The effect of light on outer segment Ca2+ in the uv–sensitive cones of zebrafish

Abstract

Abstract: : Purpose:The uv–sensitive cones of the zebrafish are insensitive to visible light and can therefore be used for Ca²⁺ measurements with fluo dyes and an argon ion laser without undue bleaching of the cone photopigment. We have used this technique to explore in detail the relationship between outer segment Ca²⁺ concentration (Ca²⁺_i) and photocurrent during a flash of light and in the presence of backgrounds. Methods:Dark–adapted zebrafish were stunned by concussion, then killed by decapitation and pithing. Cones were isolated from the retina and loaded for 30 min with 10 µM fluo 4–AM; fluorescence was measured as described previously (e.g. Matthews & Fain 552:763–776, 2003). Simultaneous photocurrent measurements were made with a suction pipette. Results: A single cone was stimulated repeatedly with a flash of constant intensity from the optical bench; after each light flash a single 50 ms laser exposure was used to evoke dye fluorescence before and at four times during the ensuing flash response. Comparison of photocurrent with fluorescence indicated that Ca²⁺_i declined after the flash and then recovered with a time course somewhat delayed but similar to that of the photocurrent. When cones were exposed to steady background light to compare the change in sensitivity and photocurrent with the change in Ca²⁺_i, we found that for dim backgrounds, photocurrent and Ca²⁺_i declined in proportion; but for brighter backgrounds, Ca²⁺_i was considerably greater than expected, probably as the result of light–induced Ca²⁺ release. This release of Ca²⁺ seemed, however, to have little effect on the sensitivity or photocurrent of the cell, which both declined monotonically in spite of the anomalous increase in Ca²⁺_i. Conclusions:Our results indicate that the Ca²⁺_i of zebrafish cones depends primarily on the level of the photocurrent during a flash or in dim backgrounds. Bright backgrounds within the physiological range can produce a significant release of Ca²⁺, but this release appears to have a mimimal effect on the sensitivity or channel opening probability of the cell. This suggests that changes in Ca²⁺_i in zebrafish cones may have only a small effect on light adaptation apart from the modulation of guanylyl cyclase.

Keywords: photoreceptors • calcium • signal transduction