The long-term goal of this project is to understand the mechanisms that control neurotransmission from rods and cones. Release of the excitatory neurotransmitter L-glutamate from photoreceptors is regulated by the activity of L-type Ca2+ channels. In the physiological voltage range, there appears to be a linear relationship between the influx of Ca2+ through these channels and the release of glutamate at the photoreceptor synapse. This linearity differs from the non-linear relationship found at most other synapses. One aim of this application is to use electrophysiological (capacitance monitoring and whole cell patch clamp recording) techniques, photolysis of caged Ca2+, and Ca2+ imaging techniques to examine the Ca2+ dependence of release from larval tiger salamander photoreceptors. One way in which a linear relationship between Ca2+ influx and release might arise is if vesicular exocytosis is initiated by the binding of only a single Ca2+ ion. If the binding of multiple Ca2+ ions is required to initiate release, then linearity between ICa and release is likely to reflect the linear summation, accompanying activation of an increasing number of Ca2+ channels, of sparsely distributed release sites with non-overlapping Ca2+ microdomains. These two possibilities will be investigated. The existence of a large number of modulators that can alter the voltage dependence or amplitude of photoreceptor ICa appear to present a challenge for photoreceptors to maintain the stable level of ICa activation necessary for stable synaptic output. The second major aim of this application is to use whole cell patch clamp recording as well as Ca2+ and Cl- imaging techniques to test the relative contribution of three specific intrinsic modulatory mechanisms to stabilizing ICa activation in rod and cone photoreceptors: (1) Ca-dependent inactivation of ICa, (2) depletion of synaptic cleft Ca2+, and (3) activation of Ca2+-activated Cl- channels. In addition to their importance in normal vision, regulation of photoreceptor ICa, intracellular Ca2+ concentration, and glutamate release are also important in pathophysiology of the retina. For example, increased intracellular Ca2+ levels in rods and cones may contribute to photoreceptor degeneration, and increased glutamate release arising from enhanced activation of ICa can have excitotoxic consequences on post-synaptic neurons. Thus, understanding the intrinsic mechanisms in rods and cones that regulate ICa, intracellular Ca2+ levels, and synaptic transmission is important for understanding the physiology of both diseased and normal retina.