Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

Abstract

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 μM, buffering becomes minimal.

Phototransduction and retinal degeneration in Drosophila

“…Extrusion of Ca 2+ via the CalX Na + /Ca 2+ exchanger Within 20 ms of light stimulation, the Ca 2+ concentration in the rhabdomeral microvilli can rise from a resting Ca 2+ level of ∼160 nM, to reach peak values higher than 200 μM, before declining to a steady-state of ∼20 μM [151][152][153]. To accomplish the dynamic changes in Ca 2+ levels during the photoresponse, a rapid Ca 2+ extrusion mechanism is required to counter the TRP-dependent influx of Ca 2+ .…”

Calcium Transients in the Rhabdomeres of Dark- and Light-Adapted Fly Photoreceptor Cells

“…Preparation and the electrophysiological methods were as described by Oberwinkler and Stavenga (1998). Briefly, the animal was immobilized and placed on the stage of a Leitz Orthoplan epifluorescence microscope (Leitz, Wetzlar, Germany).…”

“…We have recently demonstrated that the free Ca 2ϩ concentration can be measured by recording the fluorescence emitted from Ca 2ϩ indicator dyes in single photoreceptor cells of blowflies in vivo (Oberwinkler and Stavenga, 1998). In these earlier measurements, the fluorescent signal was sampled from the entire cell.…”

“…Light emitted from fluorophores in the rhabdomeres follows the reverse path and is consequently focused by the ommatidial lenses to form a beam with 1-2°divergence. Light originating from other parts of the cell would pass through the facet lenses forming a much wider beam of ϳ10° (Oberwinkler and Stavenga, 1998). This light, however, is to a very large extent absorbed by the pigment granules in the primary pigment cells (Fig.…”

“…Previous studies have shown that light stimulation causes a rapid rise in free Ca 2ϩ concentration throughout the photoreceptor cell that can exceed 10 M (Hardie, 1996a;Oberwinkler and Stavenga, 1998). The anatomy of the rhabdomere, however, suggests that the kinetics and absolute values of [Ca 2ϩ ] rh might be considerably different from those encountered in the cell body.…”