The goal of this project is a quantitative description of the mechanism of signal transmission between cone photoreceptors and Off bipolar cells in the mammalian retina. Cones make two types of synapse: ribbon and basal. Ribbon synapses mediate transmission between cones and post- synaptic horizontal and On bipolar cells. The ribbon synapses have docked synaptic vesicles, and thus resemble conventional synapses made by other neurons in the brain. Basal synapses mediate transmission between cones and post-synaptic Off bipolar cells. At basal synapses, the membranes of pre- and post-synaptic cells come into close apposition, but there are no obvious docked vesicles and no active zones. Basal synapses are structurally different from conventional synapses, and little is known of their function. It is important to know how basal synapses function, since these synapses form a crucial link in the pathway that signals decreases in bright illumination. There are at least three hypotheses for how basal synapses operate: 1) Transmitter is released by Ca/2+-dependent exocytosis at basal junctions in the absence of morphological specialization (local vesicular release); 2) Exocytosis occurs only at ribbon synapses and basal synapses receive transmitter by overflow (remote vesicular release); and, 3) Transmitter release occurs at the basal synapse by a Ca/2+-independent, transporter-mediated mechanism. The function of basal synapses will be characterized in a mammalian retinal slice preparation in which the membrane voltage of both a cone (the pre-synaptic cell) and a hyperpolarizing bipolar cell (the post-synaptic cell) are controlled simultaneously with patch- pipette voltage clamps. Specific experiments have been designed to differentiated between three mechanisms. Experiments will first determine whether transmission at the basal synapse is vesicular by characterizing its Ca/2+ and voltage dependence. Experiments will then distinguish between local and remote release by characterizing the time- and voltage-dependent rate of vesicle fusion in a cone, the shape of the quantal response, the dwell-time of transmitter in the synaptic cleft, and the properties of the post-synaptic glutamate receptors.
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