Comparative Ca2D channel contributions to intracellular Ca2D levels in the circadian clock

Abstract

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 Ca2þ, but the mechanisms are not definitively established. SCN cytosolic Ca2þ levels exhibit a peak during the day, when both action potential firing and Ca2þ 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 Ca2þ in SCN neurons. Using this technique, the Ca2þ channel subtypes contributing to intracellular Ca2þ at the peak and trough of the circadian cycle were assessed using a pharmacological approach with Ca2þ channel inhibitors. Peak (218 5 16 nM) and trough (172 5 13 nM) Ca2þ levels were quantified, indicating a 1.3-fold circadian variance in Ca2þ concentration. Inhibition of ryanodine-receptor-mediated Ca2þ release produced a larger relative decrease in cytosolic Ca2þ at both time points compared to voltage-gated Ca2þchannels. These results support the hypothesis that circadian Ca2þ rhythms in SCN neurons are predominantly driven by intracellular Ca2þ channels, although not exclusively so. The study provides a foundation for future experiments to probe Ca2þ signaling in a dynamic biological context using FLAREs.