Glutamate is the primary excitatory neurotransmitter of the vertebrate CNS and the Drosophila neuromuscular junction (NMJ). The vesicular glutamate transporter (VGLUT) is responsible for filling synaptic vesicles with transmitter at glutamatergic synapses. The glutamate content of a synaptic vesicle is a fundamental parameter controlling the strength of synaptic transmission in the healthy brain, while the misregulated release of vesicular glutamate may contribute to the pathophysiology of such diseases as stroke, ALS, and epilepsy. This proposal will test the hypothesis that the expression, trafficking, and activity of the Drosophila homolog of VGLUT (D VGLUT) regulates the function, plasticity, and development of a glutamatergic synapse by controlling the glutamate content of synaptic vesicles. Our studies will combine genetic, cell biological, and electrophysiological methods to test the function of this important protein. The Drosophila homolog of the vesicular glutamate transporter (DVGLUT) has been identified and mutants have been generated that allow for the manipulation of its expression at the synapse. Biochemical, electrophysiological, and electron microscopic techniques will be employed to investigate the physiological role of DVGLUT in the filling of synaptic vesicles with glutamate (Aim 1). Neurotransmitter not only mediates synaptic transmission, but can also regulate the development of synapses. DVGLUT mutants will be used to manipulate the levels of glutamate in synaptic vesicles and the consequences for synapse formation, glutamate receptor localization, and synaptic maturation will be investigated (Aim 2). Finally, the mechanisms regulating the membrane trafficking of DVGLUT will be determined in order to define the role of DVGLUT trafficking in the regulation of glutamatergic transmission (Aim 3). The results of these experiments will provide novel and fundamental information on the mechanisms by which VGLUT contributes to the development and function of glutamatergic synapses.
