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HR Matthews, Y Leung, GL Fain; Measurement of Ca2+i During the Flash Response Without Photopigment Bleaching in Isolated Ultraviolet-Sensitive Zebrafish Cones . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1837.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Light decreases photoreceptor Ca2+i by reducing its influx through the outer segment conductance while allowing continued efflux via Na+/Ca2+-K+ exchange. However, presently-available fluorescent Ca2+ indicators require an excitation intensity sufficiently high as to bleach almost all of the photopigment in amphibian, reptile, or mammalian photoreceptors, allowing only a single measurement from each cell. We have resolved this problem by using ultraviolet-sensitive zebrafish cones, which are most sensitive at 365 nm and are ∼105 times less sensitive to the 514 nm light of our laser spot microscope. Methods: Fluo-4 was loaded as its AM-ester. The inner segment of an isolated ultraviolet-sensitive cone was held by a suction pipette. Dye fluorescence was excited from the outer segment by an argon ion laser. The cell was exposed to a near-saturating 405 nm flash and at variable times thereafter to a 514 nm laser flash, calculated to bleach less than 0.02% of the photopigment. By repeating this protocol with different time intervals between light stimulus and laser exposure, the time course of dye fluorescence during the flash response could be investigated in a single cell. Results: The suppression of the circulating current was accompanied by a reduction in peak dye fluorescence, representing a dynamic fall in Ca2+i during the light response. The return of the photocurrent to baseline after the flash was accompanied by a rapid return of Ca2+i to its level in darkness. Dye fluorescence evoked by laser flashes in darkness before or after the flash response exhibited a component of rapid rise which was not present for laser flashes delivered at the response peak. When cells were pre-incubated with 100 µM BAPTA/AM, the amplitude and kinetics of this component were not significantly affected (Student’s T test, 5% level), suggesting that it represented a change in dye properties instead of a change in Ca2+i. When Ca2+i was measured in ultraviolet-sensitive zebrafish cones rapidly superfused with 0Ca2+/0Na+ solution, a 15 3% rise in fluorescence was observed over a time course of 1-2 s, which was abolished by BAPTA incorporation. Conclusion: These observations demonstrate directly the dynamic fall in Ca2+i during the dim flash response and suggest that light may in addition induce a release of Ca2+ in zebrafish cones, similar to that previously observed in salamander rods.
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