purinergic p2x7 receptors mediate cell death in mouse cerebellar astrocytes in culture

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1521-0103/347/3/802815$25.00 http://dx.doi.org/10.1124/jpet.113.209452 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 347:802815, December 2013 Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics Purinergic P2X7 Receptors Mediate Cell Death in Mouse Cerebellar Astrocytes in Culture Elvira Salas, Luz María G. Carrasquero, Luis A. Olivos-Oré, Diego Bustillo, Antonio R. Artalejo, Maria Teresa Miras-Portugal, and Esmerilda G. Delicado Department of Biochemistry, Faculty of Medicine, University of Costa Rica, San José, Costa Rica (E.S.); Departments of Biochemistry (L.M.G.C., M.T.M.-P., E.G.D.) and Toxicology and Pharmacology (L.A.O., D.B., A.R.A.), Faculty of Veterinary Medicine, Complutense University of Madrid, Madrid, Spain; and Neurochemistry Research Institute, Complutense University of Madrid, Madrid, Spain (L.A.O., D.B., A.R.A., M.T.M.P., E.G.D.) Received September 6, 2013; accepted October 7, 2013 ABSTRACT The brain distribution and functional role of glial P2X7 receptors are broader and more complex than initially anticipated. We characterized P2X7 receptors from cerebellar astrocytes at the molecular, immunocytochemical, biophysical, and cell physio- logic levels. Mouse cerebellar astrocytes in culture express mRNA coding for P2X7 receptors, which is translated into P2X7 receptor protein as proven by Western blot analysis and im- munocytochemistry. Fura-2 imaging showed cytosolic calcium responses to ATP and the synthetic analog 39-O-(4-benzoyl)benzoyl- ATP (BzATP) exhibited two components, namely an initial tran- sient and metabotropic component followed by a sustained one that depended on extracellular calcium. This latter component, which was absent in astrocytes from P2X7 receptor knockout mice (P2X7 KO), was modulated by extracellular Mg 21 , and was sensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)- 1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP also elicited inwardly directed nondesensitizing whole-cell ionic currents that were reduced by extracellular Mg 21 and P2X7 antagonists (BBG and calmidazolium). In contrast to that previously reported in rat cerebellar astrocytes, sustained BzATP application induced a gradual increase in membrane permeability to large cations, such as N-methyl-D-glucamine and 4-[3-methyl- 2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]di- iodide, which ultimately led to the death of mouse astrocytes. Cerebellar astrocyte cell death was prevented by BBG but not by calmidazolium, removal of extracellular calcium, or treatment with the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)- Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotic- type mechanism of cell death. Since this cellular response was not observed in astrocytes from P2X7 KO mice, this study suggests that stimulation of P2X7 receptor may convey a cell death signal to cerebellar astrocytes in a species-specific manner. Introduction The P2X7 receptor is a piece of the complex puzzle of nu- cleotide signaling, which involves various membrane recep- tors, ectoenzymes, and membrane transporters (Volonté and DAmbrosi, 2009). Among P2X nucleotide receptors, the ATP- gated cation channels, P2X7 stands out for its peculiar char- acteristics (North, 2002). This receptor exhibits low affinity for ATP, requiring at least a 100-fold higher nucleotide con- centration for activation than the other P2X receptors, and is potentiated in divalent cation-free solutions. In addition, repeated or sustained P2X7 receptor stimulation can be as- sociated with a progressive increase in plasma membrane permeability to large molecules (up to 900 Da). Structurally, P2X7 is distinguished from other members of the ionotropic family by a long intracellular C-terminal tail containing multiple protein and lipid interaction motifs (Costa-Junior et al., 2011). The first evidence suggesting the presence of P2X7 receptors in the nervous system came from the work of Deuchars et al. (2001), who observed that 39-O-(4-benzoyl)ben- zoyl-ATP (BzATP), a P2X7 receptor agonist, depolarized the membrane and increased the firing rate of spinal cord glutamatergic neurons; hence, the authors proposed that P2X7 receptors could promote transmitter release from nerve terminals. The presence of presynaptic P2X7 receptors was corroborated by many studies carried out in synaptosomes, purified neuronal cultures, and tissue slices (Sperlágh et al., 2002; Miras-Portugal et al., 2003). In addition to neurons, functional P2X7 receptors were also identified in glial cells, including astrocytes from several brain areas (Kukley et al., 2001; Panenka et al., 2001; Duan et al., 2003), Müller and This research was supported by the Spanish Ministry of Economy and Competitiveness [Grants BFU2011-26253 and BFU2011-24743] and the Spanish Ion Channel Initiative [Grant CSD2008-00005]. E.S. was the recipient of a research fellowship at the University of Costa Rica and the Fundación Carolina. dx.doi.org/10.1124/jpet.113.209452. ABBREVIATIONS: A438079, 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine; a,b-meATP, a,b-methylene-ATP; BBG, Brilliant Blue G; BzATP, 39-O-(4-benzoyl)benzoyl-ATP; BSA, bovine serum albumin; GFAP, glial fibrillary acidic protein; KO, knockout; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; NMDG, N-methyl-D-glucamine; PBS, phosphate-buffered saline; PPADS, pyridoxalphosphate-6-azophenyl- 29,49-disulfonic acid; RT-PCR, real-time polymerase chain reaction; WT, wild-type; Yo-Pro-1, 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3- (triethylammonio)propyl]diiodide; Z-DEVD-FMK, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone. 802 at Harvard University, Museum of Comparative Zoology on November 28, 2013 jpet.aspetjournals.org Downloaded from

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1521-0103/347/3/802–815$25.00 http://dx.doi.org/10.1124/jpet.113.209452THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 347:802–815, December 2013Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

Purinergic P2X7 Receptors Mediate Cell Death in MouseCerebellar Astrocytes in Culture

Elvira Salas, Luz María G. Carrasquero, Luis A. Olivos-Oré, Diego Bustillo,Antonio R. Artalejo, Maria Teresa Miras-Portugal, and Esmerilda G. DelicadoDepartment of Biochemistry, Faculty of Medicine, University of Costa Rica, San José, Costa Rica (E.S.); Departments ofBiochemistry (L.M.G.C., M.T.M.-P., E.G.D.) and Toxicology and Pharmacology (L.A.O., D.B., A.R.A.), Faculty of VeterinaryMedicine, Complutense University of Madrid, Madrid, Spain; and Neurochemistry Research Institute, Complutense University ofMadrid, Madrid, Spain (L.A.O., D.B., A.R.A., M.T.M.P., E.G.D.)

Received September 6, 2013; accepted October 7, 2013

ABSTRACTThe brain distribution and functional role of glial P2X7 receptorsare broader and more complex than initially anticipated. Wecharacterized P2X7 receptors from cerebellar astrocytes at themolecular, immunocytochemical, biophysical, and cell physio-logic levels. Mouse cerebellar astrocytes in culture expressmRNA coding for P2X7 receptors, which is translated into P2X7receptor protein as proven by Western blot analysis and im-munocytochemistry. Fura-2 imaging showed cytosolic calciumresponses to ATP and the synthetic analog 39-O-(4-benzoyl)benzoyl-ATP (BzATP) exhibited two components, namely an initial tran-sient and metabotropic component followed by a sustained onethat depended on extracellular calcium. This latter component,which was absent in astrocytes from P2X7 receptor knockoutmice (P2X7 KO), was modulated by extracellular Mg21, and wassensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP

also elicited inwardly directed nondesensitizing whole-cell ioniccurrents that were reduced by extracellular Mg21 and P2X7antagonists (BBG and calmidazolium). In contrast to thatpreviously reported in rat cerebellar astrocytes, sustained BzATPapplication induced a gradual increase in membrane permeabilityto large cations, such as N-methyl-D-glucamine and 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]di-iodide, which ultimately led to the death of mouse astrocytes.Cerebellar astrocyte cell death was prevented by BBG but not bycalmidazolium, removal of extracellular calcium, or treatment withthe caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotic-type mechanism of cell death. Since this cellular response wasnot observed in astrocytes from P2X7 KO mice, this studysuggests that stimulation of P2X7 receptor may convey a celldeath signal to cerebellar astrocytes in a species-specific manner.

IntroductionThe P2X7 receptor is a piece of the complex puzzle of nu-

cleotide signaling, which involves various membrane recep-tors, ectoenzymes, and membrane transporters (Volonté andD’Ambrosi, 2009). Among P2X nucleotide receptors, the ATP-gated cation channels, P2X7 stands out for its peculiar char-acteristics (North, 2002). This receptor exhibits low affinityfor ATP, requiring at least a 100-fold higher nucleotide con-centration for activation than the other P2X receptors, andis potentiated in divalent cation-free solutions. In addition,repeated or sustained P2X7 receptor stimulation can be as-sociated with a progressive increase in plasma membrane

permeability to large molecules (up to 900 Da). Structurally,P2X7 is distinguished from other members of the ionotropicfamily by a long intracellular C-terminal tail containingmultiple protein and lipid interaction motifs (Costa-Junioret al., 2011). The first evidence suggesting the presence ofP2X7 receptors in the nervous system came from the work ofDeuchars et al. (2001), who observed that 39-O-(4-benzoyl)ben-zoyl-ATP (BzATP), a P2X7 receptor agonist, depolarized themembrane and increased the firing rate of spinal cordglutamatergic neurons; hence, the authors proposed thatP2X7 receptors could promote transmitter release from nerveterminals. The presence of presynaptic P2X7 receptors wascorroborated by many studies carried out in synaptosomes,purified neuronal cultures, and tissue slices (Sperlágh et al.,2002; Miras-Portugal et al., 2003). In addition to neurons,functional P2X7 receptors were also identified in glial cells,including astrocytes from several brain areas (Kukley et al.,2001; Panenka et al., 2001; Duan et al., 2003), Müller and

This research was supported by the Spanish Ministry of Economy andCompetitiveness [Grants BFU2011-26253 and BFU2011-24743] and theSpanish Ion Channel Initiative [Grant CSD2008-00005]. E.S. was the recipientof a research fellowship at the University of Costa Rica and the FundaciónCarolina.

dx.doi.org/10.1124/jpet.113.209452.

ABBREVIATIONS: A438079, 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine; a,b-meATP, a,b-methylene-ATP; BBG, Brilliant Blue G;BzATP, 39-O-(4-benzoyl)benzoyl-ATP; BSA, bovine serum albumin; GFAP, glial fibrillary acidic protein; KO, knockout; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMDG, N-methyl-D-glucamine; PBS, phosphate-buffered saline; PPADS, pyridoxalphosphate-6-azophenyl-29,49-disulfonic acid; RT-PCR, real-time polymerase chain reaction; WT, wild-type; Yo-Pro-1, 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]diiodide; Z-DEVD-FMK, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone.

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Bergmann glia (Pannicke et al., 2000), oligodendrocytes(James and Butt, 2001), and microglia (Hide et al., 2000). Inastroglia, P2X7 receptors have also been related to the releaseof gliotransmitters such as glutamate and ATP, as well as tothe propagation of intercellular calcium waves (Suadicaniet al., 2006; Bennett et al., 2009). P2X7 expression appears tobe increased in brain injury related to ischemia and to inflam-mation, as well as in neurodegenerative disorders such asAlzheimer’s disease (Parvathenani et al., 2003), Huntington’sdisease (Díaz-Hernández et al., 2009), multiple sclerosis(Matute, 2011), and depression (Lucae et al., 2006). In contrastto their accepted contribution to neurodegeneration, neuro-protective actions of P2X7 receptors have also been reported(Skaper et al., 2010). In rat cerebellar granule neurons, P2X7receptors activate a survival pathway alternative to that ofthe classic neurotrophic factors, depending on protein kinaseC activation but converging on glycogen synthase kinase-3inhibition to promote neuronal survival (Ortega et al., 2010).P2X7 receptors could also be involved in neuronal develop-ment. Both the P2X7 receptor and the ectoenzyme tissue-nonspecific alkaline phosphatase are expressed in mousehippocampal neurons at early stages of differentiation, therebycontrolling, in a coordinated way, the elongation of the growthcone (Díez-Zaera et al., 2011).From the above-described, a question arises regarding

whether the plethora of P2X7 receptor–mediated actions in-volves a single type of receptor or whether several types areneeded (Sánchez-Nogueiro et al., 2005). We decided to gaininsight into this issue by investigating differences in P2X7receptor signaling between cerebellar astrocytes from tworodent species, namely the rat and the mouse. We previouslydemonstrated that rat cerebellar astrocytes express P2X7receptors coupled to both ionotropic and metabotropic sig-naling mechanisms (Carrasquero et al., 2009, 2010). More-over, treatment with BzATP for 30 minutes induced complexchanges in cell morphology but no signs of cell death. Here, wecarried out the characterization of P2X7 receptors in cere-bellar astrocytes from mice. Our results show that the stimu-lation of mouse astrocytes with millimolar ATP or micromolarBzATP concentrations elicits fast cytosolic calcium responsesand plasma membrane ion currents mediated by functionalP2X7 receptors. Sustained P2X7 receptor stimulation leads toa progressive increase in plasma membrane permeability toN-methyl-D-glucamine (NMDG1) and to an enhanced uptakeof 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethyl-ammonio)propyl]diiodide (Yo-Pro-1), which ultimately com-promised the cell viability. Interestingly, nucleotide-inducedcell death could not be elicited in astrocytes from P2X7receptor–deficient mice. These results indicate that P2X7receptor orthologs mediate a fundamentally opposed effectat the cell level (morphologic changes or cell death), thuspointing to differences in the signaling mechanisms distal toion channel opening.

Materials and MethodsReagents. Chemicals and reagents were purchased from Sigma

Chemicals (Alcobendas, Spain), Gibco (Invitrogen Barcelona, Spain),Molecular Probes (Invitrogen, Barcelona, Spain), and Merck (Madrid,Spain) unless otherwise noted.

Experimental Animals. All experiments were carried out at theComplutense University of Madrid (Madrid, Spain) following the

International Council for Laboratory Animal Science guidelines. Theassays were designed to minimize the number of mice whilemaintaining statistical validity.

Littermates (male and female) of wild-type C57B1/6J (WT) andP2X7 receptor knockout (P2X7 KO) mice were used as the source ofprimary astrocyte cultures. P2X7 KO mice were obtained from Pfizer(Groton, CT) as generated by Solle et al. (2001). P2X7 KO mice havea deletion in the region from T1527 to T1607, located at exon 13,resulting in the disruption of the C terminus of the P2X7 receptor.These mice were fertile.

Primary Cell Cultures. Primary astrocyte cultures were pre-pared from cerebella of WT and P2X7 KO mice aged 4–5 days. Six toeight cerebella were immersed in isolation buffer [140 mM NaCl, 4mM KCl, 1.2 mM Na2HPO4, 1.5 mM MgSO4, 15 mM glucose, 10 mMHEPES, and 50 mM bovine serum albumin (BSA), pH 7.4, containing50 U/ml penicillin and 50 mg/ml streptomycin] and cleaned of othertissues. After mechanical disaggregation, cerebellar tissue wasdigested in a mixture of trypsin (0.17 mg/ml) and isolation bufferover 10 minutes at 37°C, at which time the reaction was stopped with5 ml of isolation buffer containing 0.25 mg/ml soybean trypsininhibitor and 10 U/ml DNase I. Cells then were dispersed by gentlemechanical pipetting. Astrocytes were purified as previously de-scribed by Jiménez et al. (1999). In brief, cultures were depleted ofmicroglial cells by 16 hours of orbital shaking, and cerebellar cellswere plated at a density of 105 cells/cm2 in Dulbecco’s modified Eagle’smedium containing 10% (v/v) fetal bovine serum, 30 mM glucose,50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml kanamycin, and2.5 mg/ml amphotericin. Cells were maintained in culture at 37°C ina humidified atmosphere of 5% CO2/95% air until reaching confluence(approximately 10 days). The medium was replaced every 3 days. Thepurity of the cultures was determined by morphologic examinationunder phase-contrast microscopy and by selective staining of glialfibrillary acidic protein (GFAP) performed by immunocytochemistry.Cells were harvested by treating the culture flask with 0.03% trypsinand 0.0013% EDTA in phosphate-buffered saline (PBS). For cytosoliccalcium imaging, electrophysiological recordings, and immunocyto-chemical experiments, cells were plated onto 15-mm diametercoverslips precoated with poly(L-lysine) (Biochrom AG, Berlin,Germany) in 35-mm Petri dishes at a density of 104 cells/cm2. Forcell viability assays, cells were plated on 24-multiwell plates ata density of 9 � 104/well, whereas for real-time polymerase chainreaction (RT-PCR) and Western blot studies, cells were plated onto10-cm diameter Petri dishes at a density of 3 � 106 cells/plate. Withthe exception of calcium imaging and electrophysiological and immuno-cytochemical experiments, cells were routinely used 48 hours afterplating.

Immunocytochemistry. For immunofluorescence assays, cellsattached to coverslips were fixed with cold 4% (w/v) paraformaldehydefor 4 minutes, washed three times with PBS, and subsequentlyincubated at 37°C for 1 hour in blocking solution [PBS containing 3%(w/v) BSA, 0.1% (v/v) Triton X-100, and 5% (v/v) normal goat serum].Rabbit antibody against the C terminus of the P2X7 receptor (1:100dilution; Alomone Labs, Jerusalem, Israel) andmouse anti-GFAP IgG(1:200 dilution; Sigma Chemicals) were used as the primary anti-bodies. After overnight incubation at 4°C with these antibodies andthree washes in blocking solution, cells were exposed for 1 hour at37°C to anti-mouse fluorescein isothiocyanate–conjugated secondaryantibody (1:200 dilution; Sigma Chemicals) and anti-rabbit-Cy3-conjugated secondary antibody (1:400; The Jackson Laboratory, BarHarbor, ME), and washed again three times. Cell nuclei were revealedwith 49,6-diamidino-2-fenilindol (1 mM, 5 minutes). Coverslips,mounted according to standard procedures with Prolong antifademedium (Invitrogen, Barcelona, Spain), were viewed with a TE-200microscope (Nikon, Melville, NY) equipped with a Fluor-S �60/1.4 oilobjective and fluorescein, rhodamine, and 49,6-diamidino-2-fenilindolfilters. Images were obtained using a Kappa ACC1 camera controlledby Kappa image base control software from Kappa Optronics GmbH(Gleichen, Germany).

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 803

Quantitative RT-PCR. Quantitative RT-PCR experiments wereconducted as previously described (Gómez-Villafuertes et al., 2009).Total RNA was extracted from mouse cerebellar astrocyte culturesusing an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) followingthe manufacturer’s instructions. After digestion with DNase, totalRNA was quantified and reversed transcribed using M-MLV reversetranscriptase, 6 mg of random primers, and 350 mM dNTPs (all fromInvitrogen). Quantitative real-time PCR was performed using gene-specific primers and TaqMan MGB probes for mouse P2X1–5 andP2X7 receptors, as well as b-actin (all from Applied Biosystems,Foster City, CA). For the P2X6 receptor, the primers were describedpreviously and the probe design was FAM-59-CTTCCGTTCCTCTG-GC-39-MGB. Fast thermal cycling was performed using a StepOnePlusReal-Time PCR System (Applied Biosystems) as follows: denaturationfor one cycle of 95°C for 20 seconds, followed by 40 cycles each of 95°Cfor 1 second and 60°C for 20 seconds. The results were normalized byparallel amplification of the endogenous control b-actin.

Western Blotting. Western blot experiments were performed onlysates of cultured cerebellar astrocytes. Cells were washed withnormal Locke’s solution (140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2,1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM glucose, and 10 mMHEPES, pH 7.4) for 1 hour at 37°C and were subsequently scrapedfrom the dishes in 500 ml of load sample buffer containing 12.5% (v/v)glycerol, 30 mM (hydroxymethyl)aminomethane (Tris), 1% (w/v) SDS,0.02% (w/v) bromphenol blue, and 5% (v/v) b-mercaptoethanol(pH 6.8) for a total protein concentration of approximately 1 mg/ml.The resulting cell extracts were denatured at 99°C over 4minutes andprocessed (aliquots of 30 mg of protein) by SDS gel electrophoresis on10% acrylamide gels in electrophoresis buffer (25 mM Tris, 200 mMglycine, and 0.1% SDS, pH 8.3) at 4°C. The resolved proteins werethen transferred to polyvinylidene fluoride membranes (AmershamBiosciences, Piscataway, NJ) at 240 mA for 80 minutes at 4°C intransfer buffer containing 25 mM Tris, 192 mM glycine, and 20%methanol, pH 8.3. Membranes were incubated for 1 hour at roomtemperature with 1% Tween 20 in Tris buffer solution (100 mM NaCland 10 mM Tris, pH 7.5) plus 5% nonfat dry milk. Blots were in-cubated overnight at 4°C with primary antibodies against the C ter-minus of the P2X7 receptor (1:100 dilution; Alomone Labs) orglyceraldehyde 3-phosphate dehydrogenase (1:1000 dilution; Ambion,Austin, TX). Secondary anti-mouse (Santa Cruz Biotechnology, SantaCruz, CA) or anti-rabbit (Dako, Carpinteria, CA) horseradishperoxidase–conjugated antibodies were used at a 1:3000 dilution.Signals were visualized with the Super Signal Substrate (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’srecommendations and quantified by a Fluo-S Imager (Bio-Rad,Hercules, CA). To validate the suitability of the P2X7 receptor antibody,similar experiments were carried out on total lysates obtained frommouse peritoneal macrophage cultures, prepared as described byVelasco et al. (1997).

Cytosolic Calcium Imaging. Cytosolic calcium imaging experi-ments were conducted basically as previously described (Carrasqueroet al., 2005). Astrocytes from WT and P2X7 KO mice attached tocoverslips were incubated in Locke’s solution (see “Western Blotting”)supplemented with 1 mg/ml BSA and 5–7 mM fura-2/AM (Invitrogen)for 45 minutes at 37°C. Once washed in fresh Locke’s solution,coverslips were placed in a superfusion chamber and the cells wereimaged through a Nikon TE-200 microscope with a Plan Fluor �20/0.5 objective. The incoming light was alternately set at 340 and 380nm. Exposure time was 100 ms and the change in the wavelengthfrom 340 to 380 nm was carried out in less than 5 ms. Images wereobtained using an ORCA-ER C 47 42-80 camera from Hamamatsu(Hamamatsu City, Japan) controlled by MetaFluor 6.2r and PCsoftware (Universal Imaging Corp., Cambridge, UK). Fluorescencedata were acquired at a frequency of 2 Hz as the average signal froman elliptical region within each cell. For each wavelength, thebackground signal was subtracted and the ratio F340/F380 calculated.

Specific reagents were dissolved in Locke’s solution and applied tothe cells by superfusion at 37°C. Cells were challenged with pulses of

variable duration (30–120 seconds) of either ATP or BzATP. Theincrease in F340/F380 over basal values (DF340/F380) was used to definethe amplitude of the nucleotide-induced cytosolic calcium responses.Concentration-response curves were built by plotting the DF340/F380

values against the log concentration of the nucleotide. Curve fittingwas done by nonlinear regression with Origin-Pro 8 software (Origin-Lab Corporation, Northampton,MA). The effect of several P2X receptorantagonists and modulators was also assayed. Astrocytes were pre-treated with 10mMBrilliant Blue G (BBG) or 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (Donnelly-Roberts and Jarvis, 2007),two P2X7 selective antagonists, or with 30 mM pyridoxalphosphate-6-azophenyl-29,49-disulfonic acid (PPADS), a nonselective P2 antagonist(Bianchi et al., 1999; Jiang et al., 2000) for 3 minutes, and thensuperfused with 3 mM ATP in the presence of the antagonist. Calciumresponses were also evaluated after the addition of 3 mMMgSO4 to thenormal Locke’s solution (final concentration of 4.2 mM) and in theabsence of either extracellular magnesium or calcium (by the additionof 6 mM Tris/6 mM EGTA to achieve a free calcium concentration of~200 nM).

Electrophysiological Recordings. Patch-clamp experimentswere performed as described previously (Carrasquero et al., 2009).Cells seeded at low density (104 cells/cm2) were used for patch-clamprecording so that avoiding space clamp problems attributable to gapjunctions could be avoided. Electrophysiological recordings werecarried out with an EPC9 patch-clamp amplifier using Pulse/PatchMaster software (HEKA Electronic, Lambrecht, Germany).Pipettes were pulled from Kimax borosilicate glass (Witz Scientific,Holland, OH) and subsequently wax-coated and fire-polished toobtain a final resistance of 2–3 MV when filled with standardsolutions. The extracellular (bath) solution contained the following:140 mM NaCl, 1 mM CaCl2, 10 mM HEPES, and 10 glucose (pH 7.2,adjusted with NaOH; approximately 300 mOsm). Recording pipetteswere filled with a solution containing the following: 140 mMNMDG1,5 mM EGTA, and 10 mM HEPES (pH 7.2, adjusted with HCl;approximately 290 mOsm). Cells attached to glass coverslips weretransferred to a recording chamber placed in the stage of an invertedZeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany)and continuously superfused with bath solution (perfusion rate of1 ml/min).

Membrane currents were measured in the whole-cell configurationof the patch-clamp technique, filtered at 3 kHz, and sampled at 10KHz.Once established electrical access to the cytoplasm, cells were heldat a voltage (Vh) of 270 mV. Series resistance (5–10 MV) was com-pensated by 80% and monitored throughout the experiment to-gether with the cell membrane capacitance. Considering that NMDG1

has a Mr of 195.21 Da, a series resistance of 10 MV, and an astrocyte’smembrane capacitance of 17.66 1.42 pF (n5 51 cells), we estimated inapproximately 10 minutes the time required by NMDG1 to equilibratebetween the patch pipette and the cytoplasm of the recorded cell (Puschand Neher, 1988). Because of this, drug application started at least 10minutes after obtaining the whole-cell configuration, and experimentsin which series resistance changed by more than 20% or holding cur-rent exceeded 20 pAwere not analyzed. All recordings were obtained atroom temperature (21–25°C).

Ligand-gated currents were activated by P2X receptor agonistsapplied onto the cell under investigation by means of a puffer glass-pipette (3–5 mm tip diameter, MPCU; List Electronics, Darmstadt,Germany) or a gravity-driven perfusion system with five independentlines controlled by electronic valves (The Lee Company, Westbrook,CO). Stock solutions of drugs were diluted daily in extracellular salineand incorporated into the perfusion system a few minutes beforestarting the experiments.

Time-dependent changes in the relative permeability of NMDG1

with respect to Na1 (PNMDG/PNa) of P2X7 receptors were computedfrom changes in the reversal potential (Vrev) of BzATP-inducedcurrents, assuming ion activities of 0.75 for both cations (Nörenberget al., 2011), by a transform of the Goldman-Hodgkin-Katz equationfor bi-ionic conditions. Vrev values from individual cells were corrected

804 Salas et al.

for the liquid junction potential between the pipette’s solution and theextracellular solution, which was calculated to be 26.37 mV by usingthe Patcher’s Tools module included in Igor-Pro software (Wave-Metrics, Inc., Lake Oswego, OR).

Yo-Pro-1 Uptake. Measurements of Yo-Pro-1 uptake were per-formed in astrocyte cell populations. Astrocytes were collected bytrypsinization from confluent cultures, washed, and resuspended inMg21-free Locke’s solution. Measurements were made in a Perkin-Elmer LS-50B fluorometer from 1.5-ml samples containing approx-imately 300,000 cells and 1 mM Yo-Pro-1 iodide (Molecular Probes) instirred cuvettes at 37°C. Fluorescence was excited with 490-nm lightand detected through a 510-nm centered slit. BzATP was added to thecuvettes from at least 100-fold concentrated stock solutions to avoidlarge volume variations. When the effect of P2X7 receptor antagonistswas tested, cells were preincubated with the antagonist for 3 minutesbefore the addition of BzATP.

Cell Viability Assays. Cell viability was evaluated with theLIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells accordingto the manufacturer’s instructions. The kit combines two fluorescentprobes, calcein AM, and ethidium homodimer-1, allowing the simul-taneous determination of live and dead cells. Following cleavage byintracellular esterases, the polyanionic dye calcein is retained withinlive cells, producing an intense uniform green fluorescence, whereasethidium homodimer-1 enters cells with damaged membranes andupon binding to nucleic acids produces a bright red fluorescence.Confluent WT or P2X7 KO cerebellar astrocytes, seeded in 24-wellplates, were washed for 15 minutes with 1 ml normal Locke’s solutionbefore being incubated with 3 mM ATP or 500 mM BzATP for 30minutes at 37°C. At the end of the stimulation period, the incubationmedium was replaced with fresh Locke’s solution, and the labeledcells were viewed under a fluorescence microscope equipped with aconventional fluorescein long-pass filter. All experiments were donein duplicate and repeated three times.

Cell viability was also determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which assessesmitochondrial function. Confluent astrocytes were first washed for 15minutes with 1 ml of normal Locke’s solution warmed at 37°C. Cellswere then treated with 5 mM ATP or 500 mM BzATP for 60 minutes.Nonstimulated cells were used as controls. After drug treatment,incubation medium was removed and the tetrasodium salt MTT(Sigma Chemicals) was added to a final concentration of 0.5 mg/ml in

Locke’s solution and maintained for 2 hours at 37°C. An equal volumeof MTT solubilization solution (10% Triton X-100 plus 0.1 N HCl inanhydrous isopropanol) was then added, and the mix was kept for 30minutes at room temperature with orbital shaking. Cellular extractswere collected and measured spectrophotometrically at 570 nm. Insomewells, the effects of P2X7 antagonists (BBG or calmidazolium) orthe caspase 3 inhibitor II benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), administered 3 minutesbefore and during nucleotide incubation, were also evaluated. Valueswere normalized with respect to those obtained in nonstimulatedcells.

Statistical Analysis. Results are expressed as means 6 S.E.M.calculated from at least three experiments usually performed on cellsfrom different cultures. Statistical differences between two groups ofdata were assessed by the t test, and a P value, 0.05 was taken as thelimit of significance. When multiple comparisons were made, one-wayanalysis of variance was used and Dunnett’s post test analysis wasapplied only when a significant (P, 0.05) effect was indicated by one-way analysis of variance (GraphPad Prism 5; GraphPad SoftwareInc., San Diego, CA).

ResultsCharacterization of Cerebellar Astrocyte Cultures

from Neonatal Mice. Our study began by determining theage of mice rendering optimal cerebellar astrocyte cultures.Cerebellar cells obtained from P6/P7 mice grew slowly andtended to form dense multilayered aggregates. The confluentstate was reached between 15 and 20 days in vitro, when mostcells showed a dark-gray appearance and marked cytoplasmicgranularity. In addition, P7 cells displayed small and in-consistent cytosolic calcium responses to purinergic agonists.These drawbacks were solved by using younger pups (P4–P5)and by plating the cells at higher density (105 cells/cm2).Because tissues from younger animals were more delicate,it was also necessary to use a low trypsin concentration(0.17 mg/ml) to avoid cell damage. Astrocyte cultures fromP4 pups were found to have a good morphologic appearanceand exhibited prominent cytosolic calcium responses to

Fig. 1. Mouse cerebellar astrocytes in culture ex-press P2X7 receptors. (A) Immunocytochemicalidentification of astrocytes from mouse cerebellacultures. Immunofluorescence image showing anti-GFAP-FITC staining (green) in confluent cultures ofcerebellar cells. Cell nuclei were revealed with DAPI(blue). (B) Relative expression of mRNA encodingP2X subunits in mouse cerebellar astrocytes. Ex-pression of P2X subunits was normalized withrespect to the b-actin transcript, as determined byquantitative RT-PCR. (C and D) Detection of P2X7subunits in mouse cerebellar astrocytes. Purifiedcultures of cerebellar astrocytes, positive to GFAPstaining, were used to detect P2X7 receptors byboth immunocytochemistry and immunoblotting. (C)Immunofluorescence image of a mouse cerebellarastrocyte stained with anti-GFAP-FITC (green) andanti-P2X7 receptor-Cy3 (red) antibodies. (D) Repre-sentative Western blot of the P2X7 protein. Lysates(30 mg of protein) of WT or P2X7 KOmouse cerebellarastrocytes and ofmouse peritonealmacrophages (Mac)were immunoblotted with a P2X7 receptor antibodydirected against the intracellular C terminus (Alo-mone Labs). Protein loading was assessed with ananti-GAPDH antibody. DAPI, 49,6-diamidino-2-phe-nylindole; FITC, fluorescein isothiocyanate; GAPDH,glyceraldehyde 3-phosphate dehydrogenase. Scale bar,20 mm.

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 805

nucleotides. As for P6/P7 cultures, growing cells from P4 micetended to form discrete multicellular groups. Smaller cellgroups were easier to grow compared with isolated cells, whichhad slower growing and higher dying rates. The astrocytesused in this study formed a confluent layer of process-bearingcells after 10–12 days in culture. More than 98% of these cellsexpressed the astrocyte-specificmarkerGFAP (Fig. 1, A andC).The same protocol was used to obtain purified astrocytic

cultures from P2X7 KO mice. We did not observe anysignificant differences between WT and P2X7 KO cultureswith regard to growing rate and morphology.P2X7 Receptors Are Expressed in Mouse Cerebellar

Astrocytes. The expression of P2X7 receptor was investi-gated at the transcriptional and protein levels. Figure 1Bshows that WT cerebellar astrocytes express mRNAs codingfor P2X4, P2X5, and P2X7 receptor subunits. P2X4 receptormRNA was the most abundant. Other ionotropic purinergicreceptor subunits (P2X1, 2, 3, and 6) were not detected inmouse cerebellar astrocytes. The presence of P2X7 proteinwas analyzed by immunocytochemistry (Fig. 1C). Most of theGFAP-positive astrocytes exhibited a punctate immunolabel-ing, which appeared to be located at specific zones of theplasma membrane. The expression of P2X7 subunits was also

corroborated by Western blot experiments (Fig. 1D). Immu-notransference experiments in WT cerebellar astrocytesrevealed a 77-kDa band that was not detected in extractsfrom P2X7 KO cultures. To validate the anti-P2X7 receptorantibody used in immunocytochemical experiments and thespecificity of the band detected in WT cerebellar astrocyteextracts, we checked its presence in native immune cells,which express high levels of P2X7 receptors. As expected,a similar band (77 kDa) was detected on total lysates frommouse peritoneal macrophages, which would correspond tothe full length of P2X7 subunits (isoform P2X7A).Nucleotide-Evoked Calcium Responses in Mouse

Cerebellar Astrocytes: Involvement of P2X7 Receptors.Once the presence of P2X7 protein inWT cerebellar astrocyteswas established, its functional properties were assessed. Cellswere stimulated with the nucleotides a,b-methylene-ATP(a,b-meATP), ATP, CTP, ATP, UTP, and BzATP and thecorresponding calcium responses were recorded at the single-cell level. Neither a,b-meATP (100 mM), which is selective forP2X1/3 subunits, nor CTP (100 mM), which activates P2X4subunit-containing receptors, were able to modify the cyto-solic free calcium concentration in mouse cerebellar astro-cytes (data not shown), whereas practically all tested cells

Fig. 2. Cytosolic calcium responses elicited by ATP in single mouse cerebellar astrocytes. (A) Fura-2 fluorescence ratio (F340/F380) traces representativeof ATP-evoked responses from mouse cerebellar astrocytes. Cells were continuously superfused with fresh Locke’s solution and the nucleotide wasapplied only during the period indicated by horizontal bars. (B) Concentration-response curves for the two components of the ATP-evoked calciumresponse. Data were normalized with respect to the responses elicited by 5 mM ATP. Each point represents the mean6 S.E.M of the responses obtainedin three independent experiments performed with different cultures. The logistic curves were fitted by nonlinear regression.

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responded to 100 mM ATP, UTP, or BzATP stimulation, witha rise in cytosolic calcium (Figs. 2 and 3). Long stimulations(120 seconds) with 100 mM ATP produced an initial elevationof cytosolic calcium (DF340/F380 of 0.48 6 0.01; n 5 300 cells)that decayed with a biphasic time course (Fig. 2A). Interest-ingly, a 10-fold higher ATP concentration (1 mM) elicitedcalcium responses with more complex kinetics, consisting of aninitial peak (DF340/F380 of 0.65 6 0.01; n 5 300 cells) followedby a sustained phase or by a slow increase in cytosolic calcium.The amplitude of the initial peak and the maximum value ofthe second phase of the calcium responses to different ATPconcentrations (100 nM to 5 mM) were used to obtain thecorresponding concentration-response curves. The EC50 valuefor the initial transient phase was 33 6 3 mM, whereas that ofthe second component was 1.096 0.11 mM (Fig. 2B). A similarphenotype was observed in the calcium responses evoked byBzATP, a synthetic agonist that displays greater potency thanATP at the P2X7 receptors (Fig. 3A). Therefore, the EC50

values for the two components of the calcium response toBzATP were 39 6 3 and 230 6 21 mM, respectively (Fig. 3B).

A hallmark of the P2X7 receptor is its sensitivity toextracellular divalent cations (Virginio et al., 1997; Acuña-Castillo et al., 2007). To determine whether nucleotidecalcium responses were mediated by the P2X7 receptor, wefirst assessed their dependence on extracellular calcium.Removal of this cation from the perfusion medium ablatedthe second phase of calcium responses elicited by either ATP(3 mM) or BzATP (500 mM), whereas the initial transientcomponent was significantly preserved (Fig. 4). Modifying theconcentration of extracellular Mg21, either by removing orincreasing the Mg21 content in the Locke’s solution, alsoproduced a change in the shape of the calcium responsesevoked by ATP (3 mM). Removing Mg21 from the perfusionsolution was associated with a potentiation of the secondcomponent of the response, which occluded the initial transientone. On the contrary, elevating the concentration of Mg21 from1.2 to 4.2 mM produced a marked reduction of the secondcomponent (Fig. 5A). These results strongly suggest theinvolvement of P2X7 receptors in ATP-induced calciumresponses.

Fig. 3. Concentration dependence of BzATP-evoked calcium responses in single mouse cerebellar astrocytes. (A) Fura-2 fluorescence ratio (F340/F380)traces representative of BzATP-evoked responses from mouse cerebellar astrocytes. Cells were continuously superfused with fresh Locke’s solution andthe nucleotide was applied only during the period indicated by the horizontal bars. (B) Concentration-response curves for the two components of theBzATP-evoked calcium responses. Data were normalized with respect to the response elicited by 500 mMBzATP. Each point represents the mean6 S.E.M.of the responses obtained in three independent experiments performed with different cultures. The logistic curves were fitted by nonlinear regression.

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 807

We next analyzed the effect of P2X7 receptor antagonists oncalcium responses evoked by 3 mM ATP. Preincubation ofcerebellar astrocytes for 3 minutes with 10 mMBBG abolishedthe second component of calcium responses, whereas thetransient component (DF340/F380 5 0.436 0.01; n5 300 cells)was mostly preserved (Fig. 5B). Another P2X7 antagonist, 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079;10 mM), also depressed the persistent component of the cal-cium response to ATP while barely affecting the initialtransient component. These data corroborated that the ex-tracellular calcium-dependent sustained component of thecalcium response to ATP wasmediated by P2X7 receptors. Wealso tested the effect of the broad P2 antagonist, PPADS.Preincubation with PPADS (30 mM) strongly depressed ATPcalcium responses by reducing both the initial and the sus-tained phases of the response (Fig. 5B).P2X7 Receptors Mediate Membrane Current Res-

ponses Evoked by Nucleotides in Mouse CerebellarAstrocytes. To get direct evidence supporting the involve-ment of P2X7 receptors in nucleotide-evoked calcium responses

from mouse cerebellar astrocytes, we conducted patch-clampexperiments to detect the ion flux mediated by this sort ofionotropic receptor. In the first series of experiments, cells(n 5 9) were consecutively challenged with 100 mM BzATP,100 mM ATP, and 1 mM ATP (3-second pulses, at 2-minuteintervals). As expected from calcium imaging results, 100 mMBzATP and 1 mM ATP applications were associated with theappearance of inwardly directed currents in all cells tested,whereas 100 mM ATP was not able to evoke any net inwardcurrent, thus implying a pure metabotropic action at thisconcentration on mouse cerebellar astrocytes (Fig. 6A).Currents evoked by 1 mM ATP were less than half (72.85 621.2 pA; n 5 9 cells) of those activated by BzATP in the samecells (171.90 6 36.47 pA), suggesting a lower potency of theendogenous agonist at this ionotropic purinergic receptor(Young et al., 2008; Nörenberg et al., 2010; Oliveira et al.,2011). Peak currents evoked by BzATP in the entire group ofcells tested were of 170.11 6 18.30 pA (n 5 42 cells), whichconsidering an averagemembrane capacitance of 16.86 0.9 pF,implies a mean current density of approximately 10 pA/pF.The contribution of P2X7 receptors to BzATP-induced cur-rents was assayed by evaluating their sensitivity to BBG(10 mM), calmidazolium (1 mM), and Mg21 (1 mM) added tothe perfusion medium. Each of these three maneuvers (applied2 minutes before and during agonist administration) reducedby approximately 75% the amplitude of the nucleotide-evokedcurrent, hence pointing to the P2X7 receptor as the under-lying channel (Fig. 6B).Sustained Stimulation of P2X7 Receptors Increases

Plasma Membrane Permeability to Large Cations inMouse Cerebellar Astrocytes. The P2X7 receptor is re-markable from the biophysical point of view in that briefstimulation opens a nonselective channel permeable to smallions (Na1, K1, Ca21) (Carrasquero et al., 2009), but prolongedstimulation leads to a progressive increase in membranepermeability with the opening of a pore through which largecations, such as NMDG1 (Mr 5 196) and Yo-Pro-1 (Mr 5 629),can pass in and out of the cells (Chaumont and Khakh, 2008;Richler et al., 2008). Therefore, we set out to investigatewhether an increase in membrane permeability to NMDG1

occurred during prolonged stimulation (90 seconds) of P2X7receptors with BzATP (100 mM) bymonitoring the shift in Vrev

under bionic conditions (Na1 and NMDG1 being the onlycharge carriers in the extracellular and intracellular solution,respectively). Vrev was estimated from the current response tovoltage ramps (from2100 to150mV over 1 second) repetitivelyapplied every 3 seconds. Current ramps were leak-subtractedwith the averaged current to 3 voltage ramps generated priorto BzATP application, and the measured Vrev were correctedby the liquid junction potential between the pipette’s solutionand the extracellular solution (26.37 mV) (Fig. 6C). In thecontinuous presence of BzATP,Vrev shifted from 29.716 2.09mVto a steady state value of 15.87 6 0.16 mV, and this co-rresponding to a change in the permeability ratio PNMDG/PNa

from 0.26 6 0.02 to 0.49 6 0.003 (n 5 8 cells). Thus, long-lasting stimulation of P2X7 receptors is associated with theopening of a large-cation-permeable pore in mouse cerebellarastrocytes.An alternative way of testing the increase in large-cation

permeability brought about by a sustained stimulation of P2Xreceptors is to measure the cellular uptake of cationic dyes,such as Yo-Pro-1. Fig. 7, A and B, shows that mouse cerebellar

Fig. 4. Dependence on extracellular calcium of cytosolic calcium res-ponses induced by high concentrations of ATP or BzATP in single mousecerebellar astrocytes. Representative traces of changes in fura-2 fluores-cence ratio (F340/F380) evoked by 3 mM ATP or 500 mM BzATP in thepresence and absence of extracellular calcium. Astrocytes were challengedtwice with the specified nucleotide (continuous horizontal lines). Whereindicated, cells were superfused with a Ca2+-free Locke’s solution (dottedlines) 5 minutes before and during the second nucleotide application.Extracellular calcium was diminished by adding a Tris/EGTA mixture toLocke’s solution to obtain a free calcium concentration of approximately200 nM. Recordings represent typical responses observed in 270 cells fromthree different cultures.

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astrocytes took up Yo-Pro-1 upon stimulation with 500 mMBzATP in a time-dependent manner. To confirm the in-volvement of P2X7 receptors in Yo-Pro-1 uptake, we examinedthe effect of BBG and calmidazolium. As shown in Fig. 7C,10 mM BBG had no effect on basal uptake but completelyblocked Yo-Pro-1 uptake elicited by BzATP. Interestingly andin contrast with its inhibitory effect on BzATP-evoked currents(Fig. 6B), 1 mMcalmidazolium did not affect Yo-Pro-1 uptake inmouse cerebellar astrocytes, indicating that this cation dye wasentering into the cell through nonselective membrane pores,which were formed after P2X7 receptor stimulation.To further prove that Yo-Pro-1 uptake is an event secondary

to P2X7 receptor stimulation, we investigated nucleotideresponses in P2X7 KO cerebellar astrocytes. Figure 8 showscalcium responses and Yo-Pro-1 uptake obtained in cerebellarastrocytes from WT and P2X7 KO mice. As could be expectedfromWestern blot experiments (Fig. 1D), P2X7 KO cerebellarastrocytes bathed in normal Locke’s solution exhibitedcytosolic calcium responses to ATP (3 mM) or BzATP (500 mM)that displayed only a transient component (Fig. 8A). Peakvalues were similar for ATP (DF340/F380 of 0.43 6 0.01; n 5100 cells) and BzATP stimulations (DF340/F380 of 0.40 6 0.01;n 5 120 cells), and compared well with those observed in

WT cells (0.45 6 0.02 for 3 mM ATP and 0.48 6 0.03 for500 mM BATP; n 5 150 cells). Likewise, P2X7 KO astrocytesresponded to UTP with cytosolic calcium increases similar tothose observed in WT cells (results not shown). In P2X7 KOcells, application of BzATP (500 mM, 60 minutes) did notinduce Yo-Pro-1 uptake. No difference was observed betweendata obtained from nonstimulated cells and cells treated withthe nucleotide. These results further support the involvementof the P2X7 receptor in changes in plasma membranepermeability triggered by BzATP in mouse cerebellarastrocytes.P2X7 Receptors Mediate Cell Death in Mouse Cere-

bellar Astrocytes. Considering that prolonged activation ofP2X7 receptors increased the membrane permeability to largemolecules in mouse cerebellar astrocytes, we decided to testwhether P2X7 receptor stimulation could also affect the via-bility of the cultures. As shown in Fig. 8C, treatment of WTcerebellar astrocytes with 3 mM ATP or 500 mM BzATP for30 minutes considerably decreased cell viability as determinedwith the LIVE/DEAD Viability/Cytotoxicity Assay Kit. Impor-tantly, similar treatments did not affect the viability of P2X7KO cells, hence indicating that P2X7 receptors were engaged inthe nucleotide-induced death of WT cerebellar astrocytes.

Fig. 5. P2X7 receptors mediate the second component of the calcium responses evoked by ATP in mouse cerebellar astrocytes. (A) Modulation byextracellular Mg2+ of calcium responses induced by ATP. Cells were superfused with normal Mg2+ (1.2 mM), Mg2+-free (0 Mg2+), or high Mg2+ (4.2 mM)Locke’s solution for at least 5 minutes before being stimulated with 3 mM ATP. ATP was applied during the time indicated by horizontal bars.Recordings represent typical responses observed in more than 180 cells from at least three different cultures. (B) Effect of the P2 antagonists on calciumresponses evoked by ATP. Cells were exposed to BBG (10 mM), A438079 (10 mM), or PPADS (30 mM) 3 minutes before (dotted lines) and during thestimulation with 3 mM ATP (continuous lines). Recordings represent typical responses observed in over 100 cells from three different cultures.

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 809

Fig. 6. P2X7 receptors mediate nucleotide-induced ionic currents in mouse cerebellar astrocytes. (A) The left panel shows representative whole-cellcurrents in response to the consecutive application of 100 mM BzATP, 100 mM ATP, and 1 mM ATP to a mouse cerebellar astrocyte. Nucleotides werefocally applied onto the cell for 3 seconds (horizontal bar) at 2-minute intervals. Vh = 270 mV. The right panel shows a histogram representing peakcurrent responses to nucleotide application from nine experiments similar to that depicted in the left panel. (B) Effects of P2X7 antagonists andmodulators on currents evoked by BzATP. The left panel shows representative whole-cell currents evoked by two consecutive applications (I1 and I2) of100 mM BzATP, 4 minutes apart. The PX7 receptor antagonists BBG (10 mM; upper row), calmidazolium (1 mM; middle row), and the PX7 receptormodulator, Mg2+ (1 mM; lower row) were present in the perfusion medium 2 minutes before and during I2. Vh = 270 mV. The right panel showsa histogram summarizing the results from experiments similar to those depicted in the left panel. The columns represent the ratio of peak currents

810 Salas et al.

To more easily quantify the cell death response to nu-cleotides, we used the MTT assay, which estimates the num-ber of viable cells by measuring the cellular metabolic state.As shown in Fig. 9A, a 60-minute treatment of astrocytes withhigh concentrations of ATP (5 mM) or BzATP (500 mM) led toa 40% reduction in cell viability. Importantly, the P2X7antagonist BBG (10 mM), administered 3 minutes before andduring the subsequent 60-minute incubation, prevented celldeath induced by 500 mM BzATP, and markedly attenuatedthe diminution in cell viability produced by 5 mM ATP. Atvariance, calmidazolium (1 mM) was unable to inhibit celldeath induced by either BzATP or ATP. It should be notedthat neither BBG nor calmidazolium affected cerebellar as-trocytes viability in the absence of nucleotides. Cell deathelicited by 500 mM BzATP did not depend on either ex-tracellular calcium or the activation of caspase 3 since it wasnot modified by removing calcium from the Locke’s solution orincubation with 30 mM Z-DEVD-FMK, a caspase-3 inhibitor(Fig. 9B). Altogether, our results point to a necrotic-typemechanism for the cell death elicited by P2X7 receptorstimulation in mouse cerebellar astrocytes (Fig. 9B).

DiscussionThe central message of this study is that mouse cerebellar

astrocytes express functional P2X7 receptors, which areultimately responsible for a nucleotide-induced cell deatheffect. This conclusion is supported by the following findings.First, the P2X7 subunit 77-kDa protein was detected byimmunocytochemistry and Western blot analysis. Second,both ATP and BzATP induced membrane ionic currents andionotropic cytosolic calcium responses, with BzATP beinga more potent agonist. Third, nucleotide-induced membranecurrents and inotropic calcium responses were sensitive toextracellular Mg21 and to P2X7 receptor antagonists. Fourth,prolonged exposure of cerebellar astrocytes to high concen-trations of nucleotides increased the permeability of plasmamembrane to large cations and reduced cell viability, and botheffects were prevented by treatment with BBG. Finally,cellular responses attributed to P2X7 receptors were absentin P2X7 KO cerebellar astrocytes.This study also reports the preparation of purified astrocyte

cultures from mice cerebella. In our hands, P4 was the bestage from which cerebellar cells could be dissociated to initiateastrocyte cultures. Isolated cells typically reached confluenceafter 10–12 days in culture, and thereafter consistentlyexhibited cytosolic calcium responses to purinergic agonists.Likewise, we did not observe any obvious difference betweenWT and P2X7 KO cultures.Mouse cerebellar astrocytes responded to ATP and BzATP

stimulations with cytosolic calcium elevations whose kinetics

depended on the nucleotide concentration used. Low concen-trations of nucleotides (#100 mM) induced transient calciumresponses, which declined in the presence of the agonist, butbiphasic calcium responses were obtained when the agonistconcentrations were increased. The two phases of the calciumresponse evoked by ATP differed in their sensitivity toexternal divalents (Ca21 and Mg21) and to P2X7 receptorantagonists. The initial transient phase was mainly metab-otropic in nature and was practically insensitive to P2X7receptor antagonists, which indicates the involvement of aP2Y metabotropic receptor, at which ATP and BzATP wereequipotent. In fact, similar calcium responses were elicited byUTP (Wildman et al., 2003; León-Otegui et al., 2011). Bycontrast, the second, sustained or slowly rising phase of thecalcium response, observed only at high (millimolar) concen-trations of ATP, depended on extracellular Ca21, and wasinhibited by extracellular Mg21 and P2X7 receptor antago-nists (BBG and A438079); all of these features support theinvolvement of P2X7 receptors in its generation (Virginioet al., 1997; Hibell et al., 2001; Acuña-Castillo et al., 2007).Moreover, the EC50 values estimated for this component of thecalcium response (1.09 mM and 230 mM for ATP and BzATP,respectively) agree well with those reported for the clonedmouse P2X7 receptor (Chessell et al., 1998; Hibell et al., 2001;Young et al., 2007). Confirmatory evidence for the participa-tion of P2X7 receptors was obtained in astrocytes from P2X7KO mice: nucleotide calcium responses recorded from P2X7KO cells lack the second component, such that their shaperesembled calcium responses obtained inWT astrocytes in theabsence of extracellular calcium.Biphasic nucleotide calcium responses observed in mouse

astrocytes were similar to those elicited by BzATP in ratcerebellar astrocytes (Carrasquero et al., 2009). In the ratmodel, however, ATPwas unable to evoke a sustained calciumelevation like that elicited by the synthetic P2X7 agonist.Considering that the rat P2X7 receptor is more sensitive toATP than the mouse ortholog (Young et al., 2007), our resultssuggest a reduced expression level of P2X7 receptors in rat cer-ebellar astrocytes compared with their murine counterparts.The electrophysiological results also corroborate the in-

volvement of P2X7 receptors in nucleotide responses. Inmouse cerebellar astrocytes, nondesensitizing currents wereelicited by the two nucleotides, and, as expected, BzATPdisplayed higher potency than ATP (Duan et al., 2003;Nörenberg et al., 2010, 2011). The magnitude and the currentdensity of BzATP-induced currents in mouse cerebellarastrocytes (approximately 30 pA/pF) were much higher thanthose from rat cerebellar astrocytes (approximately 3 pA/pF)(Carrasquero et al., 2009). Another distinctive feature of P2X7receptor–mediated currents from mouse cerebellar astrocytesis the progressive increase in the fractional permeability to

during I2 with respect to I1. The effects of drug-free perfusion solution (control; n = 8 cells), BBG (10 mM; n = 7 cells), calmidazolium (1 mM; n = 13 cells),and Mg2+ (1 mM; n = 6 cells) are shown. *P , 0.05; ***P , 0.005, with respect to control. (C) Prolonged stimulation of P2X7 receptors increasesmembrane permeability to NMDG+. The left panel shows representative currents in response to voltage ramps (from 2100 to +50 mV, 1-secondduration) applied at 3-second intervals in the continuous presence of BzATP (100 mM). A 90-second application of BzATP (100 mM) evoked a whole-cellcurrent that slowly declined over the stimulation period. Circles correspond to currents sampled during the 100 ms preceding each voltage ramp at theVh of270 mV (upper inset). Current ramps were leak-corrected by subtracting the averaged current response to 3 ramps applied immediately before theadministration of BzATP. The point of intersection of the corrected current ramp with the voltage axis was used to determine the apparent reversalpotential (Vrev) of the P2X7 receptor–mediated current. After subtraction of the liquid junction potential (26.37 mV), the true Vrev was calculated andplotted against time (lower inset). The right panel shows the time course of the fractional NMDG+ permeability (PNMDG/PNa) for experiments similar tothose shown in the left panel. Data represent the mean 6 S.E.M. (n = 8 cells). Cz, calmidazolium.

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 811

NMDG1 during prolonged BzATP stimulation, which con-trasts with the lack of such an increase observed in HEK-293cells expressing rat P2X7 receptors when the extracellular

solution contains the normal Na1 concentration (Jiang et al.,2005; Yan et al., 2008). This difference likely rests on thedivergence in the sequence of the C-terminal tail regionbetween the rat and mouse P2X7 receptor orthologs and onthe accepted role of this intracellular domain in controllingplasma membrane permeability after P2X7 receptor stimula-tion (Chessell et al., 1998; Costa-Junior et al., 2011). Speciesdifference between rat and mouse P2X7 receptors may also beresponsible for differences in the ability to accumulateYo-Pro-1 between rat and mice cerebellar astrocytes (Smartet al., 2003). Our results clearly show that Yo-Pro-1 uptakedevelops rapidly in mice cerebellar astrocytes upon BzATPstimulation. In contrast, we found no dye uptake in ratcerebellar astrocytes under the same experimental conditions(Carrasquero et al., 2009). Most likely, opening of amembranepore permeable to large molecules underlies the cytotoxiceffect of prolonged P2X7 receptor stimulation in micecerebellar astrocytes. Three complementary lines of evidencesupport this assertion. First, neither accumulation of Yo-Pro-1nor reduction of cell viability was observed in P2X7 KOcerebellar astrocytes upon incubation with BzATP. Second,in WT cerebellar astrocytes, BBG inhibited BzATP-inducedionic currents, as well as Yo-Pro-1 uptake and cell death.Third, calmidazolium, which is known to block P2X7receptor–associated small cation channels without affectingthe formation of large membrane pores (Virginio et al., 1997),had no effect on both Yo-Pro-1 uptake and cell death despiteinhibiting BzATP-activated ionic currents in WT astrocytes.Hence, altogether these results point to a close relationshipbetween P2X7 pore formation and cytotoxicity of mice cerebellarastrocytes. Moreover, cell death evoked by P2X7 receptorstimulation appears to be independent of extracellular Ca21

and presumably Ca21 entry as well and is not related to caspase3 activation, thereby suggesting a necrotic-type mechanismof cell death. Necrotic death has long been considered anunregulated form of cell death, resulting in rupture of theplasma membrane and a rapid lysis of the cell. However,recent studies revealed that necrosis can also be regulatedthrough specific intrinsic cellular programs, involving a newserine/threonine kinase family, the receptor interactingprotein kinases (Festjens et al., 2007). In this respect, sig-naling pathways initiated by P2X7 receptor stimulation andleading to cell death in mouse cerebellar astrocytes willcertainly deserve additional investigations.Results obtained in mouse cerebellar astrocytes also con-

trast with those reported for other noncerebellar rat astro-cytes. In rat cortical astrocytes, stimulation of P2X7 receptorsinduces a state of reversible growth arrest rather than celldeath (Neary et al., 2008). Likewise, injection of BzATP intorat brain did not cause a decrease in the number of eitherGFAP-positive cells or GFAP/BrdU-proliferating cells (Frankeet al., 2007). As suggested by our studies in rat and mousecerebellar astrocytes, different cellular consequences of P2X7receptor stimulation may be due to different receptor expres-sion levels (Hickman et al., 1994; Narcisse et al., 2005;Nagasawa et al., 2009), but certainly also to differences inamino acid sequence at the C-terminal tail domain with en-suing differences in the ability to induce membrane permeabi-lization either directly or through the interaction with othersignaling proteins expressed in an species-dependent manner(Costa-Junior et al., 2011).

Fig. 7. Stimulation of P2X7 receptors induces Yo-Pro-1 uptake in mousecerebellar astrocyte populations. (A) Yo-Pro-1 uptake by cerebellarastrocytes after the addition of 500 mM BzATP. The trace represents thechange in fluorescence expressed in arbitrary units of a cell suspension ofapproximately 300,000 cells, in a stirred cuvette, as described inMaterialsand Methods. The arrow indicates the time of addition of 500 mM BzATP.(B) Time course of Yo-Pro-1 accumulation induced by 500 mM BzATP inmouse cerebellar astrocytes. Cell suspensions were incubated with thenucleotide for different times at 37°C and the Yo-Pro-1 fluorescence wasmeasured. Data were normalized to Yo-Pro-1 uptake at the specified times(5, 15, 30, 45, and 60 minutes) in the absence of BzATP. Values are means6 S.E.M. of three independent experiments performed in triplicate usingdifferent cultures. (C) Effect of P2X7 antagonists on Yo-Pro-1 uptake bymouse cerebellar astrocytes. Cell populations were incubated with 10 mMBBG or 1 mM calmidazolium for 3 minutes before and during thetreatment with BzATP (500 mM, 60 minutes), and the Yo-Pro-1fluorescence was measured as described in Materials and Methods. Datawere normalized to Yo-Pro-1 uptake in the absence of drugs (basal).Results are means 6 S.E.M. of the results obtained in two differentexperiments performed in triplicate using different cell cultures. ***P ,0.0001 with respect to basal. AU, arbitrary unit; Cz, calmidazolium.

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Mouse models (WT) and protein-null animals (KOmice) areextensively used to better understand the function andpathophysiological implications of proteins. Studies carriedout with P2X7 KO mice have revealed that P2X7 receptorsplay important roles under physiologic and pathologic con-ditions (Solle et al., 2001; Chessell et al., 2005; Fulgenzi et al.,2008), and have also suggested the existence of analogousproteins that escape from gene inactivation in the nervoussystem. Previous studies conducted in mouse cerebellargranule neurons from P2X7 KOmice revealed the existence offunctional P2X7-like receptors, which probably correspond toa functional splice variant that obviates gene inactivation(Sánchez-Nogueiro et al., 2005; Nicke et al., 2009). The datapresented here clearly demonstrate that WT cerebellarastrocytes express functional P2X7 receptors, which areabsent from P2X7 KO cells. P2X7 KO cerebellar astrocytesdo not express the P2X7 receptor protein nor exhibit anyresponse that could be attributed to this receptor. Thisvalidates the P2X7 KO mouse model in astroglia (Suadicaniet al., 2006).

A question that still remains open is the role of P2X7receptors in astrocyte physiology. Although high extracellularATP concentrations sufficient to activate P2X7 receptors canbe reached at the extracellular space, the crucial issue is howlong they remain high enough. Under physiologic conditions,ectoenzymes present in the same or surrounding cells willrapidly breakdown ATP, thereby terminating their actions onP2X7 receptors. However, under pathologic situations, suchas brain ischemia or trauma there is a long-lasting ATP ac-cumulation, leading to persistent activation of their receptors(Verkhratsky et al., 2009; Franke et al., 2012). Undoubtedly,brief stimulation or persistent activation of P2X7 receptorshas different cellular implications. Data reported here showthat persistent P2X7 stimulation might result in a species-related manner in a cell death signal to mouse cerebellarastrocytes. In addition to provide novel insight into P2X7receptor signaling in cerebellar astroglia, our results maythus serve as a reminder of the importance of consideringspecies differences when selecting experimental models forpathophysiological and drug-screening research programs. In

Fig. 8. Nucleotides induce sustained calciumresponses, Yo-Pro-1 uptake, and cell death in WTbut not in P2X7 KO cerebellar astrocytes. (A)Calcium responses to ATP (3 mM) and BzATP(500 mM) in WT and P2X7 KO cerebellar astrocytes.Fura-2 fluorescence ratio (F340/F380) increases weretaken as indicative of rises in intracellular calciumconcentration. Astrocytes were stimulated with nucleo-tides as indicated by the horizontal bars. Recordingsrepresent typical responses observed in over 120 cellsfrom three different cultures of WT or P2X7 KOastrocytes. (B) Yo-Pro-1 uptake induced by BzATP insuspensions of WT or KO P2X7 cerebellar astrocytes.Astrocytes were incubated in the presence or absenceof 500 mMBzATP for 60 minutes at 37°C and Yo-Pro-1fluorescence measured as described in the Materialsand Methods. Data were normalized with respect toYo-Pro-1 uptake in the absence of BzATP (basal).Results are the means 6 S.E.M. of results obtained intwo experiments performed in triplicate from differentcultures. ***P , 0.0001 versus basal. (C) Effect ofprolonged stimulation with nucleotides on cellularviability of WT and KO cerebellar astrocytes. WT orP2X7 KO cells were incubated with 3 mM ATP or 500mM BzATP for 30 minutes and cell viability wasmeasured with the LIVE/DEAD Viability/CytotoxicityAssay Kit that allows simultaneous determination oflive and dead cells by combining calcein AM andethidium homodimer-1. Live cells can be identified bytheir green fluorescence, whereas dead cells emit inred.

Functional P2X7 Receptor in Mouse Cerebellar Astrocytes 813

this regard, one should keep in mind that heterologouslyexpressed human, mouse, and rat P2X7 receptors all havebeen reported to show differences at the associated ionchannel level with respect to agonist and antagonist potenciesand with regard the kinetics of formation of the largemembrane pores, which could be translated into largerdifferences in cellular responses as a function of the particulartissue in which they are naturally expressed (Chessell et al.,1998).

Authorship Contributions

Participated in research design: Carrasquero, Artalejo, Miras-Portugal, Delicado.

Conducted experiments: Salas, Carrasquero, Olivos-Oré, Bustillo,Delicado.

Performed data analysis: Salas, Carrasquero, Olivos-Oré, Bustillo,Artalejo, Miras-Portugal, Delicado.

Wrote or contributed to the writing of the manuscript: Salas,Artalejo, Miras-Portugal, Delicado.

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Fig. 9. P2X7 receptors mediate cell death in mouse cerebellar astrocytes.(A) Astrocytes were treated with 5 mM ATP or 500 mM BzATP for 60minutes and the effect on cell viability was analyzed by the MTT assay.Where indicated, cells were preincubated with 10 mM BBG or 1mMcalmidazolium for 3 minutes before and during the subsequent 60-minuteincubation in the presence or the absence of nucleotides. Data arepresented as the percentage of cell viability under control conditions,when similar incubations were made in the absence of drugs. Values aremeans6 S.E.M. of at least three experiments performed in duplicate fromdifferent cultures. **P, 0.004; ***P, 0.0001 for the conditions indicatedby line-brackets. (B) Effect of extracellular Ca2+ removal and caspase 3inhibition on cell death induced by P2X7 receptor stimulation of mousecerebellar astrocytes. Cell were preincubated with Ca2+-free Locke’ssolution (Ca2+ was replaced by 6mMTris/6 mMEGTA) or 30 mMZ-DEVD-FMK-caspase 3 inhibitor for 20 minutes before and during the subsequent60-minute incubation in the presence or the absence of 500 mM BzATP.Data are presented as the percentage of cell viability under controlconditions, where similar incubations were made in the absence of drugs.Values are the means6 S.E.M. of at least three experiments performed induplicate using different cell cultures. ***P , 0.0001 for the conditionsindicated by line-brackets. Cz, calmidazolium; FMK, Z-DEVD-FMK-caspase 3 inhibitor.

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Address correspondence to: Dr. Esmerilda García Delicado, Department ofBiochemistry, Faculty of Veterinary Medicine, Complutense University ofMadrid, 28040 Madrid, Spain. E-mail: [email protected]

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