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.

Department of Cellular and Molecular Neuroendocrinology Institute of Physiology of the Czech Academy of Sciences Prague Czechia

Department of Physiology Faculty of Science Charles University Prague Czechia

Zobrazit více v PubMed

Abbracchio M. P., Burnstock G. (1994). Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol. Ther. 64, 445–475. 10.1016/0163-7258(94)00048-4 PubMed DOI

Abe M., Herzog E. D., Yamazaki S., Straume M., Tei H., Sakaki Y., et al. . (2002). Circadian rhythms in isolated brain regions. J. Neurosci. 22, 350–356. PubMed PMC

Anderson C. M., Bergher J. P., Swanson R. A. (2004). ATP-induced ATP release from astrocytes. J. Neurochem. 88, 246–256. 10.1111/j.1471-4159.2004.02204.x PubMed DOI

Balázs B., Dankó T., Kovács G., Kóles L., Hediger M. A., Zsembery A. (2013). Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods. Cell. Physiol. Biochem. 32, 11–24. 10.1159/000350119 PubMed DOI

Ballerini P., Rathbone M. P., Di Iorio P., Renzetti A., Giuliani P., D'Alimonte I., et al. . (1996). Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release. Neuroreport 7, 2533–2537. 10.1097/00001756-199611040-00026 PubMed DOI

Bhattacharya A., Vavra V., Svobodova I., Bendova Z., Vereb G., Zemkova H. (2013). Potentiation of inhibitory synaptic transmission by extracellular ATP in rat suprachiasmatic nuclei. J. Neurosci. 33, 8035–8044. 10.1523/JNEUROSCI.4682-12.2013 PubMed DOI PMC

Brancaccio M., Patton A. P., Chesham J. E., Maywood E. S., Hastings M. H. (2017). Astrocytes control circadian timekeeping in the suprachiasmatic nucleus via glutamatergic signaling. Neuron 93, 1420.e5–1435.e5. 10.1016/j.neuron.2017.02.030 PubMed DOI PMC

Burkeen J. F., Womac A. D., Earnest D. J., Zoran M. J. (2011). Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes. J. Neurosci. 31, 8432–8440. 10.1523/JNEUROSCI.6576-10.2011 PubMed DOI PMC

Carrasquero L. M., Delicado E. G., Bustillo D., Gutiérrez-Martín Y., Artalejo A. R., Miras-Portugal M. T. (2009). P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes. J. Neurochem. 110, 879–889. 10.1111/j.1471-4159.2009.06179.x PubMed DOI

Cervetto C., Alloisio S., Frattaroli D., Mazzotta M. C., Milanese M., Gavazzo P., et al. . (2013). The P2X7 receptor as a route for non-exocytotic glutamate release: dependence on the carboxyl tail. J. Neurochem. 124, 821–831. 10.1111/jnc.12143 PubMed DOI

Coddou C., Yan Z., Obsil T., Huidobro-Toro J. P., Stojilkovic S. S. (2011). Activation and regulation of purinergic P2X receptor channels. Pharmacol. Rev. 63, 641–683. 10.1124/pr.110.003129 PubMed DOI PMC

Colwell C. S. (2000). Rhythmic coupling among cells in the suprachiasmatic nucleus. J. Neurobiol. 43, 379–388. 10.1002/1097-4695(20000615)43:4<379::AID-NEU6>3.0.CO;2-0 PubMed DOI PMC

Contreras J. E., Sánchez H. A., Véliz L. P., Bukauskas F. F., Bennett M. V., Sáez J. C. (2004). Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res. Brain Res. Rev. 47, 290–303. 10.1016/j.brainresrev.2004.08.002 PubMed DOI PMC

Di Cesare Mannelli L., Marcoli M., Micheli L., Zanardelli M., Maura G., Ghelardini C., et al. . (2015). Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: a pain mechanism mediated by Pannexin 1. Neuropharmacology 97, 133–141. 10.1016/j.neuropharm.2015.05.037 PubMed DOI

Donnelly-Roberts D. L., Namovic M. T., Surber B., Vaidyanathan S. X., Perez-Medrano A., Wang Y., et al. . (2009). [3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors. Neuropharmacology 56, 223–229. 10.1016/j.neuropharm.2008.06.012 PubMed DOI

Duan S., Anderson C. M., Keung E. C., Chen Y., Chen Y., Swanson R. A. (2003). P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J. Neurosci. 23, 1320–1328. PubMed PMC

Fumagalli M., Brambilla R., D'Ambrosi N., Volonté C., Matteoli M., Verderio C., et al. . (2003). Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors. Glia 43, 218–203. 10.1002/glia.10248 PubMed DOI

Groos G., Hendriks J. (1982). Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro. Neurosci. Lett. 34, 283–288. 10.1016/0304-3940(82)90189-6 PubMed DOI

Guilding C., Hughes A. T., Brown T. M., Namvar S., Piggins H. D. (2009). A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus. Mol. Brain 2:28. 10.1186/1756-6606-2-28 PubMed DOI PMC

Hamilton N., Vayro S., Kirchhoff F., Verkhratsky A., Robbins J., Gorecki D. C., et al. . (2008). Mechanisms of ATP- and glutamate-mediated calcium signaling in white matter astrocytes. Glia 56, 734–749. 10.1002/glia.20649 PubMed DOI

Haydon P. G. (2001). GLIA: listening and talking to the synapse. Nat. Rev. Neurosci. 2, 185–193. 10.1038/35058528 PubMed DOI

Hong Y., Zhao T., Li X. J., Li S. (2016). Mutant huntingtin impairs BDNF release from astrocytes by disrupting conversion of Rab3a-GTP into Rab3a-GDP. J. Neurosci. 36, 8790–8801. 10.1523/JNEUROSCI.0168-16.2016 PubMed DOI PMC

Iglesias R., Dahl G., Qiu F., Spray D. C., Scemes E. (2009). Pannexin 1: the molecular substrate of astrocyte “hemichannels.” J. Neurosci. 29, 7092–7097. 10.1523/JNEUROSCI.6062-08.2009 PubMed DOI PMC

Illes P., Khan T. M., Rubini P. (2017). Neuronal P2X7 receptors revisited: do they really exist? J. Neurosci. 37, 7049–7062. 10.1523/JNEUROSCI.3103-16.2017 PubMed DOI PMC

Illes P., Verkhratsky A., Burnstock G., Franke H. (2012). P2X receptors and their roles in astroglia in the central and peripheral nervous system. Neuroscientist 18, 422–438. 10.1177/1073858411418524 PubMed DOI

Inouye S. T., Kawamura H. (1979). Persistence of circadian rhythmicity in a mammalian hypothalamic island containing the suprachiasmatic nucleus. Proc. Natl. Acad. Sci. U.S.A. 76, 5962–5966. 10.1073/pnas.76.11.5962 PubMed DOI PMC

Jacomy H., Burlet A., Bosler O. (1999). Vasoactive intestinal peptide neurons as synaptic targets for vasopressin neurons in the suprachiasmatic nucleus. Double-label immunocytochemical demonstration in the rat. Neuroscience 88, 859–870. 10.1016/S0306-4522(98)00259-0 PubMed DOI

Jiang L. H., Mackenzie A. B., North R. A., Surprenant A. (2000). Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors. Mol. Pharmacol. 58, 82–88. 10.1124/mol.58.1.82 PubMed DOI

Kamatsuka Y., Fukagawa M., Furuta T., Ohishi A., Nishida K., Nagasawa K. (2014). Astrocytes, but not neurons, exhibit constitutive activation of P2X7 receptors in mouse acute cortical slices under non-stimulated resting conditions. Biol. Pharm. Bull. 37, 1958–1962. 10.1248/bpb.b14-00000 PubMed DOI

Khakh B. S., Lester H. A. (1999). Dynamic selectivity filters in ion channels. Neuron 23, 653–658. 10.1016/S0896-6273(01)80025-8 PubMed DOI

Khakh B. S., Sofroniew M. V. (2015). Diversity of astrocyte functions and phenotypes in neural circuits. Nat. Neurosci. 18, 942–952. 10.1038/nn.4043 PubMed DOI PMC

Kretschmannova K., Svobodova I., Zemkova H. (2003). Day-night variations in zinc sensitivity of GABAA receptor-channels in rat suprachiasmatic nucleus. Brain Res. Mol. Brain Res. 120, 46–51. 10.1016/j.molbrainres.2003.09.017 PubMed DOI

Li M., Kawate T., Silberberg S. D., Swartz K. J. (2011). Pore-opening mechanism in trimeric P2X receptor channels. Nat. Commun. 1:44. 10.1038/ncomms1048 PubMed DOI PMC

Li S., Bjelobaba I., Yan Z., Kucka M., Tomic M., Stojilkovic S. S. (2011). Expression and roles of pannexins in ATP release in the pituitary gland. Endocrinology 152, 2342–2352. 10.1210/en.2010-1216 PubMed DOI PMC

Locovei S., Scemes E., Qiu F., Spray D. C., Dahl G. (2007). Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. FEBS Lett. 581, 483–488. 10.1016/j.febslet.2006.12.056 PubMed DOI PMC

Marpegan L., Swanstrom A. E., Chung K., Simon T., Haydon P. G., Khan S. K., et al. . (2011). Circadian regulation of ATP release in astrocytes. J. Neurosci. 31, 8342–8350. 10.1523/JNEUROSCI.6537-10.2011 PubMed DOI PMC

Michel A. D., Chambers L. J., Clay W. C., Condreay J. P., Walter D. S., Chessell I. P. (2007). Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding. Br. J. Pharmacol. 151, 103–114. 10.1038/sj.bjp.0707196 PubMed DOI PMC

Michel A. D., Chambers L. J., Walter D. S. (2008). Negative and positive allosteric modulators of the P2X(7) receptor. Br. J. Pharmacol. 153, 737–750. 10.1038/sj.bjp.0707625 PubMed DOI PMC

Moore R. Y., Card J. P. (1985). Visual pathways and the entrainment of circadian rhythms. Ann. N.Y. Acad. Sci. 453, 123–133. 10.1111/j.1749-6632.1985.tb11805.x PubMed DOI

Moore R. Y., Eichler V. B. (1972). Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res. 42, 201–206. 10.1016/0006-8993(72)90054-6 PubMed DOI

Muller M. S., Taylor C. W. (2017). ATP evokes Ca2+ signals in cultured foetal human cortical astrocytes entirely through G protein-coupled P2Y receptors. J. Neurochem. 142, 876–885. 10.1111/jnc.14119 PubMed DOI PMC

Narcisse L., Scemes E., Zhao Y., Lee S. C., Brosnan C. F. (2005). The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes. Glia 49, 245–258. 10.1002/glia.20110 PubMed DOI PMC

Nörenberg W., Hempel C., Urban N., Sobottka H., Illes P., Schaefer M. (2011a). Clemastine potentiates the human P2X7 receptor by sensitizing it to lower ATP concentrations. J. Biol. Chem. 286, 11067–11081. 10.1074/jbc.M110.198879 PubMed DOI PMC

Nörenberg W., Schunk J., Fischer W., Sobottka H., Riedel T., Oliveira J. F., et al. . (2011b). Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia. Br. J. Pharmacol. 160, 1941–1952. 10.1111/j.1476-5381.2010.00736.x PubMed DOI PMC

North R. A. (2002). Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013–1067. 10.1152/physrev.00015.2002 PubMed DOI

Pan H. C., Chou Y. C., Sun S. H. (2015). P2X7 R-mediated Ca(2+) -independent d-serine release via pannexin-1 of the P2X7 R-pannexin-1 complex in astrocytes. Glia 63, 877–893. 10.1002/glia.22790 PubMed DOI

Pangrsic T., Potokar M., Stenovec M., Kreft M., Fabbretti E., Nistri A., et al. . (2007). Exocytotic release of ATP from cultured astrocytes. J. Biol. Chem. 282, 28749–28758. 10.1074/jbc.M700290200 PubMed DOI

Pascual O., Casper K. B., Kubera C., Zhang J., Revilla-Sanchez R., Sul J. Y., et al. . (2005). Astrocytic purinergic signaling coordinates synaptic networks. Science 310, 113–116. 10.1126/science.1116916 PubMed DOI

Pelegrin P., Surprenant A. (2006). Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J. 25, 5071–5082. 10.1038/sj.emboj.7601378 PubMed DOI PMC

Pellegatti P., Falzoni S., Pinton P., Rizzuto R., Di Virgilio F. (2005). A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion. Mol. Biol. Cell. 16, 3659–3665. 10.1091/mbc.E05-03-0222 PubMed DOI PMC

Pennartz C. M., de Jeu M. T., Bos N. P., Schaap J., Geurtsen A. M. (2002). Diurnal modulation of pacemaker potentials and calcium current in the mammalian circadian clock. Nature 416, 286–290. 10.1038/nature728 PubMed DOI

Reppert S. M. (1998). A clockwork explosion! Neuron 21, 1–4. PubMed

Schenk U., Westendorf A. M., Radaelli E., Casati A., Ferro M., Fumagalli M., et al. . (2008). Purinergic control of T cell activation by ATP released through pannexin-1 hemichannels. Sci. Signal. 1:ra6. 10.1126/scisignal.1160583 PubMed DOI

Schousboe A., Bak L. K., Waagepetersen H. S. (2013). Astrocytic control of biosynthesis and turnover of the neurotransmitters glutamate and GABA. Front. Endocrinol. 4:102. 10.3389/fendo.2013.00102 PubMed DOI PMC

Sperlágh B., Vizi E. S., Wirkner K., Illes P. (2006). P2X7 receptors in the nervous system. Prog. Neurobiol. 78, 327–346. 10.1016/j.pneurobio.2006.03.007 PubMed DOI

Stephan F. K., Zucker I. (1972). Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. Natl. Acad. Sci. U.S.A. 69, 1583–1586. 10.1073/pnas.69.6.1583 PubMed DOI PMC

Stout C. E., Costantin J. L., Naus C. C., Charles A. C. (2002). Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J. Biol. Chem. 277, 10482–10488. 10.1074/jbc.M109902200 PubMed DOI

Suadicani S. O., Iglesias R., Wang J., Dahl G., Spray D. C., Scemes E. (2012). ATP signaling is deficient in cultured Pannexin1-null mouse astrocytes. Glia 60, 1106–1116. 10.1002/glia.22338 PubMed DOI PMC

Surprenant A., Rassendren F., Kawashima E., North R. A., Buell G. (1996). The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272, 735–738. 10.1126/science.272.5262.735 PubMed DOI

Svobodova I., Vanecek J., Zemkova H. (2003). The bidirectional phase-shifting effects of melatonin on the arginine vasopressin secretion rhythm in rat suprachiasmatic nuclei in vitro. Brain Res. Mol. Brain Res. 116, 80–85. 10.1016/S0169-328X(03)00254-7 PubMed DOI

van den Pol A. N., Finkbeiner S. M., Cornell-Bell A. H. (1992). Calcium excitability and oscillations in suprachiasmatic nucleus neurons and glia in vitro. J. Neurosci. 12, 2648–2664. PubMed PMC

Vavra V., Bhattacharya A., Zemkova H. (2011). Facilitation of glutamate and GABA release by P2X receptor activation in supraoptic neurons from freshly isolated rat brain slices. Neuroscience 188, 1–12. 10.1016/j.neuroscience.2011.04.067 PubMed DOI

Verkhratsky A., Krishtal O. A., Burnstock G. (2009). Purinoceptors on neuroglia. Mol. Neurobiol. 39, 190–208. 10.1007/s12035-009-8063-2 PubMed DOI

von Kügelgen I., Wetter A. (2000). Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch. Pharmacol. 362, 310–323. 10.1007/s002100000310 PubMed DOI

Wang C. M., Chang Y. Y., Kuo J. S., Sun S. H. (2002). Activation of P2X(7) receptors induced [(3)H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl(-)/HCO(3)(-)-dependent mechanism. Glia 37, 8–18. 10.1002/glia.10004 PubMed DOI

Watanabe K., Koibuchi N., Ohtake H., Yamaoka S. (1993). Circadian rhythms of vasopressin release in primary cultures of rat suprachiasmatic nucleus. Brain Res. 624, 115–120. 10.1016/0006-8993(93)90067-W PubMed DOI

Welsh D. K., Reppert S. M. (1996). Gap junctions couple astrocytes but not neurons in dissociated cultures of rat suprachiasmatic nucleus. Brain Res. 706, 30–36. 10.1016/0006-8993(95)01172-2 PubMed DOI

White R. J., Reynolds I. J. (1997). Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones. J. Physiol. 498 (Pt 1), 31–47. 10.1113/jphysiol.1997.sp021839 PubMed DOI PMC

Womac A. D., Burkeen J. F., Neuendorff N., Earnest D. J., Zoran M. J. (2009). Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes. Eur. J. Neurosci. 30, 869–876. 10.1111/j.1460-9568.2009.06874.x PubMed DOI PMC

Yamazaki S., Ishida Y., Inouye S. (1994). Circadian rhythms of adenosine triphosphate contents in the suprachiasmatic nucleus, anterior hypothalamic area and caudate putamen of the rat–negative correlation with electrical activity. Brain Res. 664, 237–240. 10.1016/0006-8993(94)91978-X PubMed DOI

Yan E., Li B., Gu L., Hertz L., Peng L. (2013). Mechanisms for L-channel-mediated increase in [Ca(2+)]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels. Cell Calcium 54, 335–342. 10.1016/j.ceca.2013.08.002 PubMed DOI

Zhao H., Zhang X., Dai Z., Feng Y., Li Q., Zhang J. H., et al. . (2016). P2X7 receptor suppression preserves blood-brain barrier through inhibiting RhoA activation after experimental intracerebral hemorrhage in rats. Sci. Rep. 6:23286. 10.1038/srep23286 PubMed DOI PMC

Zimmermann H. (2000). Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs. Arch. Pharmacol. 362, 299–309. 10.1007/s002100000309 PubMed DOI

Dual PI3Kδ/γ Inhibitor Duvelisib Prevents Development of Neuropathic Pain in Model of Paclitaxel-Induced Peripheral Neuropathy

The Circadian Rhythms of STAT3 in the Rat Pineal Gland and Its Involvement in Arylalkylamine-N-Acetyltransferase Regulation

The day/night difference in the circadian clock's response to acute lipopolysaccharide and the rhythmic Stat3 expression in the rat suprachiasmatic nucleus