The ultimate goal of this work is to develop a comprehensive understanding of how glutamate is released and eliminated at ribbon synapses of the retina. The mechanisms underlying vesicular release of glutamate at these synapses remain largely unknown. Miniature excitatory postsynaptic currents (MEPSCs) have traditionally been thought to arise from the exocytosis of single presynaptic vesicles, but preliminary results of this investigator suggest that at photoreceptor ribbon synapses they actually arise from the exocytosis of a cluster of vesicles. Furthermore, the contents of vesicles within such a cluster appear to be released in a series of sequential bursts, and multidimensional analysis of MEPSC shape suggests that the effects of individual vesicles within a cluster sum in a highly nonlinear and cooperative manner. The initial focus of the proposed research will be to gather additional evidence in support of these hypotheses. This work will use MEPSCs observed in off-center bipolar cells as an assay for transmitter release from rods and cones. The shapes of MEPSCs will be analyzed in a novel manner in order to determine an underlying subunit structure. The effects of agents such as calcium and osmotic pressure, which may affect the timing of subunit release, will be examined. The processes of glutamate uptake and glutamate receptor desensitization will also be studied by examining how agents that block these processes affect MEPSC time course. These data, plus data from an analysis of light-suppressed current fluctuations in bipolar cells will be used to generate a detailed model of synaptic transmission at photoreceptor ribbon synapses. Although the proposed work is primarily basic research, focused towards solving a fundamental problem in neurobiology, it could also have important implications for clinical ophthalmology Glutamate excitotoxicity is thought to contribute to degenerative conditions such as glaucoma. A better understanding of the unknown steps between presynaptic calcium influx and neurotransmitter exocytosis could lead to new approaches for limiting the rate of glutamate release at ribbon synapses, such as the synapse between bipolar cells and ganglion cells. A better understanding of the dynamics of glutamate diffusion, uptake, and binding to postsynaptic receptors within the synaptic cleft might also suggest better strategies to prevent glutamate-induced retinal degeneration.
