Characterization of Zebrafish Green Cone Photoresponse Recorded with Pressure-Polished Patch Pipettes Yielding Efficient Intracellular Dialysis

摘要

The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca²⁺ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca²⁺-buffering capacity of the outer segment. Importantly, the Ca²⁺-buffer action preserves the fast response rising phase, when the Ca²⁺-dependent negative feedback is activated by the light-induced decline in intracellular Ca²⁺.