RESEARCH ARTICLE Astrocytes Inhibit Nitric Oxide-Dependent Ca 21 Dynamics in Activated Microglia: Involvement of ATP Released Via Pannexin 1 Channels Juan A. Orellana*, Trinidad D. Montero, and Rommy von Bernhardi* Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca 21 and produce high amounts of nitric oxide (NO). However, whether NO, Ca 21 dynamics, and gliotransmitter release are reciprocally modulated is not fully under- stood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO-dependent Ca 21 dynamics and ATP release in LPS-stimulated microglia. Gri- ess assays and Fura-2AM time-lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca 21 ] i that depended on the sequential activation of iNOS, COXs, and EP 1 receptor. TGFb1 released by astrocytes inhibited the abovementioned responses and also abolished LPS-induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS-stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS-stimulated microglia, exogenous ATP triggered activation of purinergic P2Y 1 receptors resulting in Ca 21 release from intracellular stores. Interestingly, TGFb1 released by astrocytes inhibited ATP- induced Ca 21 response in LPS-stimulated microglia to that observed in control microglia. Finally, COX/EP 1 receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS- stimulated microglia. Thus, Ca 21 dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies. GLIA 2013;00:000–000 Key words: neuroinflammation, calcium, glia Introduction A n increased body of evidence shows that the innate immune response exerts a dichotomous role in the cen- tral nervous system (CNS). Under physiological conditions, microglia exhibit a resting phenotype associated with produc- tion of anti-inflammatory and neurotrophic factors, whereas in response to a wide variety of insults they shift to an acti- vated phenotype (Block et al., 2007). In this state, rather than serving protective functions, microglia activation possibly becomes a detrimental process leading to further recruitment of other cells involved in the innate immune response (von Bernhardi, 2007) that promote disease progression, character- ized by synaptic dysfunction and even cell death as occur in chronic neurodegenerative disorders (Block et al., 2007; Ram- irez et al., 2008). Under these conditions, microglia exhibit an elevated intracellular Ca 21 concentration ([Ca 21 ] i ) and increased production of nitric oxide (NO) and prostaglandins (Farber and Kettenmann, 2006; Saha and Pahan, 2006), processes associated with neuronal damage in neuroinflamma- tory conditions (Anrather et al., 2011; Ha et al., 2008). On the contrary, most of evidence indicate that astrocytes, the largest glial cell population of the brain, could support View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22573 Published online Month 0, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Oct 18, 2012, Accepted for publication Aug 19, 2013. Address correspondence to Juan A. Orellana, Departamento de Neurolog  ıa, Escuela de Medicina, Pontificia Universidad Cat  olica de Chile, Marcoleta 391, Santiago, Chile. E-mail: jaorella@uc.cl or Rommy von Bernhardi, Departamento de Neurolog  ıa, Escuela de Medicina, Pontificia Universidad Cat  olica de Chile, Marcoleta 391, Santiago, Chile. E-mail: rvonb@med.puc.cl From the Departamento de Neurolog  ıa; Escuela de Medicina, Pontificia Universidad Cat  olica de Chile, Santiago, Chile. Additional Supporting Information may be found in the online version of this article. V C 2013 Wiley Periodicals, Inc. 1