The long-term objective of this project is to elucidate the molecular mechanisms underlying synaptic transmission from photoreceptors to second-order neurons in the vertebrate retina. This problem is being studied in the retina of the tiger salamander, principally through the use of electrophysiological and pharmacological techniques. During the next grant period, the focus will be on the role of chloride and hydrogen ions in shaping the light responses of photoreceptors and horizontal cells and in modulating signal transfer between these cells.
The specific aims are: (1) to measure the activities of chloride and hydrogen ions in photoreceptors and horizontal cells through intracellular recording from these cells in the intact retina with double-barrel Cl-sensitive and pH-sensitive microelectrodes as well as through optical recording from isolated cells with the fluorescent pH indicator BCECF, with the objective to determine whether chloride is distributed passively or is pumped actively in these neurons, (2) to measure extracellular pH in the outer retina in various physiological states, with the objective to determine whether there are significant activity-dependent pH changes which might modulate neuronal function, (3) to uncover the mechanism(s) involved in regulating internal chloride levels in photoreceptors and horizontal cells, and in particular, to use specific pharmacological blockers and the method of acid challenge to test for the presence of the major known chloride pumps, C1/HCO3 exchange and Na/K/2C1 cotransport, in these cells, and (4) to clarify the specific functions of chloride conductances in photoreceptors and horizontal cells through experiments which alter the movement of chloride ions across the plasma membrane such as chloride substitution and the application of chloride channel blockers and chloride transport blockers.
