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