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Vesicular glutamate transporters as molecular regulators of neural communication

Final Report Summary - SYNVGLUT (Vesicular glutamate transporters as molecular regulators of neural communication)

The vesicular glutamate transporter (VGLUT) protein plays a crucial role in communication between neurons. At the synapse, VGLUTs fill synaptic vesicles with the excitatory neurotransmitter, glutamate, which is subsequently released in response to stimulation of the neuron. However, in this project, we determined additional, fundamental roles that VGLUTs play in synaptic transmission. One additional role that VGLUTs play in shaping synaptic transmission is by affecting synaptic vesicle release probability. We determined that this role of VGLUTs occurs by two means: the specific isoform of VGLUT expressed on the vesicle (Weston et al., 2011) and the number of VGLUTs expressed on a vesicle (Herman et al., 2014). Through the meticulous characterization of the mechanisms responsible for these phenomena, we found that the isoform-specific effects of VGLUT on release probability is due to an interaction of the VGLUT1 isoform with a vesicle retrieval-related protein, endophilin (Westin et al., 2011). Further investigation into this pathway led to our development of a novel electron microscopy technique, which uses optogenetic control of neuronal activity combined with precisely timed fixation by high pressure freezing (Watanabe et al., 2013, 2014). This technique has led to great insight into mechanisms of endocytosis occurring immediately following neurotransmitter release and will provide information about the interactions of membrane and proteins occurring during the cycle of neurotransmission at the synapse. The mechanism of VGLUT protein number effects on release probability was determined to be by way of the neurotransmitter fill-state of synaptic vesicles (Herman et al., 2014). This novel mechanism affecting release probability suggests a fundamental, biophysical role of the intra-vesicle environment on vesicle fusion. Additional work has shown that a 50% decrease in VGLUT protein level by genetic manipulation leads to deficits in synaptic vesicle release characteristics (Herman et al., in preparation), possible describing the underlying synaptic mechanisms leading to the implication reduced functional VGLUT proteins in a number of disease states. Finally, we investigated the role of the isoform VGLUT3 in packaging different types of neurotransmitters into the same vesicle, a phenomenon that is hypothesized to occur at select synapses in the brain. We found that expression of VGLUT3 in cultured, mammalian inhibitory neurons caused co-packaging and co-release of glutamate and the inhibitory neurotransmitter, GABA, from synaptic vesicles (Zimmermann et al., in preparation). Overall, the goals of this project were to identify multiple functions for a vital protein in synaptic transmission, VGLUT. Our findings contribute to the basic understanding of fundamental processes occurring in the brain.