Abstract
The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5′-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.
Highlights
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the principal circadian pacemaker in mammals (Moore and Eichler, 1972; Stephan and Zucker, 1972)
Organotypic SCN cultures maintain the clear organization of SCN cells along the dorsoventral axis (Figure S1) and exhibit circadian rhythm in secretion of arginine vasopressin (AVP) for up to 2 weeks in vitro, with a phase that is consistent with the light regime experienced by the donor animal (Svobodova et al, 2003)
In the remaining 6% of cultures (n = 4), either no rhythm was observed or the peak did not occur between 24:00 and 04:00 h; these cultures were discarded. These data show that circadian rhythm of adenosine 5′-triphosphate (ATP) release peaks at an opposite phase to the previously described AVP secretory rhythm that peaked at ∼12:00 h in similar organotypic SCN cultures (Svobodova et al, 2003)
Summary
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the principal circadian pacemaker in mammals (Moore and Eichler, 1972; Stephan and Zucker, 1972). In most species, including rat, the SCN has two subdivisions that differ in neuronal input and neuropeptide contents. Circadian rhythm in AVP secretion parallels rhythm of electrical activity in SCN neurons (Inouye and Kawamura, 1979; Groos and Hendriks, 1982; Pennartz et al, 2002) and both these rhythms are mediated by the expression of clock genes (Reppert, 1998). The SCN generates a circadian rhythm in extracellular adenosine 5′-triphosphate (ATP) accumulation, which negatively correlates with the electrical activity and AVP secretion rhythms (Yamazaki et al, 1994; Womac et al, 2009). The pathway by which ATP travels from the cytosol of SCN cells to the extracellular space is still unknown
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