Comparative Ca(2+) channel contributions to intracellular Ca(2+) levels in the circadian clock

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 Ca(2+), but the mechanisms are not definitively established. SCN cytosolic Ca(2+) levels exhibit a peak during the day, when both action potential firing and Ca(2+) 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 Ca(2+) in SCN neurons. Using this technique, the Ca(2+) channel subtypes contributing to intracellular Ca(2+) at the peak and trough of the circadian cycle were assessed using a pharmacological approach with Ca(2+) channel inhibitors. Peak (218 ± 16 nM) and trough (172 ± 13 nM) Ca(2+) levels were quantified, indicating a 1.3-fold circadian variance in Ca(2+) concentration. Inhibition of ryanodine-receptor-mediated Ca(2+) release produced a larger relative decrease in cytosolic Ca(2+) at both time points compared to voltage-gated Ca(2+)channels. These results support the hypothesis that circadian Ca(2+) rhythms in SCN neurons are predominantly driven by intracellular Ca(2+) channels, although not exclusively so. The study provides a foundation for future experiments to probe Ca(2+) signaling in a dynamic biological context using FLAREs.