Calcium modulation and dynamics in adult zebrafish cone photoreceptors

Abstract

Purpose: Ca²⁺ regulates a variety of cellular processes including signal transduction, energy production, and mitochondrial function. Because photoreceptors (PRs) use significant quantities of energy, consuming more ATP/minute per gram tissue than even the brain, our goal is to understand how PRs respond to external fluctuations of Ca²⁺, and how intracellular Ca²⁺ dynamics influence energy metabolism. We have previously shown that larval zebrafish PRs display heterogeneous Ca²⁺ transients. In this study we investigate Ca²⁺ dynamics in adult zebrafish PRs.

Methods: Adult zebrafish retinas expressing the genetically encoded fluorescent Ca²⁺ sensor GCaMP in cone PRs were dissected, flat-mounted, and sliced into 400-µm sections. Retinas were stained with propidium iodide or BODIPY and slices were imaged in a temperature-controlled chamber. Retinas were perfused with 0 mM or 3 mM Ca²⁺ Ringer’s solution with and without 10 mM isridipine. GCaMP fluorescence was monitored by confocal microscopy.

Results: Ca²⁺ transients heterogeneous in amplitude and duration are prevalent in both the synapse and cell body of adult PRs. When Ca²⁺ is removed from the media, intracellular GCaMP fluorescence initially decreases, but then transiently increases, suggesting a compensatory Ca²⁺ release from internal stores. When extracellular Ca²⁺ is restored, intracellular fluorescence increases, first in the synapse and then throughout the cell. To understand how PRs take up Ca²⁺ from extracellular stores, we treated retinas with isridipine to block Ca²⁺ entry through L-type Ca²⁺ channels in the synapse. Preliminary experiments show that isridipine slows recovery of GCaMP fluorescence, suggesting PRs take up Ca²⁺ predominately through the synapse. Further, repeated transients released from intracellular stores may compensate for this block in external Ca²⁺ uptake.

Conclusions: Transgenic adult zebrafish retinas with GCaMP in cone PRs display robust Ca²⁺ dynamics. Intracellular Ca²⁺ responds to changes in external Ca²⁺. PRs take up Ca²⁺ predominantly from the synapse and then distribute it to the cell body. Insufficient external Ca²⁺ or a block in Ca²⁺ uptake elicits compensatory release of Ca²⁺ into the cytoplasm from internal stores. Understanding how PRs regulate Ca²⁺ homeostasis will provide a framework for understanding how PRs use Ca²⁺ to modulate metabolic processes such as NADH and ATP production, to regulate energy production and maintain cell health.