Plante Amber E, Rao Vishnu P, Rizzo Megan A, Meredith Andrea L
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
Biophys Rep (N Y). 2021 Sep 8;1(1). doi: 10.1016/j.bpr.2021.100005. Epub 2021 Jul 21.
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.
哺乳动物的昼夜节律由位于视交叉上核（SCN）的大脑中央时钟协调。多种分子和细胞信号在SCN神经元内呈现昼夜变化，包括细胞内钙，但具体机制尚未明确确立。SCN胞质钙水平在白天达到峰值，此时动作电位发放和钙通道活性均增加，而在夜间降低，这与发放率的降低相关。在本研究中，我们采用单色荧光各向异性报告基因（FLARE）、金星FLARE-钙指示剂，并结合偏振反转选择性平面照明显微镜来测量SCN神经元胞质钙的节律性变化。使用该技术，通过使用钙通道抑制剂的药理学方法评估了在昼夜周期的峰值和谷值时对细胞内钙有贡献的钙通道亚型。对峰值（218±16 nM）和谷值（172±13 nM）的钙水平进行了量化，表明钙浓度存在1.3倍的昼夜变化。与电压门控钙通道相比，ryanodine受体介导的钙释放的抑制在两个时间点均导致胞质钙的相对更大幅度降低。这些结果支持以下假设，即SCN神经元中的昼夜钙节律主要由细胞内钙通道驱动，尽管并非完全如此。该研究为未来使用FLAREs在动态生物学背景下探究钙信号的实验奠定了基础。
