmitter release. However, it has been difficult to precisely define this cascade of events. One essential protein is rab3, and by determining its role in the synapse this study will provide necessary information to put into place a critical building block in the emerging picture of the molecular events linking calcium to the release of neurotransmitter.
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