A multidisciplinary approach involving intracellular recording and dye injection, optic nerve backfilling and postembed light and electron microscopic immunocytochemistry will be employed to study the synaptic organization of identified neurons in the retinas of goldfish and tiger salamander. 1) The relative proportion and spatial distribution of inhibitory and excitory inputs on the dendrites of ON, OFF and ON-OFF cells is not known. Amacrine cells and ganglion cells will be recorded, classified as to their response to light, injected with HRP and processed for EM to determine their synaptic organization with respect to the inhibitory transmitter, GABA and glycine and excitatory transmitters, ACh and glutamine. Also, we plan to develop a retinal slice preparation in goldfish to combine transmitter pharmacology, dye injection and EM analysis of identified ganglion cells. 2) Determine the distribution of GABA receptors (monoclonal antibody against GABA/A/benzodazepine receptor complex) in the retina of goldfish and other vertebrate species and compare this distribution with ohter GABAergic markers. This mAB labels photoreceptor terminals in nonmammals, and analysis will provide information about sites of feedback transmission from horizontal cells to photoreceptors. 3) Determine the organization of synaptic terminals of mixed rod/cone bipolar cells in goldfish. Electrophysiological evidence suggest that ON (mb-type) and OFF (ma-type) differ on the basis of direct vs indirect GABAergic input. There is little known about other synaptic inputs or about the output targets of these large bipolar cells. 4) Determine if Na-K ATPase staining in the goldfish IPL is correlated with whether the transmitter at a synapase is excititory or inhibitory. This study could provide an independent means to predict the ionic conductance gated at a given synapase (i.e., sodium vs potassium or chloride). Also, test my hypothesis that the Na-K ATPase is involved in the dopamine-mediated inhibition of transmitter GABA release from goldfish horizontal cells. This study would provide a mechanism for the control of transmitter release that does not involve calcium-dependent vesicular exocytosis. These studies will provide detailed transmitter connectivity of physiologically identified retinal neurons. The transmitters under study are sufficiently conserved across species that these data should lead to a general understanding of the role of these transmitters in visual encoding.
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