Characterization of calcium release in zebrafish visible cone outer segments

Abstract

Abstract: : Purpose: Bright illumination produces light–activated release of Ca²⁺ in the outer segments of zebrafish visible cones. This Ca²⁺ release can be accompanied by a photocurrent, as shown in nof mutants lacking cone transducin (Brockerhoff et al., J. Neurosci., 23, 470–480, 2003). We have characterized this release in greater detail in an attempt to understand its physiological significance. Methods:Electrical recordings and calcium measurements were made from fluo–4AM loaded double cones whose inner segments were held in a suction electrode. Outer segments were rapidly perfused with a 0Ca²⁺/0Na⁺ solution to minimize surface membrane Ca²⁺ fluxes, and intracellular free [Ca²⁺]i was measured with laser spot microscopy. Results: WT visible cones loaded with fluo–4 and stimulated in Ringer responded to the bright light of the laser with a decrease in fluorescence having 2 time constants of approximately 0.5 and 3.8 s, representing a decrease in [Ca²⁺]i. This time course could be accelerated by previous laser exposure in 0Ca²⁺/0Na⁺ solution, indicating a component of light–activated release in Ringer solution. This release could be seen more clearly in 0Ca²⁺/0Na⁺ solution, in which the fluorescence increased as a result of laser exposure. The rate and amplitude of this increase was diminished by pre–incorporation of BAPTA. The fraction of the pool released increased monotonically with increasing bleaching intensity, much as for salamander rods. The release pool recovered rapidly after the turning off of the light, and even after a 50% bleach in an isolated cone the pool was completely recovered in 2 min. In nof cones, the Ca²⁺ increase was accompanied by a slow outward photocurrent blocked by L–cis diltiazem.Conclusions:Light–activated Ca²⁺ release in zebrafish cones is similar to that in salamander rods (Matthews and Fain, J. Physiol. 552, 763–776, 2003). Since however cones operate in much brighter light than rods and recover more quickly after a bleach, it is more likely that release plays a role in the response of the cell within the physiological range of light intensities.

Keywords: calcium • photoreceptors • electrophysiology: non–clinical