UP-REGULATION OF P2X2, P2X4 RECEPTOR AND ISCHEMIC CELLDEATH: PREVENTION BY P2 ANTAGONISTS

F. CAVALIERE,a F. FLORENZANO,a S. AMADIO,a,b

F. R. FUSCO,a M. T. VISCOMI,a N. D’AMBROSI,a,b

F. VACCA,a,e G. SANCESARIO,a,b G. BERNARDI,a,b

M. MOLINARIa,d AND C. VOLONTEa,c*aIRCCS Santa Lucia Foundation, Rome, ItalybUniversity of Rome “Tor Vergata,” Department of Neuroscience,Rome, ItalycInstitute of Neurobiology and Molecular Medicine, C.N.R., Rome, ItalydInstitute of Neurology, Catholic University, Rome, ItalyeDepartment of Human Physiology and Pharmacology, University ofRome La Sapienza, Rome, Italy

Abstract—In the present work we examined the involvementof selected P2X receptors for extracellular ATP in the onset ofneuronal cell death caused by glucose/oxygen deprivation.The in vitro studies of organotypic cultures from hippocam-pus evidenced that P2X2 and P2X4 were up-regulated byglucose/oxygen deprivation. Moreover, we showed that isch-emic conditions induced specific neuronal loss not only inhippocampal, but also in cortical and striatal organotypiccultures and the P2 receptor antagonists basilen blue andsuramin prevented these detrimental effects. In the in vivoexperiments we confirmed the induction of P2X receptors inthe hippocampus of gerbils subjected to bilateral commoncarotid occlusion. In particular, P2X2 and P2X4 proteins be-came significantly up-regulated, although to different extentand in different cellular phenotypes. The induction was con-fined to the pyramidal cell layer of the CA1 subfield and to thetransition zone of the CA2 subfield and it was coincident withthe area of neuronal damage. P2X2 was expressed in neuro-nal cell bodies and fibers in the CA1 pyramidal cell layer andin the strata oriens and radiatum. Intense P2X4 immunofluo-rescence was localized to microglia cells.

Our results indicate a direct involvement of P2X receptorsin the mechanisms sustaining cell death evoked by metabo-lism impairment and suggest the use of selected P2 antago-nists as effective neuroprotecting agents. © 2003 IBRO. Pub-lished by Elsevier Science Ltd. All rights reserved.

Key words: microglia, organotypic cultures, gerbil, carotidocclusion.

Brain damage resulting from oxygen/glucose depletionduring an ischemic insult arises from complex cellular andmolecular events leading to both acute and delayed neu-

ronal death (Banasiak et al., 2000; Lee et al., 1999; Nico-tera et al., 1998; Choi, 1996; Nitatori, et al., 1995). Onecrucial step in the toxic consequences evoked by oxygen/glucose deprivation is the release of threshold quantities ofglutamate, namely excitotoxicity, which occurs throughionotropic channel opening (Choi, 1996; Michaelis, 1998).Another critical event is the functional expression of theglutamate receptors. Whereas activation of the metabo-tropic receptors of groups II and III has neuroprotectiveroles (Bond et al., 1999; Henrich-Noack et al., 2000), ex-cessive stimulation of group I receptors would often resultin neurotoxicity (Pellegrini-Giampietro et al., 1999; Nicolettiet al., 1996). Similarly, several studies described a bene-ficial action of adenosine and A1 receptor agonists in bothin vitro and in vivo ischemia (Abbracchio and Cattabeni,1999), in pure glial cultures and in enriched neuronal cells(Jurkowitz et al., 1998). Conversely, the function of the A2

and A3 receptor subtype is still not fully understood (Ab-bracchio et al., 1997; Pedata et al., 2001). Along with theinvolvement of glutamate and adenosine, a direct partici-pation of extracellular ATP receptors in ischemic stress isnow gaining momentum (Phyllis et al., 1993; Juranyi et al.,1999; Cavaliere et al., 2001a,b). In particular, P2 receptorswere previously shown to directly mediate ischemic signal-ing in CNS neurons in primary culture (Cavaliere et al.,2001a,b, 2002) and in carotid afferent neurons (Prasad etal., 2001). Moreover, despite the strict control exerted byectonucleotidases degrading extracellular ATP into aden-osine (Braun et al., 2000), extracellular ATP per se wasdemonstrated to be toxic for primary neuronal cultures,inducing both necrotic and apoptotic cell loss (Amadio etal., 2002). In fact, several P2 receptor antagonists didabolish the cell death fate of primary neurons exposed toexcessive glutamate (Volonte and Merlo, 1996), to serum/potassium deprivation (Volonte et al., 1999) and to hypo-glycemia or chemical hypoxia (Cavaliere et al., 2001a,b).

ATP released in vivo and in vitro under physiological orpathological conditions (Phyllis et al., 1993; Neary, 1996;Vizi and Sperlagh, 1999; Burnstock, 2002) from neuronsand/or glial cells (Chakfe et al., 2002), can elicit eitherphysiological functions, behaving as a neurotransmitter,neuromodulator and growth factor (Rathbone et al., 1999;Burnstock, 1999; D’Ambrosi et al., 2001), or detrimentaleffects, acting as a toxic agent (Vizi and Sperlagh, 1999;Amadio et al., 2002). Moreover, ATP can activate micro-glial cells, that are involved either in the integrity protectionof the CNS or in the establishment of a number of patho-logical conditions (Kreutzberg, 1996; Minghetti et al.,1999). These actions are mediated by ligand-gated P2X

*Correspondence to: C. Volonte, CNR/IRCCS Santa Lucia Founda-tion, Via Ardeatina 354, 00179 Rome, Italy. Tel: �39-06-5150-1557;fax: �39-06-5150-1556.E-mail address: cinzia@in.rm.cnr.it (C. Volonte).Abbreviations: BB, basilen blue; CLSM, confocal laser scanningmicroscopy; GFAP, glial fibrillary acid protein; GFM, glucose-freemedium; IB4, isolectin B4; MBP, myelin basic protein; NeuN, neuronalnuclei; NFL, neurofilament L; PB, phosphate buffer; Syn,synaptophysin.

Neuroscience 120 (2003) 85–98

0306-4522/03$30.00�0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.doi:10.1016/S0306-4522(03)00228-8

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(responsible for ATP-induced depolarization and Ca2� in-flux), as well as metabotropic P2Y nucleotide receptors(responsible for G protein-mediated signals) expressed onboth neurons and microglial cells (Illes et al., 1996; Fieldsand Stevens, 2000).

In the present work, we investigated the modulation ofP2X receptors, particularly P2X2 and P2X4 proteins, fol-lowing ischemic insults. Experiments were carried out bothin vitro, using glucose/oxygen-deprived organotypic cul-tures, and in vivo, using a model of brain ischemia in gerbil.For this purpose, we combined immunohistochemistry,confocal microscopy and Western blotting techniques.

EXPERIMENTAL PROCEDURES

Organotypic cultures

Hippocampus. Organotypic hippocampal slice cultureswere prepared using a modification of the method by Stoppini etal. (1991). Briefly, Wistar rat pups (8–10 days old) were killed andbrain removed. Hippocampi were excised (Fig. 1A), cut on a McIlwain tissue chopper (400 �m) and separated into cold HBSS(0.185 mg/ml CaCl2, 0.1 mg/ml MgSO4, 0.4 mg/ml KCl, 0.06mg/ml KH2PO4, 8 mg/ml NaCl, 0.05 mg/ml Na2HPO4, 0.35 mg/mlNaHCO3, 1 mg/ml glucose). Four slices were plated on eachMillicell CM culture inserts (Millipore, Rome, Italy) and maintained

in organotypic maintenance medium (50% BME, 25% HBSS, 25%heat-inactivated horse serum, supplemented with 4.5 mg/ml glu-cose, 1 mM glutamine) at 37 °C, 100% humidity and 5% CO2.Medium was changed every 3–4 days and experiments per-formed after 14 days in vitro.

Cortex–striatum. Double cultures were prepared using amodification of the method by Plenz and Kitai (1996). Wistar ratpups (2–3 days old) were killed and the brains were removed.Coronal sections of 350 �m were cut with a vibratome and dis-sected for dorso-lateral cortical and striatal tissues (Fig. 1B).Slices were plated in serial order on Millicell CM culture insertsand maintained in 1 ml of organotypic maintenance medium. After3 days, 10 �M of the mitosis inhibitor cytosine �-D-arabinofurano-side (Sigma, Mi-Italy) was added for 24 h and the medium waschanged every 3–4 days. Experiments were generally performedafter 14 days in vitro.

In vitro ischemia

The Millicell CM inserts with organotypic slices were placed in 1 mlof glucose-free medium (GFM), consisting of Earle’s balanced saltsolution (Sigma) and 5 �g/ml propidium iodide (Sigma), with orwithout basilen blue (BB) or suramin (Sigma). Before the use, theGFM was saturated with 95% N2 and then maintained with thecultures at 37 °C for different times, in a N2-saturated environ-ment. The GFM was then replaced with the organotypic mainte-nance medium and the cultures were kept with 5 �g/ml propidium

Fig. 1. Schematic view of organotypic slice preparations. (A) Section used to prepare hippocampal organotypic cultures and microphotograph of theculture at 14 DIV. (B) Schematic drawing of the three cuts used to isolate the cortico/striatal organotypic cultures and microphotograph of the cultureat 14 days in vitro. CX, cortex; CP, caudate-putamen.

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iodide under normoxic conditions for additional 18 h, before eval-uating cell death. For normoxic conditions, the medium consistedof GFM supplemented with 1 mg/ml glucose.

Evaluation of neuronal death

Neuronal damage was assessed 20 h after ischemia by propidiumiodide incorporation on organotypic cultures. The extent of celldeath was quantified with a fluorescence microscope, using thedensitometry software Neuroscion Image for Windows obtainedfrom the NIH public domain.

Antisense oligonucleotide

Experiments with antisense oligonucleotides were performed withthe antisense X2AS (5'-CAAGCGCCGGACCATGGCCGC-3') orsense X2S (5'-GCGGCCATGGTCCGGCGCTTG-3'), both fromRoche Diagnostic, Mi, Italy. The oligonucleotides were diluted inOptimem (Gibco, Paisley, Scotland, UK) at 3 �M and added to theorganotypic cultures for up to 3 days. The cellular damage wasevaluated 20 h after ischemia, by propidium iodide incorporation.Oligonucleotides correspond to nucleotide 31–51 of rat P2X2

mRNA (GeneBank accession number U14414).

Total protein extraction from organotypic cultures

Organotypic cultures were maintained under ischemic conditionsin the simultaneous presence or absence of 100 �M suramin.After different lengths of time, four organotypic slices from eachexperimental condition were extracted in RIPA buffer (PBS sup-plemented with 1% NP-40, 0.5% sodium deoxycholate, 0.1%SDS, 0.5 �M PMSF, 10 �g/ml leupeptin, all from Sigma) andhomogenized. They were maintained for 1 h on ice, sonicated andcentrifuged at 4 °C at 10000�g for 10 min. Protein quantificationwas performed in the supernatants by Bradford colorimetric assay(Biorad, Mi, Italy).

Western blot

Equal amounts of total protein from each sample (50 �g) wereseparated by SDS-PAGE on a 12% polyacrylamide gel and trans-ferred overnight onto a nitrocellulose membrane Hybond C (Am-ersham, Cologno Monzese, Mi, Italy). The filters were pre-wettedin 5% non-fat milk in TBS-T (10 mM Tris pH 8, 150 mM NaCl, 0.1%Tween 20) and hybridized for 3 h with anti-P2X1,2,4,7 antisera(1:200; Alomone, Jerusalem, Israel). All antisera were immuno-detected with an anti-rabbit HRP conjugated antibody and devel-oped by ECL chemioluminescence (Santa Cruz, Mi, Italy). Quan-tification of the specific bands was performed in a linear range ofdetection, by using Kodak 1D 3.5.3 software. Statistical differ-ences were evaluated by t-test analysis of the data, with P valuesat least �0.05.

In vivo ischemia

Six adult Mongolian gerbils (Meriones unguiculatus; Morini, S.Polo D’Enza RE, Italy) were used for this study. The experimentswere conducted under institutional approval and in accordancewith the National Institutes of Health Guide for the Care and Useof Laboratory Animals. All efforts were made to minimize thenumber of animals used and their suffering.

As previously described (Sancesario et al., 1994), three ani-mals were anesthetized for 5 min in an induction chamber, with amixture of 5% halothane in N2O/O2 (70:30); they were placed ona heating pad, to control their temperature, and the anesthesiawas maintained with a mixture of 1.5% halothane in N2O/O2

70:30. After a ventral neck incision along the midline, the commoncarotid arteries were exposed, isolated and clamped with microa-neurism clips. During the carotid occlusion, the anesthetic gas

was not administered. The clips were removed after 10 min andthe skin incision was sutured. Three additional animals wereanesthetized with the same procedure and sham operated byexposing their common carotid arteries. Five days following sur-gery, gerbils were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and transcardially perfused with 100 ml of saline solutionat room temperature, followed by 100 ml of 4% paraformaldehyde(weight/volume) in 0.1 M phosphate buffer (PB). The brains wereremoved, maintained in the same fixative for 2 h, washed threetimes in PB and cryo-protected in a solution at 30% sucrose/PB at4 °C. High levels of reproducibility and consistency was obtainedwithin each experimental group.

Histology

Transverse sections 40 �m thick were cut throughout the hip-pocampus on a freezing microtome and collected in seven series.The first set was processed for Cresyl Violet staining, in order toassess the extent and features of cellular degeneration. The re-maining six series were processed for conventional immunohisto-chemistry and for double immunofluorescence studies.

Conventional microscopy. Antisera against the purinergicreceptors P2X2,4 (Alomone) were used diluted 1:500 for 24 h, inPB containing 2% normal goat serum (Vector Laboratories, Bur-lingame, CA, USA) and 0.3% Triton X-100. All P2 antisera wereaffinity purified and raised against highly purified peptides (identityconfirmed by mass spectrography and amino acid analysis), cor-responding to specific epitopes not present in any other knownprotein (P2X2: 457–472; P2X4: 370–388). Specificity for eachsignal was also tested by immunoreactions in the presence ofneutralizing peptides (ratio 1:1 between peptide and antiserum).After three rinses (5 min each) in 0.1 M PB, sections were incu-bated with goat anti-rabbit biotinylated secondary IgG (1:200, 2 hat room temperature, Vector Laboratories). After three 5-minrinses in 0.1 M PB, sections were incubated in avidin-biotin com-plex for 1.5 h (Vector Laboratories), in 3,3' diaminobenzidine(0.05%) for 10 min, mounted on gelatinized glass microscopeslides. Subsequently, the sections were dehydrated in ethanolascending series, cleared in xylene and coverslipped with Entel-lan. The material was analyzed under bright-field illumination witha Zeiss Axioscope light microscope. Digital images were takenwith a digital camera (CoolPix 990, Nikon) and arranged in plateswith CorelDRAW 9 (Corel Corporation).

Confocal microscopy. Four sets of serial adjacent trans-verse sections were used for double immunofluorescence label-ing. Each set was incubated in a mixture of primary antisera for24 h in 0.3% Triton X-100 in 2% normal donkey serum in PB.Rabbit anti-P2X4 (1:100) was used in combination with mouseanti-NeuN (neuronal nuclei, 1:100, Chemicon International, Inc.,Temecula, CA, USA), or biotinylated IB4 (isolectin B4 from Griffo-nia Simplicifolia seeds, 10 �g/ml, Sigma), or mouse anti-GFAP(glial fibrillary acidic protein, 1:100, Sigma) or mouse anti-MBP(myelin basic protein, 1:100, Boehringer Mannheim, Monza Mi,Italy), or goat anti-NFL (neurofilament-L protein, 1:100; SantaCruz), or mouse anti-Syn (synaptophysin, 1:100, Sigma, St. Louis,MO, USA). The antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunore-search, West Baltimore Pike, PA, USA; red immunofluorescence)or Cy2-conjugated donkey anti-mouse IgG (1:100, Jackson Im-munoresearch; green immunofluorescence). Cy2-conjugated ex-travidin (1:100, Jackson Immunoresearch) was used for IB4 hist-ofluorescence. The sections were washed in PB three times for 5min each and then incubated in a solution containing a mixture ofthe secondary antibodies. After rinsing, the sections weremounted on slide glasses, allowed to air dry and coverslipped withGel/Mount anti-fading medium (Biomeda, Foster City, CA, USA).

Double label immunofluorescence was analyzed by means of

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a confocal laser scanning microscope (CLSM; LSM 510, Zeiss,Arese Mi, Italy) equipped with an argon laser emitting at 488 nmand a helium/neon laser emitting at 543 nm. Stacks of opticalsections separated by 1 �m each were collected and analyzed.Plates were generated with CorelDRAW 9.

RESULTS

In vitro ischemia in organotypic cultures

Here we have investigated whether the expression ofselected P2X receptors for extracellular ATP was al-tered during in vitro and in vivo models of cerebralischemia. We first adopted organotypic cultures (Fig. 1),since this cellular system resembles the in vivo modelbetter than dissociated cultures. Individual cells are infact in tight contact with each other, maintain their orga-notypic architecture and preserve neuron– glia interac-tions, tissue-specific transport and ion diffusion sys-tems. Moreover, the organotypic cultures represent anefficient cellular model to overcome the in vivo impedi-ments of the blood– brain barrier. Hippocampal organo-typic slices were therefore excised from rat brain (Fig.1A) and subjected to oxygen/glucose deprivation fordifferent lengths of time. Total protein was analyzed byquantitative Western blot and immunoreactions wereperformed in the presence of various P2X commerciallyavailable antisera (Fig. 2A). P2X1 antisera did not showpositive reactions with hippocampal organotypic slices(data not shown). P2X2 and P2X4 receptor proteins weremaximally 2.5-fold up-regulated (P�0.05, by t-test anal-ysis) in 20 h of reperfusion following 3 h of ischemia (Fig.2A). The P2 antagonist suramin (100 �M), not discrim-inating among P2 receptor subunits, partially preventedthis induction (30% inhibition, P�0.05, Fig. 2A), withoutaffecting basal levels of P2 protein expression (data notshown). The P2 antagonist BB was similarly effective(data not shown). P2X7 receptor protein was also re-markably modulated by ischemic conditions and P2X3

was not tested in the present study since it was notmodulated in dissociated primary cerebellar granule cul-tures during hypoglycemia (Cavaliere et al., 2002). Byperforming immunoreactions in the presence of neutral-izing peptides for each P2X receptor subunit we posi-tively verified the specificity of each signal (data notshown).

We then tested the action of P2 antagonists on theextent of neuronal damage caused by ischemic condi-tions. Consistently with previous observations (Moroniet al., 2001; Laake et al., 1999), we proved by propidiumiodide incorporation in hippocampal organotypic slicesthat glutamate caused widespread cellular loss (Fig.2B), hypoglycemia induced neurodegeneration only inthe CA1 region (data not shown), and ischemia evokedvisible damage in the entire CA1–3 region (Fig. 2B).Furthermore, cell loss was precisely obtained within thesame time course required for P2 receptors up-regula-tion (20 h). The presence of suramin during the ischemicinsult totally prevented cell death (Fig. 2B), also reduc-ing P2 receptor overexpression (Fig. 2A). Similarly, an-tisense oligonucleotides for P2X2 were able to decrease

the ischemic damage, as detected by reduced incorpo-ration of propidium iodide (Fig. 2B).

The up-regulation of P2X4 isoform under ischemiawas also positively confirmed by immunofluorescence(Fig. 3A). Moreover, double immunofluorescence al-lowed to characterize P2X4 cellular expression (Fig. 3B).Colocalization was found for P2X4 and IB4 (marker formicroglia), but not for P2X4 and MBP (marker for oligo-dendrocytes) or GFAP (marker for astrocytes; Fig. 3B).Double immunostaining of P2X4 and NeuN (marker forneurons) showed that P2X4 was only lightly expressedon hippocampal neurons (see merged images in Fig.3B).

In order to confirm and extend our results, we testedslice cultures from additional brain regions and an addi-tional P2 antagonist (Fig. 4). BB (100 �M) significantlyreduced the cellular damage confined to the CA1–3region of the hippocampus (Fig. 4A). Moreover, massivecellular loss was observed in the dorso-lateral region ofthe cortex and in the caudate putamen in double orga-notypic cultures from cortex/striatum undergoing oxy-gen-glucose deprivation for 40 min (Fig. 4B, C).Whereas BB totally prevented striatal cell degeneration(Fig. 4C), it only partially reduced (25%) cortical celldeath (Fig. 4B), thereby confirming previous results ob-tained in vitro with dissociated primary neuronal cultures(Amadio et al., 2002).

In vivo ischemia in gerbil

In order to validate the results obtained in vitro (Figs. 2–4),we tested also in vivo the modulation of P2X2 and P2X4

receptors during ischemia. As expected, in the hippocam-pus of sham-operated animals no cellular damage wasobserved in the pyramidal cell layer of Nissl-stained sec-tions (Fig. 5A). No immunostaining for P2X2 or P2X4 re-ceptors was observed in these animals in DAB-reactedmaterial (Fig. 5C, E), with the only exception of somerounded cell bodies intensely P2X4-immunopositive, eitherjuxtaposed to or inside the blood vessels lightly immuno-stained for P2X4 (data not shown). These cells morpho-logically resembled pericytes and mast cells, which areknown to be in strict contact with blood–brain vessels.Conversely, in Nissl-stained sections of hippocampus ofischemic animals we observed a conspicuous cellular lossmainly in the CA1 subfield and in the transition zone of theCA2 subfield (Fig. 5B). The spatial extent of the neuronaldamage was consistent among different cases, although asmall variability in the degree of neuronal loss was ob-served. In the ischemic immunoreactive tissue, an intenseinduction of both P2X2 and P2X4 was detected (Fig. 5D,F–I). This induction was confined to the pyramidal celllayer of the CA1 and to the transition zone of the CA2 andit was coincident with the area and intensity of the neuronaldamage.

In particular, in the ischemic sections processed forP2X2 receptor subunit, we observed immunopositive fibersextending throughout the CA1–CA2 pyramidal cell layerand strata oriens and radiatum (Fig. 5D, G). The immuno-reactive signal was homogeneously distributed over fibers

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of medium to small diameter and the immunolabeled fibersformed a delicate plexus with many spiny branches andpuncta sparse or moderately aggregated within the stra-tum oriens, where the pyramidal neurons give off to thebasal dendritic arborizations. However, some positive fi-

bers, likely corresponding to the apical dendrites of thepyramidal cells, were detected also into the stratum radia-tum pointing toward the dentate gyrus (Fig. 5G). Occasion-ally, fine branches emerged at right angles from the afore-mentioned fibers.

Fig. 2. In vitro up-regulation of P2X2,4 proteins by ischemia and protection from cellular damage in the presence of P2 antagonist or P2X2 antisenseoligonucleotides. (A) Hippocampal organotypic cultures were maintained for 40 min under ischemic conditions, in the simultaneous presence orabsence of 100 �M suramin. Twenty hours after ischemia, the slices were homogenized and subjected to Western blot analysis using P2X2,4 antisera.(B) Hippocampal slices maintained for 40 min under ischemic conditions, without (Ischemia) or with 100 �M suramin (Isch/Suramin), or in the presenceof P2X2 sense (3 �M P2X2-S) or antisense (3 �M P2X2-AS) oligonucleotides were labeled with propidium iodide for 20 h. Cellular damage wasvisualized by fluorescence microscopy. Control cultures (CTRL) were maintained in the presence of 1 mg/ml glucose or exposed to 1 mM glutamatefor 20 h (Glut). Results are representative of at least three independent experiments.

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In the ischemic sections processed for P2X4 recep-tor subunit, the immunolabeling was strictly confined tothe pyramidal cell layer of the CA1 and to the transitionzone of the CA2 subfield (Fig. 5F). The staining patternwas characterized by highly to moderately intense im-

munostaining in puncta and in short and thick cellularprocesses surrounding unstained structures, likely cor-responding to unlabeled cellular bodies (Fig. 5H).

In order to prove the nature of the P2X2 immunopo-sitive structures, double label immunofluorescence ex-

Fig. 3. P2X4 receptor is localized on microglia in organotypic hippocampal cultures. (A) Organotypic cultures were subjected to oxygen–glucosedeprivation for 40 min. Immunofluorescence was performed 1 h later with P2X4 antisera and visualized with a 5� magnification. (B) Doubleimmunostaining between P2X4 and several cellular markers (IB4 for microglia; MBP for oligodendrocytes; GFAP for astrocytes; NeuN for neuronalbodies) was performed on ischemic slices and analyzed by CLSM. P2X4–IB4 double staining: Note the colocalization of the two markers, as shownin the magnified image. P2X4–MBP: No P2X4 staining was observed on oligodendrocytes (green fluorescence) that presented processes in strictcontact with P2X4 positive structures (see magnified image). P2X4–GFAP: No colocalization was observed between P2X4 and the astrocyte marker.P2X4–NeuN: Processes with intense P2X4 staining were NeuN negative; faint P2X4 positivity was also observed in the cytoplasm of NeuN positivecells (see white arrow in the magnified image).

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periments were performed using P2X2 antiserum cou-pled to antisera raised against several cellular markers(Fig. 6). P2X2 immunofluorescence was coupled eitherto immunofluorescence for NFL, NeuN, Syn (markers forneuronal fibers, cell bodies and synaptic structures; Fig.6A–C) or MBP (an oligodendrocyte marker; Fig. 6E) orto IB4 histofluorescence (a marker for microglia; Fig.6D). The P2X2 signal was observed both in the fibers(confirming the results of the DAB-reacted material) andin the neuronal cell bodies of the CA1 and CA2 transitionzone (Fig. 6A; P2X2). Moreover, colocalization of P2X2

and NFL or NeuN (Fig. 6A, B) confirmed the neuronalorigin of the P2X2 expression. The lack of P2X2 and Syndouble labeling suggested the nonsynaptic localizationof the P2X2 receptor (Fig. 6C). The specificity of theP2X2 neuronal expression was further confirmed by the

lack of colocalization between P2X2 and IB4 or MBP glialmarkers (Fig. 6D, E).

In order to confirm in vivo the nature of the P2X4

immunopositive structures, double label immunofluores-cence experiments were performed using P2X4 anti-serum coupled to several cellular markers. P2X4 immu-nofluorescence (Fig. 7C, D) was coupled either to IB4

histofluorescence (Fig. 7A, E), or to immunofluores-cence respectively for NeuN (Fig. 7B, F), for MBP (Fig.8A), or for GFAP (Fig. 8B). In sham- (data not shown)and ischemic-operated animals, the immunofluores-cence for P2X4 receptor was light and diffuse in hip-pocampal neurons, as shown by colocalization withNeuN (Fig. 7B, D, F–N). In addition, only in ischemicanimals, an intense signal was observed in short, thickand branched processes with a high degree of IB4 and

Fig. 4. The P2 antagonist BB prevents ischemic damage occurring in various brain regions. Hippocampal (A), cortical (B) and striatal (C) slices at 14(A) and 12 (B, C) days in vitro were deprived of oxygen/glucose for 40 min, in the absence (Isch) or the simultaneous presence of 100 �M BB (Isch/BB).Control slices (CTRL) were maintained in the presence of 1 mg/ml glucose. Neuronal damage was evaluated and quantified 20 h after ischemia bypropidium iodide incorporation. Results are representative of at least three independent experiments.

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P2X4 colocalization (Fig. 7A, C, E). These latter struc-tures have been thus identified as reactive and hyper-trophic microglia expressing P2X4. No colocalizationwas observed in P2X4 and MBP or in P2X4 and GFAPdouble-labeled preparations (Fig. 8A, B). Taken to-

gether, these data confirm the results from in vitro stud-ies and from DAB-stained sections both supporting themicroglial origin of the P2X4 up-regulation after ischemia(Figs. 3 and 5). Finally, CLSM observations of P2X4/NeuN double labeling allowed us to characterize the

Fig. 5. In vivo up-regulation of P2X2,4 proteins in gerbil hippocampus after ischemia. Nissl staining of the hippocampus of a sham-operated (A) orischemic animal (B); P2X2 immunostaining of a sham-operated (C) or ischemic animal (D); P2X4 immunostaining of a sham-operated (E) or ischemicanimal (F); arrows: CA1–CA2 transition zone. (G) Higher magnification of P2X2-immunolabeling of the CA1 region of an ischemic animal; arrows:network of fibers in the pyramidal cell layer and apical dendrites. (H) Higher magnification of P2X4-immunolabeling of the CA1 pyramidal cell layer ofan ischemic animal; arrows: processes surrounding an unstained cellular body (marked by asterisk). (I) Higher magnification of P2X2-immunolabelingof the CA1 pyramidal cell layer of an ischemic animal. Meshwork of fibers and puncta surrounding unstained cellular bodies marked by asterisks. Scalebars�100 �m (A–F); (G)�40 �m; (H, I)�10 �m.

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Fig. 6. (Caption overleaf).

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anatomical relationships between P2X4-positive acti-vated microglia and P2X4 faintly labeled hippocampalneurons. We distinctively observed P2X4 immunola-beled activated glial processes which surrounded“healthy” NeuN-positive neurons and, occasionally,NeuN-negative structures, likely corresponding to de-generating neurons and/or glial cells (Fig. 7G–N).

DISCUSSION

Purinergic signaling can now be considered a relevantmeans to control cell fate. Under this perspective andalong with previous results establishing that cell deathevoked by extracellular ATP in different neuronal popula-tions occurs with direct up-regulation of several P2 recep-tor proteins (Amadio et al., 2002; Cavaliere et al., 2002),from our present results several observations emerge onthe expression of P2X2,4 isoforms during ischemic celldeath.

First, up-regulation of P2X2,4 takes place in topo-graphic as well as temporal accordance with neuronal lossinduced in the hippocampus by ischemic conditions. Thisevent was demonstrated both in vitro and in vivo, as doc-umented by immunohistochemistry, confocal microscopyand Western blotting. Moreover, ischemia-evoked P2X2,4

up-regulation and hippocampal cell death are prevented bythe use of P2 antagonists as well as by P2X2 antisenseoligonucleotides, therefore reinforcing a cause/effect rela-tionship between the expression of these receptors andthe ischemic damage. Nevertheless, considering that com-plex biological events such as survival/cell death workthrough multiple receptor/effector systems, it is obviousthat additional P2X/Y isoforms (similarly susceptible to P2antagonists), and additional receptor machinery such asglutamate/GABA, for example (Peoples and Li, 1998;Sperlagh et al., 2002), could conjointly participate to theischemic signaling. Moreover, P2X2,4 could influence theischemic cascade at multiple target sites.

The second consideration emerging from our results isthat up-regulation of P2X receptors evoked in vivo bycerebral ischemia is observed not only in pyramidal neu-rons (P2X2), thereby corroborating previous results on thedirect involvement of P2 proteins in neuronal cell death(Cavaliere et al., 2001a,b, 2002; Amadio et al., 2002), butalso in glial cells (P2X4). Knowing that all types of gliapossess membrane receptors for extracellular ATP(Franke et al., 2001a,b), we furthermore identified herethat P2X4 is not modulated in oligodendrocytes or astro-cytes but specifically in microglial cells, known to respondto ischemic insults with beneficial or detrimental effects(Berezovskaya et al., 1995; Bruccoleri and Harry, 2000;Carenini et al., 2001). This is in agreement with previousresults showing that P2X4 (together with P2X7; Visentin etal., 1999) is indeed activated by toxic ATP in microglialcells.

P2X2 and P2X4 isoforms are thus independently butsimultaneously regulated during ischemia and P2 recep-tors’ up-regulation could either be cause or consequenceof neuronal cell death/microglial activation and be relatedto detrimental and/or beneficial effects. At present somehypothesis can be provided. As P2X4 is expressed inten-sively in microglial cells, it seems rather a compensatorychange than the direct cause of neuronal death. The evi-dence that P2X2 is expressed in neurons destined to dieand that antisense oligonucleotides prevent cellular dam-age suggests instead a mechanistic role for P2X2 in thedeath process. Moreover, neurons and glia also sharesignaling and/or communicating mechanisms during me-tabolism impairment and both these cell types can releaseATP in an activity-dependent manner (Chakfe et al., 2002).ATP released extracellularly from glia can induce fast ex-citatory responses and Ca2� signals in neurons (Fieldsand Stevens, 2000), and can induce redistribution (Khakh,2001) or activation of P2 receptors in both neurons (Ama-dio et al., 2002) and glia (Ferrari et al., 1997). Finally,hypoxia itself (as acidosis, osmotic shock, membrane de-polarization) can induce ATP release (King et al., 1997;Vizi and Sperlagh, 1999). Hence, our new data wouldsustain the concept that ischemia-evoked neural impulsesand synaptic activity can influence glial function and thatglial cells can also respond to these signals in ways that inturn regulate neuronal responsiveness (Blanc et al., 1999).Moreover, they reinforce the idea that ATP flowed outduring ischemia/cellular damage or extruded by constitu-tive release (Morigiwa et al., 2000) can indeed behave asthe intercellular signaling molecule acting between neu-rons and glia (Fields and Stevens, 2000).

Although neurons injured by stroke or trauma in theadult mammalian CNS normally fail to regenerate, duringischemia a microglial reaction often involves proliferation,expression of immunomolecules at the cellular surface,secretion of cytokines or growth factors and differentiationinto brain phagocytes (Morigiwa et al., 2000). Some ofthese events may mediate tissue damage, while othersmay play an important role in limiting the extent of damageand even in promoting tissue repair (Imai et al., 1999; Streitet al., 1999; Raivich et al., 1999). In a previous study it wasobserved that the microglial processes surround apoptoticnuclei in the hippocampus of gerbils subjected to briefischemia (Nitatori et al., 1995). Our data complete thoseobservations, suggesting that microglial cells can also sur-round living neuronal cells (Streit et al., 1999), thus sup-porting the double scavenger and nourishing function ofmicroglia. Evidence available from in vitro studies in differ-ent models of brain damage has previously demonstratedthat purinergic receptors are modulated in response toinjury as well as regeneration (Ciccarelli et al., 2001).Recently, we have shown that P2X2,3,4 and P2Y2 areinvolved in neuritogenesis and survival of PC12 cells

Fig. 6. P2X2 protein is localized on hippocampal neurons. CLSM images (double label immunofluorescence) of CA1 pyramidal cell layer in ischemicanimals. (A) P2X2/NFL double labeling showing colocalization on cellular bodies and basal dendrites of pyramidal neurons. (B) P2X2/NeuN doublelabeling showing colocalization of the two markers. No colocalization was observed between P2X2 and the other markers Syn (C), Ib4 (D), MBP (E).

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Fig. 7. (Caption overleaf).

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(D’Ambrosi et al., 2001) whereas P2X7 and P2Y4 in celldeath of cerebellar granule neurons (Amadio et al., 2002).Moreover, we have shown in vivo that up-regulation ofP2X1,2 subunits is associated with regenerative efforts ofaxotomized precerebellar neurons (Florenzano et al.,2002). The present work demonstrates in vivo that hip-pocampal damage after ischemia is associated withP2X2,4 proteins’ up-regulation. Whereas it is conceivableto suggest from our data a potential role for P2X2 in neu-ronal degeneration, it is not similarly obvious whether P2X4

participates to beneficial or detrimental functions. The as-sociation of decreased cell death and reduced P2X4 up-regulation in the presence of P2 antagonists favors thedetrimental hypothesis.

Finally, it is worth noting that our data do not allow todirectly link different P2 receptor subunits with ischemiainsult. Damage can activate different P2X expression pat-terns and a cell phenotype generally co-expresses several“redundant” P2X/Y and heteromeric P2 subclasses. It isconceivable that modulation of extra receptor subunitsmight occur to reinforce and guarantee a specific biologicalfunction. Moreover, the selective activation by differentinjuries of different combinations of P2 subunits may evenconstitute an important and novel mechanism for generat-ing specificity.

In conclusion, the present results sustain that the roleof P2X receptors in mediating CNS and PNS responses toinjury is complex. Depending on the cell phenotype, the P2subunits repertoire, either degenerative or regenerativeefforts can be combined. A current challenge in this field isnow to define the mechanisms that after pathological in-sults regulate P2 subunits’ expression, P2 receptors’ inter-play and extracellular ATP signaling in the brain.

Acknowledgements—We thank Dr. Fiamma Peruginelli for valu-able help and suggestions with organotypic culture preparations.The professional style editing of Prof. J. H. Lynch is gratefullyacknowledged. The present research was supported by ProgettiFinalizzati Ministero della Sanita’ RA0086V to C.V. and RA0085Mto M.M., by Cofinanziamento MURST 2001 Purinoceptors andNeuroprotection and by Finanziamento ISS n. 1AC/F3 to C.V.

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(Accepted 5 March 2003)

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