RESEARCH ARTICLE P2X4 Receptors Control the Fate and Survival of Activated Microglia Nuria V azquez-Villoldo, 1 Mar  ıa Domercq, 1 Abraham Mart  ın, 2 Jordi Llop, 3 Vanessa G omez-Vallejo, 3 and Carlos Matute 1 Microglia, the resident immune cells of the central nervous system, responds to brain disarrangements by becoming activated to contend with brain damage. Here we show that the expression of P2X4 receptors is upregulated in inflammatory foci and in activated microglia in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) as well as in the optic nerve of multiple sclerosis patients. To study the role of P2X4 receptors in microgliosis, we activated microglia with LPS in vitro and in vivo. We observed that P2X4 receptor activity in vitro was increased in LPS-activated microglia as assessed by patch-clamp recordings. In addition, P2X4 receptor blockade significantly reduced microglial membrane ruffling, TNFa secre- tion and morphological changes, as well as LPS-induced microglial cell death. Accordingly, neuroinflammation provoked by LPS injection in vivo induced a rapid microglial loss in the spinal cord that was totally prevented or potentiated by P2X4 receptor blockade or facilitation, respectively. Within the brain, microglia in the hippocampal dentate gyrus showed particular vulnerability to LPS-induced neuroinflammation. Thus, microglia processes in this region retracted as early as 2 h after injec- tion of LPS and died around 24 h later, two features which were prevented by blocking P2X4 receptors. Together, these data suggest that P2X4 receptors contribute to controlling the fate of activated microglia and its survival. GLIA 2014;62:171–184 Key words: microglia, P2X4 purinergic receptor, EAE and multiple sclerosis, cell death Introduction M icroglial cells are the main effectors of the innate immune response after CNS injury (Kreutzberg, 1996). The precursors of adult microglia are yolk sac progenitors, which enter the embryo around E8 and colonize the CNS at E10 (Ginhoux et al., 2010). Microglia can be found postna- tally in all regions of the CNS, in the parenchyma and in perivascular spaces in a nonoverlapping territorial fashion, and comprise a large proportion of the total cellular makeup of the CNS, estimated to be as high as 12% of all cells. Per- haps owing to this origin, microglia share many commonal- ties with resident tissue macrophages in peripheral organ systems (Ransohoff and Perry, 2009). Like their peripherals macrophage counterparts, microglia displays a remarkable range in both morphology and activity, depending in part on the state of the surrounding tissue (Ransohoff and Perry, 2009). In their resting state, microglia continuously monitor the tissue for injury or pathological changes by extending and retracting highly motile processes on a time scale of minutes (Davalos et al., 2005; Nimmerjahn et al., 2005; reviewed in Kettenmann et al., 2013). Exogenous factors (infectious agents) as well as endogenous factors or neurotransmitters released by damaged cells or tissues induce progressive changes in microglia including altered expression of cell sur- face markers and inflammation related genes, process retrac- tion and acquisition of an ameboid morphology, migration of cell bodies, proliferation and increased phagocytic rate (Ket- tenmann et al., 2011). The rapidly diffusible neurostransmitter ATP is one of the main regulators of microglial functions. Low ATP concen- trations almost exclusively activate chemotaxis, through P2Y12 receptor activation, in order to recruit cells at the site View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22596 Published online November 19, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received May 23, 2013, Accepted for publication Oct 16, 2013. Address correspondence to Carlos Matute, Departamento de Neurociencias, Universidad del Pa  ıs Vasco, E-48940 Leioa, Spain. E-mail: carlos.matute@ehu.es or Maria Domercq, Departamento de Neurociencias, Universidad del Pa  ıs Vasco, E-48940 Leioa, Spain. E-mail: maria.domercq@ehu.es From the 1 Departamento de Neurociencias, Universidad del Pa  ıs Vasco-UPV/EHU, Barrio Sarriena s/n, 48940, Leioa, Spain, Achucarro Basque Center for Neuro- science-UPV/EHU, 48170, Zamudio, Spain, Instituto de Salud Carlos III, Centro de Investigaci  on Biom edica en Red de Enfermedades Neurodegenerativas (CIBERNED), 48940, Leioa, Spain; 2 Molecular Imaging Unit, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebasti an, Spain; 3 Radiochemistry Department, Molecular Imaging Unit, CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebasti an, Spain. Additional Supporting Information may be found in the online version of this article. V C 2013 Wiley Periodicals, Inc. 171