Comparative Ca2+ channel contributions to intracellular Ca2+ levels in the circadian clock

Abstract

Circadian rhythms in mammals are coordinated by the central clock in the brain, located in the suprachiasmatic nucleus (SCN). Multiple molecular and cellular signals display a circadian variation within SCN neurons, including intracellular Ca²⁺, but the mechanisms are not definitively established. SCN cytosolic Ca²⁺ levels exhibit a peak during the day, when both action potential firing and Ca²⁺ channel activity are increased, and are decreased at night, correlating with a reduction in firing rate. In this study, we employ a single-color fluorescence anisotropy reporter (FLARE), Venus FLARE-Cameleon, and polarization inverted selective-plane illumination microscopy to measure rhythmic changes in cytosolic Ca²⁺ in SCN neurons. Using this technique, the Ca²⁺ channel subtypes contributing to intracellular Ca²⁺ at the peak and trough of the circadian cycle were assessed using a pharmacological approach with Ca²⁺ channel inhibitors. Peak (218 ± 16 nM) and trough (172 ± 13 nM) Ca²⁺ levels were quantified, indicating a 1.3-fold circadian variance in Ca²⁺ concentration. Inhibition of ryanodine-receptor-mediated Ca²⁺ release produced a larger relative decrease in cytosolic Ca²⁺ at both time points compared to voltage-gated Ca²⁺channels. These results support the hypothesis that circadian Ca²⁺ rhythms in SCN neurons are predominantly driven by intracellular Ca²⁺ channels, although not exclusively so. The study provides a foundation for future experiments to probe Ca²⁺ signaling in a dynamic biological context using FLAREs.