Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes.

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca(2+) and ATP accumulation and have demonstrated that intracellular Ca(2+) levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca(2+) levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca(2+). In contrast, our finding that peak mitochondrial Ca(2+) occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca(2+) uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca(2+) mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes.

The master circadian pacemaker located within the suprachiasmatic nucleus (SCN) of the mammalian brain controls system-level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. ATP is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferin-luciferase chemiluminescence assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (tau) of 23.7 h in untreated as well as vehicle-treated and forskolin-treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to 12 h of light and 12 h of dark or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23 h. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.