The present project proposes to examine light-modulated neurotransmitter release from isolated vertebrate photoreceptors. Recent evidence suggests that the acidic amino acid, glutamate, may act as the neurotransmitter from photoreceptors in a variety of species, including goldfish. However, light-modulated release of endogenous glutamate from visual cell terminals has not yet been demonstrated. We propose to construct a glutamate-sensitive electrode to be used in the detection of glutamate release from goldfish photoreceptors that have been enzymatically dissociated from the retina. Two methods are described with respect to the construction of such electrodes, both of which rely on patch-clamp technology and the formation of gigaseals containing glutamate receptors. One method involves the use of biochemically isolated glutamate receptors from brain; the other employs outside-out patches from glutamate-sensitive membranes of isolated horizontal cells. The sensitivity, selectivity, and stability of the electrodes will be characterized by comparing the effects produced by glutamate and its analogues on the response properties of glutamate channels, i.e., their conductance, mean open time, and percentage time spent in the closed state. The electrodes, placed in apposition to the photoreceptor terminals, will then be used to determine (1) the degree to which the endogenous transmitter mimics the effects of glutamate on channel properties, (2) the effects of variation in photic intensity on transmitter release, and (3) the mechanisms whereby transmitter release is controlled, e.g., calcium dependence, ouabain sensitivity, etc. The results of this study will have direct bearing upon the nature of synaptic transmission by neurons in general and the processing of visual information by retinal cells in particular.
