Molecular Basis of Circadian Cytosolic Calcium Rhythms

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The hypothalamic suprachiasmatic nucleus (SCN) has a pivotal role for the mammalian circadian clock system. We have demonstrated circadian rhythms in cytosolic Ca^(2+) concentrations in cultured SCN neurons and proposed that intracellular Ca^(2+) is a candidate messenger which mediates cellular input/output signals to/from the molecular loop. However, essential regulatory genes to drive circadian Ca^(2+) rhythms were currently unknown. The present research aims to elucidate the molecular mechanism underlying the generation of circadian Ca^(2+) rhythms in clock cells. First, we examined effects of mPer1/mPer2 antisense mRNAs on SCN neurons whereas failed to observe their effects on circadian rhythms in cytosolic Ca^(2+) and action potential firings. Second, we used several gene knockout mice lacking clock genes. The SCN neurons from the Cry1/Cry2 double knockout mice represented arrhythmic Ca^(2+) oscillations and those from the RORα knockout mice represented reduced amplitude Ca^(2+) o … More scillations. Thus, we further over-expressed native mouse Bmal1 or dominant negative Bmal1 into the SCN. These treatments resulted in a significant reduction in circadian Ca^(2+) oscillations in SCN neurons. Therefore, we concluded that endogenous expression of BMAL1 is essential for the generation of circadian Ca^(2+) rhythms and thus for the physiological activity rhythms in SCN neurons.The present study also analyzed process of photic inputs to SCN neurons regarding the mobilization of cytosolic Ca^(2+) and expression of clock genes. We found light-pulse-induced mPer1/2 gene expression in the SCN and behavioral-phase-shifts were significantly reduced in cholecystokinin (CCK)-A receptor knockout mice. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells, but not retino-recipient SCN neurons. Moreover, Ca^(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8s, mobilized intracellular Ca^(2+) in amacrine cells but not retino-recipent SCN neurons. These data indicate a novel function of CCK-A receptors as part of the cellular mechanism underlying circadian photo-entrainment via amacrine-cell-mediated signal transduction pathways. Less