Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation

abstract

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca²⁺([Ca²⁺]_i), and neuronal firing rate. [Ca²⁺]_i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca²⁺]_i rhythms in the SCN, as well as the SCN neuronal network-dependence of [Ca²⁺]_i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca²⁺ indicator, into SCN neurons from PER2::LUC knockin reporter mice.Then, PER2 and [Ca²⁺]_iwere imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca²⁺]_i both exhibited cell autonomous circadian rhythms, but [Ca²⁺]_i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model, in which clock gene rhythms drive [Ca²⁺]_i rhythms. As predicted by the model, PER2 and [Ca²⁺]_irhythms were both stronger in SCN slices than in dispersed cells, and weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca²⁺]_i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca²⁺]_i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca²⁺]_i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.

Significance Statement Intracellular calcium ([Ca²⁺]_i) oscillates with a circadian (∼ 24 h) rhythm within the SCN, the brain’s master circadian pacemaker. However, what drives these [Ca²⁺]_i rhythms is not well understood. To test how rhythmic circadian clock gene expression and the SCN neuronal network influence [Ca²⁺]_irhythms, we monitored [Ca²⁺]_i and PER2 (a rhythmic clock gene) in individual SCN neurons in dispersed and slice cultures by fluorescence and bioluminescence imaging, respectively. We also tested the effects of blocking neuronal firing or knocking out Bmal1, an essential clock gene. We found that the [Ca²⁺]_i rhythm requires intact clock gene rhythms, and that it does not require the SCN neuronal network (so is cell autonomous) but is reinforced and modulated by the neuronal network.