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Neuropharmacology 81 (2014) 142e152

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Neuropharmacology

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The P2X7 receptor antagonist Brilliant Blue G attenuates contralateralrotations in a rat model of Parkinsonism through a combined controlof synaptotoxicity, neurotoxicity and gliosis

Marta R.S. Carmo a,b, Ana Paula F. Menezes a, Ana Carla L. Nunes a, Anna Pliássova b,e,Anabela P. Rolo b,c, Carlos M. Palmeira b,d, Rodrigo A. Cunha b,e,*, Paula M. Canas b,1,Geanne M. Andrade a,1

aDepartment of Physiology and Pharmacology, Federal University of Ceará, BrazilbCNC e Center for Neurosciences and Cell Biology, University of Coimbra, PortugalcDepartment of Biology, University of Aveiro, PortugaldDepartment of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugale FMUC e Faculty of Medicine, University of Coimbra, Portugal

a r t i c l e i n f o

Article history:Received 16 June 2013Received in revised form11 January 2014Accepted 27 January 2014

Keywords:ATPP2X7 receptorParkinson’s diseaseSynaptotoxicityNeuroprotectionMicrogliaAstrocytesDopamine

Abbreviations: BBG, Brilliant Blue G; CD11b, cluste11B; DA, dopamine; DAT, dopamine transporters; Dacetic acid; GFAP, glia fibrillary protein; 6-OHDA, 6-hdehydrogenase; MTT, 3-(4,5-dimethylthiazole-2-yl)-mide; PD, Parkinson’s disease; P2X7R, P2X7 receptsaline; RT, room temperature; SN, substantia nigra; T* Corresponding author. Center for Neurosciences a

Coimbra, Rua Larga, 3004-517 Coimbra, Portugal. Tel.:239822776.

E-mail address: cunharod@gmail.com (R.A. Cunha1 Shared the co-coordination of this study.

0028-3908/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2014.01.045

a b s t r a c t

Parkinson’s disease (PD) involves an initial loss of striatal dopaminergic terminals evolving into adegeneration of dopaminergic neurons in the substantia nigra (SN), which can be modeled by 6-hydroxydopamine (6-OHDA) administration. Since ATP is a danger signal acting through its P2X7 re-ceptors (P2X7R), we now tested if a blood-brain barrier-permeable P2X7R antagonist, Brilliant Blue G(BBG), controlled the 6-OHDA-induced PD-like features in rats. BBG (45 mg/kg) attenuated the 6-OHDA-induced: 1) increase of contralateral rotations in the apomorphine test, an effect mimicked by anotherP2X7R antagonist A438079 applied intra-cerebroventricularly; 2) short-term memory impairment in thepassive avoidance and cued version of the Morris Water maze; 3) reduction of dopamine content in thestriatum and SN; 4) microgliosis and astrogliosis in the striatum. To grasp the mechanism of action ofBBG, we used in vitro models exploring synaptotoxicity (striatal synaptosomes) and neurotoxicity(dopamine-differentiated neuroblastoma SH-SY5Y cells). P2X7R were present in striatal dopaminergicterminals, and BBG (100 nM) prevented the 6-OHDA-induced synaptosomal dysfunction. P2X7R werealso co-localized with tyrosine hydroxylase in SH-SY5Y cells, where BBG (100 nM) attenuated the 6-OHDA-induced neurotoxicity. This suggests that P2X7R contribute to PD pathogenesis through a tripleimpact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7Rantagonists in PD.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Parkinson’s disease (PD) is a motor disease characterized clini-cally by bradykinesia, rigidity, postural instability and tremor at rest

r of differentiation moleculeOPAC, 3,4 dihydroxyphenyl-yroxydopamine; LDH, lactate2,5-diphenyltetrazolium bro-or; PBS, phosphate bufferedH, tyrosine hydroxylase.nd Cell Biology, University ofþ351 304502904; fax: þ351

).

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(Agid, 1991). It mostly affects the striatal dopaminergic system,damaging the dopaminergic neurons in the substantia nigra(Bernheimer et al., 1973; Bézard et al., 2001). The symptoms andneurodegeneration characteristic of this neurodegenerative diseaseare preceded by an initial affection of synaptic contacts in thestriatum (Day et al., 2006), leading to the loss of striatal dopami-nergic nerve terminals (synaptotoxicity), which evolves to the overtloss of dopaminergic neurons (neurotoxicity) (Berendse et al.,2001; Bézard et al., 2001; Forno et al., 1994). Accordingly, animalmodels of PD, such as the exposure to 6-hydroxydopamine (6-OHDA) or to mitochondrial toxins (MPTP or rotenone), are basedon the destruction of dopaminergic nerve terminals which thenevolve to an overt dopaminergic cell loss in the nigra and theemergence of motor symptoms (Smeyne and Jackson-Lewis, 2005;Simola et al., 2007). This evolution from striatal synaptotoxicity to

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152 143

nigra dopaminergic cell loss is accompanied by an abnormalfunction of glia cells, typified by the astrogliosis and microgliosisthat are observed both in the striatum and in the nigra in PD(Teismann and Schulz, 2004; Halliday and Stevens, 2011). Accord-ingly, strategies aimed either at preserving the viability of nerveterminals or attenuating gliosis or preventing neuronal death canprovide a limited ability to counteract the appearance or serious-ness of the motor dysfunction characteristic of PD (Meissner et al.,2011). However, due to the multi-factorial and evolving nature ofthis neurodegenerative disease, the available therapeutic strategieshave a time-limited efficacy and drugs targeting the different fea-tures of PD still need to be identified.

ATP is an extracellular signal, operating a large family of re-ceptors, encompassing both ionotropic (P2X) and metabotropic(P2Y) receptors (Ralevic and Burnstock, 1998). Due to its largeintracellular concentration and ubiquitous presence in all celltypes, the release of ATP to the extracellular medium acts as adanger signal (Di Virgilio, 2000). ATP fulfills this function mainlythrough the activation of P2X7 receptors (P2X7R), which have aparticular property of forming cytotoxic pores and triggering celldeath (Volonté et al., 2012). This role of P2X7R has been docu-mented in diverse situations of brain damage, such as ischemia(Melani et al., 2006; Arbeloa et al., 2012), traumatic brain injury(Kimbler et al., 2012), spinal cord injury (Peng et al., 2009), epilepsy(Engel et al., 2012), experimental autoimmune encephalomyelitis(Matute et al., 2007; Sharp et al., 2008), Alzheimer’s disease (Ryuand McLarnon, 2008; Díaz-Hernández et al., 2012), prion disease(Iwamaru et al., 2012), Huntington’s disease (Díaz-Hernández et al.,2009) or depression (Basso et al., 2009). A possible role of ATP in theetiopathology of PD is heralded by the combined observations that:1) P2X7R are expressed in the dopaminergic areas affected in PD(Amadio et al., 2007; Yu et al., 2008; Able et al., 2011); 2) P2Rcontrol the release of dopamine (Trendelenburg and Bültmann,2000; Krügel et al., 2003); 3) P2R control striatal-related function(Kittner et al., 2001, 2004); 4) P2R control striatal neuronal damage(Ryu et al., 2002); 5) P2X7R control the damage of dopaminergiccells (Jun et al., 2007; Orellano et al., 2010). However, the fewstudies enquiring for a potentially therapeutic role of P2X7R an-tagonists in models of PD only looked at either morphological orneurochemical correlates of PD (Marcellino et al., 2010; Hracskóet al., 2011) and failed to simultaneously appreciate the differentcharacteristic of PD, namely the ability to prevent the behavioralmotor abnormalities. Since P2X7R can affect synaptic function(Diáz-Hernández et al., 2008; León et al., 2008; Ortega et al., 2011),astrocytic function (Ferrari et al., 2006; Gandelman et al., 2010),microgliosis (Bianco et al., 2006; Monif et al., 2009) and neuronaldamage (Cavaliere et al., 2004; Skaper et al., 2006), we now com-bined in vivo and in vitro approaches to test the impact of a selectiveand blood-brain barrier-permeable P2X7R antagonist in the maindifferent features of PD. Thus, we tested the impact of Brilliant BlueG (BBG) on the motor impairments, the loss of striatal and nigradopamine, the loss of dopaminergic markers such as tyrosine hy-droxylase and the astrogliosis and microgliosis caused by 6-OHDA;additionally, we used purified nerve terminals and differentiateddopaminergic cells to test if BBG could control synaptotoxicity and/or neurotoxicity induced by 6-OHDA.

2. Materials and methods

2.1. Animals and drug administration

Adult maleWistar rats (200e250 g) were obtained from either the animal houseof the Federal University of Ceará or from Charles River (Barcelona, Spain) and weremaintained at 23e25 �C, with 12 h light-12 h dark cycle and standard diet and tapwater ad libitum. All procedures in this study were conducted in accordancewith theprinciples and procedures outlined as “3Rs” in the guidelines of the European Union(2010/63/EU), FELASA and ARRIVE, and were approved by the Portuguese EthicalCommittee (DGAV) and by the Ethics Committee on Animal Experimentation of the

Federal University of Ceará (CEPA). Rats were anesthetized with a combination ofketamine (100 mg/kg, i.p.) and xylazine (20 mg/kg, i.p.), and unilateral intrastriatal6-OHDA injections (18 mg/3 mL in a 0.02% ascorbate-saline solution) were performedthrough a 5 mL Hamilton� syringe using a stereotaxic apparatus (Stoelting, USA) atthe following coordinates (mm): site 1: L: �2.5, AP: þ0.5, V: þ5.0; site 2: L: �3,AP: �0.5, V: þ6,0; and site 3: L: �3.7, AP: �0.9, V: þ6.5 from the bregma, accordingto the Atlas of Paxinos andWatson (2006). Sham-operated rats (SO) received vehicleandwere used as controls. The blood-brain barrier-permeable and efficacious P2X7Rantagonist Brilliant Blue G (BBG, 45 mg/kg dissolved in saline; from Sigma-Aldrich,Portugal) (see Ryu and McLarnon, 2008; Díaz-Hernández et al., 2009; Arbeloa et al.,2012; Kimbler et al., 2012) or saline were administered intraperitoneally 2 h aftersurgery and every 48 h for two weeks. This same dose of BBG has previously beenshown to yield a brain concentration of 200e220 nM (Díaz-Hernández et al., 2012),which is within the effective and selective range of BBG towards central P2X7R(Donnelly-Roberts and Jarvis, 2007). Behavioral tests were carried out from the 15thuntil the 18th days after surgery and animals were sacrificed on the 19th day.Importantly, the lack of effect of BBG in control animals in the first series of ex-periments prompted the recommendation from the Ethical Committee to compareall modifications with saline injected control animals to stop using an additionalgroup of control animals injected with only BBG.

In an additional group of experiments, we tested another selective P2X7Rantagonist, A438079 (3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridinehydrochloride, from Tocris, Bristol, United Kingdom) (Donnelly-Roberts and Jarvis,2007). A-438079 (10 mM) was administered (0.25 mL/h for 15 days) directly intothe right lateral ventricle through osmotic minipumps (Model 1002; Alzet Corpo-ration, CA), placed in a subcutaneous pocket in the dorsal region and connected viapolyethylene tubing to an intracranial cannulae (Alzet Brain Infusion Kit II) placed inthe following coordinates relative to bregma: 1.5 mm posterior; 1.0 mm lateral;3.7 mm below the horizontal plane of bregma. Control animals received a similar icvadministration of vehicle (saline) for 2 weeks and behavioral testing was carried outon the 15th day.

2.2. Rotational behavior

Eighteen days after surgery (after other behavior tests), animals were tested forrotational behavior after receiving an intraperitoneal bolus injection of apomor-phine hydrochloride (0.6 mg/kg; from Sigma-Aldrich). Rotational testing was con-ducted following the methodology initially described by Ungerstedt (1971). Briefly,animals were placed inside a cylindrical container (33 cm diameter and 35 cmheight) and contralateral rotations (the number of 360� contralateral turns) werecounted for 60 min in a quiet isolated room.

2.3. Open field test

The rats were tested for locomotor activity 15 days after surgery using an openfield apparatus as previously described (e.g. Canas et al., 2009), which consisted of ablack acrylic chamber (50� 50 cm), with 50 cm highwalls, and the floor was dividedinto four squares of equal size (Broadhurst, 1957). Each rat was positioned in thecenter of the arena and allowed to explore freely. The number of crossings andrearings were scored for 5 min. The arena was cleaned with 20% ethyl alcohol toremove any odors after testing each animal.

2.4. Passive avoidance test

Seventeen days after surgery, aversive memory was assessed as previouslydescribed (Dall’Igna et al., 2007) using a two-compartment apparatus(50� 22 � 27 cm; length xwidth x height) from Ugo Basile (Italy). In the acquisitiontrial, each rat was placed individually in the light compartment, and when the an-imal entered the dark compartment, a foot shock of 0.5 mA was delivered throughthe grid floor. The latency time to enter the dark compartment wasmeasured, with acutoff time of 300 s (baseline). The animal was removed from the apparatus, and thetrial was repeated 15 min later (early memory). After 24 h, the retrieval trial wasperformed in the same manner, but without subjecting rats to foot shocks (latememory).

2.5. Cued water maze test

On the 15th day after surgery, a separate group of rats was submitted to the cuedversion of the Morris water maze task (Morris, 1984), following the modificationsintroduced by Packard and McGaugh (1992) to model habit learning. The test wasperformed in a circular swimmingpoolwith 180 cm indiameter and60 cmdeep,filledwith opaque water. It consisted of four training days with four consecutive trials perday, during which the animals were placed in the pool and allowed to swim freely tothe escape platform placed in the center of one of the four imaginary quadrants of thepool. The escape platform was submerged and its position was cued by a 7-cmdiameter white ball attached to the top of the platform and protruding above thewater. The position of the escape platformwas always changed during each trial of theday. Several distal visual cueswere placed on thewalls of the room. The initial positioninwhich the animal was left in the tank was one of the four vertices of the imaginaryquadrantsof thepool, and thiswasvaried among trials in apseudo-randomway. If a ratdid not find the platform during a period of 60 s, it was gently guided to it. The animal

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152144

wasallowed to remainon theplatformfor20 s and then removed fromthe tank for 30 sbefore beingplaced in the next random initial position. The scores for latencyof escapefrom the starting point to the platformwere measured.

2.6. Determination of monoamine levels

The measurement of dopamine (DA) and 3,4-dihydroxyphenylacetic acid(DOPAC) in striatal and mesencephalic tissue (n ¼ 6 per group) was carried out byHPLC, as previously described (Borycz et al., 2007). Tissue homogenates (10% w/v)were sonicated in 0.1 M HClO4 for 30 s, centrifuged at 4 �C for 15 min at 15,000 rpm,and the supernatant was filtered (0.2 mm, Millipore). A 20-mL sample was theninjected into the C-18 HPLC column. The mobile phase was 0.163 M citric acid (pH3.0), containing 0.02 mM NaCl with 0.69 mM sodium octanesulfonic acid as the ionpairing reagent, 4% v/v acetonitrile and 1.7% v/v tetrahydrofuran. DA and DOPACwere electrochemically detected, using an amperometric detector (Shimadzu,Japan). The amount of monoamines was determined by comparison with freshlyprepared standards, and their concentrations were expressed as ng/mg of tissue.

2.7. Immunohistochemical analysis

Immunohistochemistry analysis was essentially carried out as previouslydescribed (Rebola et al., 2011; Gonçalves et al., 2013). Briefly, the animals were anes-thetized with sodium thiopental and transcardially perfused with ice-cold salinephosphate buffer (PBS) followed by 4% paraformaldehyde in PBS. The brains wereremoved, post-fixed in 4% paraformaldehyde for 16e24 h, and cryoprotected in 30%sucrose for 48h at 4 �C. Thebrainwas then frozen indry ice and50mmcoronal sectionswere prepared using a cryostat (Leica CM3050 S, Heidelberg, Germany) at �21 �C.Tyrosine hydroxylase (TH) immunodetection was performed in a one-in-six series of50 mm free-floating sections. The sections were washed 3 times for 10 min with PBSand incubated with PBS supplemented with 10% methanol and 1.05% hydrogenperoxide for 40 min at room temperature (RT), to block endogenous peroxidase-likeactivities. After washing 3 times for 10 min with PBS and blocking endogenous pro-teins with 10% normal goat serum in PBS supplemented with Triton X-100 (blockingsolution) for two hours at RT, the sections were incubated with the primary antibody(rabbit anti-TH, 1:1000, Millipore) diluted in blocking solution at 4 �C for 48 h. Thesections were thenwashed with PBS before incubation for 2 h at RT with a secondaryantibody (goat biotinylated anti-rabbit, 1:200, Vector Labs) diluted in blocking solu-tion, andwashedwithPBS. TheABCmethod (Vector Labs)wasused for 40minatRT foramplification of the signal which was revealed with DAB Peroxidase Substrate Kit(Vector labs). The reaction was stopped by washing with PBS before mounting ongelatin-coated slides, dried, dehydrated by a gradient of ethanol and cleared withxylene. Finally the sections were coverslipped with Eukitt (Fluka-Sigma).

Double staining forGFAP(rabbitpolyclonal,1:1000;DAKO,Glostrup,Denmark) andCD11b (rat monoclonal 5C6, 1:200; AbD Serotec, Oxford, UK) was carried out in free-floating sections by incubation at 4 �C for 48 h, whereas Iba-1 staining (rabbit poly-clonal, 1:1000; WAKO, Osaka, Japan) was carried out in free-floating sections by incu-bation at 4 �C for 72 h, both in blocking solutionwith the primary antibodies. Sectionswere washed 3 times and incubated for 2 h at RT with the corresponding secondaryantibodies coupled to fluorophores, namely goat anti-mouse or goat anti-rabbit AlexaFluor 488 or Alexa Fluor 594 (1:200e1:1000; Molecular Probes e Invitrogen, Eugene,OR) diluted in the blocking solution. The sections were washed 3 times and thenmounted with Dako fluorescent medium (Dako, USA) on gelatin-coated slides.

The stained brain sections were visualized using an epi-fluorescent microscopeZeiss Imager Z2. Immunoreactivity was measured by semi-quantitative densito-metric analysis using an image-analysis program (ImageJ software). With freehandselection we measured the optical density of both striata (ipsilateral and contra-lateral) using the anterior commissure as a negative control. The values obtained forthe sham-operated group were averaged and the values of other groups calculatedas a percentage of that mean.

2.8. Evaluation of P2X7 receptors and 6-OHDA-induced neurotoxicity in SH-SY5Ycells

SH-SY5Y cells (LGC Promochem) were cultured in a 1:1 mixture of Ham’s F12and Dulbecco Modified Eagle Medium (DMEM) (Invitrogen) supplementedwith 10%fetal bovine serum (FBS, Invitrogen) and a mix of antibiotic and antimycotic (Invi-trogen), in a humidified atmosphere of 5% of CO2 in air at 37 �C. Differentiation wasinduced by lowering the FBS in culturemedium to 1% and adding 10 mM retinoic acidduring 7 days (Lopes et al., 2010). After differentiation, cells were treated with BBG(100 nM) for 30 min before exposure to 30 mM 6-OHDA during 24 h. 6-OHDA wasfreshly prepared in 0.1% ascorbic acid to avoid oxidation.

The activity of cellular dehydrogenases, taken as an index of cellular dysfunction,was estimated by the quantification of the ability of the cells to reduce 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to form a blue for-mazan product. At the end of the exposure to drugs, cells were incubated with 1 mgof MTT (from a recently prepared stock of 5 mg/mL in PBS) and the cultures werereturned to the incubator for 3 h to allow MTT reduction to proceed. After the in-cubation media was dumped off, the purple formazan crystals were dissolved in1 mL isopropanol during 30 min on a shaking table. The resulting purple solutionwas spectrophotometrically measured at 540 nm.

Cellular damage was additionally evaluated by measuring the amount of cyto-plasmic lactate dehydrogenase (LDH) released into the medium, using an assaybased on the reduction of NADþ by the action of LDH. At the end of the treatments,the medium from each well was transferred to clean flat-bottom plate to proceedwith enzymatic analysis accordingly to the in vitro toxicology assay kit (HospitexDiagnosis).

For immunocytochemical analysis, the cells were fixed in 4% paraformaldehydein PBS at RT for 20 min, washed three times with PBS and blocked for 40 min withPBS containing 1% bovine serum albumin and 0.1% Triton X-100. Cells were thenincubated with rabbit anti-P2X7R antibody (1:500, from Alomone Labs) and mouseanti-TH antibody (1:1000, from Sigma) diluted in blocking solution at RT for onehour, washed three times in PBS with 0.1% Triton X-100, and incubated at RT for40 min with donkey AlexaFluor-594-conjugated anti-rabbit IgG and donkeyAlexaFluor-488 conjugated anti-mouse IgG (1:500, from Molecular Probes) in PBScontaining 0.5% albumin. After rinsing 3 times for 10 min in PBS, nuclei were stainedwith DAPI (2 mg/mLe fromMolecular Probes) for 10min. The coverslips were rinsed3 times in PBS and were mounted with Dako fluorescent medium (Dako, USA) ongelatin-coated slides. Cells were analyzed in a laser scanning confocal microscope(LSM 510 META, Zeiss). We confirmed that there was no labeling of SH-SY5Y cellswhen each of the primary antibodies was omitted in the assay (data not shown).

2.9. Evaluation of P2X7 receptors and 6-OHDA-induced toxicity in striatalsynaptosomes

Synaptosomes were prepared from the striatum using a sucrose/Percoll-basedseries of centrifugations, as previously described (Canas et al., 2009). The synapto-somes, with less than 2% of glial contaminations (see Cunha et al., 1992), wereplatted and fixed to carry out immunocytochemical double labeling experiments, aspreviously described (Rodrigues et al., 2005; Quiróz et al., 2009), to define the co-localization of P2X7R with either tyrosine hydroxylase (TH) or dopamine trans-porters (DAT). P2X7R were labeled with a rabbit anti-P2X7R antibody (1:500, fromAlomone Labs), which was co-incubated for 1 h at RT with a mouse anti-TH antibody(1:1000, from Sigma) or a mouse anti-DAT antibody (1:500, from Millipore), fol-lowed by incubation with donkey AlexaFluor-594-conjugated anti-rabbit IgG anddonkey AlexaFluor-488 conjugated anti-mouse IgG (1:500, from Molecular Probes).The preparations were examined under an a Zeiss Imager Z2 fluorescence micro-scope equipped with an AxioCam HRm and 63� Plan-ApoChromat oil objective (1.4numerical aperture) and analyzed with ImageJ 1.37v software (NIH, Bethesda, MD).Each coverslip was analyzed by counting three different fields and in each field atotal amount of 500 individualized elements, as previously described (Rodrigueset al., 2005). We confirmed that there was no labeling of synaptosomes wheneach of the primary antibodies was omitted in the assay (data not shown).

For the toxicity assays, the synaptosomes were resuspended in Krebs’ buffer(140 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM EDTA, 10 mM glucose, pH 7.4) tomeasure the redox status of synaptosomes, a measure of their functionality that isknown to be affected by exposure to toxins used to model PD (Park et al., 2002;Fonck and Baudry, 2003). This was carried out using Alamar blue (from Sigma), aspreviously described (Springer et al., 1998). Briefly, synaptosomes were aliquotedinto 96-well plates (100 mL per well, with 50 mg of protein) and incubated at 37 �Cwith 100 nM BBG or vehicle and, 15 min later, 30 mMOHDA or vehicle was added for2 h. After that, 95 mL of synaptosomes in Locke’s buffer (154 mM NaCl, 5.6 mM KCl,2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM HEPES, pH 7.4) were collectedand mixed with 10.5 mL of Alamar Blue dye. The mixture was incubated for 100 minat 37 �C and then centrifuged at 2200 g for 5 min. The supernatant was carefullyremoved into a 96-well plate and it was read at 570 nm. Values were expressed asthe percentage of optical density of control synaptosomes, in the absence of addeddrugs. Protein determination was performed with the BCA method.

3. Results

3.1. BBG attenuates the 6-OHDA-induced rotational behaviorwithout affecting spontaneous locomotion

6-OHDA-lesioned rats display a significant increase of thenumber of apomorphine-induced contralateral rotations (measuredduring 60min) compared to sham-operated rats (sham: 3.4�1.3 in60 min; 6-OHDA: 237.0 � 27.6 in 60 min; n ¼ 8, p < 0.05); this wasattenuated by nearly 50% upon treatment with the P2X7 receptor(P2X7R) antagonist Brilliant Blue G (BBG, 45 mg/kg) (6-OHDA þ BBG: 137.4 � 27.4; n ¼ 8, p < 0.05), whereas BBG wasdevoid of effects in sham-operated rats (Fig. 1A). Another selectiveP2X7R antagonist A-438079 (10 mM), administered at 0.25 mL/hourfor 15 days directly into the right lateral ventricle through osmoticminipumps, also significantly (p < 0.05; n ¼ 8e9) attenuatedapomorphine-induced contralateral rotations in 6-OHDA-lesioned

Fig. 1. P2X7 receptor blockade attenuates the asymmetrical turning behavior but not spontaneous locomotion in rats treated with 6-hydroxydopamine and challenged withapomorphine. (A) The P2X7 receptor antagonist Brilliant Blue G (BBG, 45 mg/kg, i.p) attenuated the apomorphine-induced rotational behavior in 6-OHDA-lesioned rats, whereas itwas devoid of effects in saline-injected rats, as gaged by the number of contralateral rotations counted for 60 min, evaluated 19 days after 6-OHDA striatal administration. (B)Another P2X7 receptor antagonist A438079 (10 mM, applied intra-cerebroventricularly through a minipump) also decreased the number of contralateral rotations counted for60 min, evaluated 15 days after 6-OHDA striatal administration. Both BBG (C) and A438079 (D) failed to modify the exploration activity of 6-OHDA-injected rats, as gaged by thenumber of crossings and rearings counted for 5 min, evaluated 15 days after 6-OHDA striatal administration. Data are mean � S.E.M. with 6e8 rats per group. *p < 0.05 vs. sham-operated, ##p < 0.05 vs. 6-OHDA, using a two-way ANOVA followed by Tukey’s post-hoc test.

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152 145

rats and was devoid of effects in sham-operated rats (Fig. 1B).Notably, the exploratory behavior (measured during 5min) was notaffected by either 6-OHDA administration and/or BBG or A-438079treatments, when evaluated 15 days after surgery as the number ofcrossings and rearings in the open field test (Fig. 1CeD).

Since BBG was devoid of effects in non-challenged rats in the 3initial behavioral tests we carried out, ethical constraints on theusage of experimental animals forced us limit the use of the groupof control animals treated with BBG and to compare all experi-mental groups with saline-treated control rats in all subsequentin vivo studies.

3.2. BBG attenuates memory deficits in 6-OHDA-lesioned rats

Aversive memory was significantly impaired in 6-OHDA-lesioned rats (early memory: 100.8 � 34.9 s; late memory:120.8 � 32.8 s; n ¼ 8/group) compared to the sham-operated group(earlymemory: 299.0� 0.0 s; latememory: 219.3� 32.6 s; n¼ 6e8/group, p < 0.05). The treatment with BBG tended to ameliorate theperformance in this aversive test, since both early and late memoryscores were not significantly different from sham-operated rats (asoccurred for 6-OHDA-treated rats), albeit the performance of ratschallenged with 6-OHDA and treated with BBG (early memory:217.7 � 54.0 s; late memory: 157.3 � 63.8 s, n ¼ 8/group) was alsonot statistically different from 6-OHDA-lesioned rats (Fig. 2A). Asimilar result was observed in a cued version of the Morris watermaze task, in which 6-OHDA caused a worsening of performanceand BBG treatment tended to attenuate this deficit since the meanescape latency throughout the training days of 6-OHDA-challengedrats treated with BBG was no longer different from control (as 6-OHDA-treated rats), but also failed to be statistically different fromthe performance of 6-OHDA-challenged rats (Fig. 2B).

3.3. BBG attenuates the loss of monoamine in 6-OHDA-lesioned rats

As shown in Table 1, the 6-OHDA administration decreased dopa-mine (DA) levels in the striatum (44.9 � 21.7 ng/mg tissue) andmesencephalon (14.2 � 4.1 ng/mg tissue) when compared to sham-operated rats (striatum: 1332.0 � 349.3; mesencephalon:298.5 � 142.8 ng/mg tissue; n ¼ 5, p < 0.05) as well as a decrease ofDOPAC in the striatum (56.2� 16.8 ng/mg tissue) andmesencephalon(6.5 � 3.0 ng/mg tissue) when compared to controls (striatum:471.4 � 99.9; mesencephalon: 53.0 � 34.6 ng/mg tissue; n ¼ 5,p< 0.05). BBG treatment prevented the 6-OHDA-induced decrease inthe content of DA and DOPAC in the striatum andmesencephalon anddidnotcausesignificantchanges (p>0.05) in theshamgroup(Table1).

3.4. BBG attenuates the loss of tyrosine hydroxylaseimmunoreactivity in 6-OHDA-lesioned rats

As shown in Fig. 3, 6-OHDA-treated rats displayed a significantdecrease of the area of tyrosine hydroxylase (TH) staining in theipsilateral striatum compared to sham-operated (control) rats (%mean of controls: 6-OHDA¼ 54.6� 0.4%, n¼ 4, p< 0.05) aswell as inthe substantianigra (%meanof controls: 6-OHDA¼ 46.2�1.8%,n¼ 4,p < 0.05). BBG treatment significantly attenuated the impact of 6-OHDA on TH immunoreactivity in the striatum (% mean of controls:6-OHDAþ BBG¼ 71.7�6.7%) and a similar tendencywasobserved inthe SN, since TH immunoreactivity was 11% larger (% mean of con-trols: 6-OHDA þ BBG ¼ 51.2 � 8.4%) when compared with 6-OHDA-treated rats (Fig. 3). The contralateral striatumwas not affected by 6-OHDA administration, and no significant differences were observedbetween the control and treated groups whereas in the SN a signifi-cant difference of TH immunoreactivity was observed betweengroups (Fig. 3), as noted by others (reviewed inDeumens et al., 2002).

Fig. 2. P2X7 receptor blockade attenuates the memory impairment displayed by ratschallenged with 6-hydroxydopamine. (A) The P2X7 receptor antagonist Brilliant Blue G(BBG, 45 mg/kg, i.p) attenuated the aversive memory in 6-OHDA-lesioned rats, asgaged by the latency time to enter the dark side of a passive avoidance apparatusduring a 300 s session, which was evaluated 17 days after 6-OHDA striatal injectioneither with an inter-trial interval of 15 min (short-term memory) or of 24 h (long-termmemory). (B) BBG (45 mg/kg, i.p) attenuated the 6-OHDA-induced impairment of theperformance in a cued version of the water maze task. The latency to escape to asubmersed platform was evaluated during four training days, with four consecutivetrials per day. Escape latencies for the individual trials were averaged by day. Data aremean � S.E.M. of 6e8 rats per group. *p < 0.05 vs. sham-operated, using a two-wayANOVA followed by Tukey’s post-hoc test.

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3.5. BBG attenuates microgliosis and astrogliosis in the striatum of6-OHDA-lesioned rats

As illustrated in Fig. 4, rats challenged with 6-OHDA displayedan increased density of GFAP-labeled astrocytes (in green; % overcontrol: 1726 � 150, p < 0.05) and CD11b-labeled microglia (inred; % over control: 1310 � 166, p < 0.05) in the striatum(p < 0.05) when compared to sham-operated (control) rats (n ¼ 5).The treatment with BBG significantly decreased the 6-OHDA-induced increase of both GFAP and CD11b immunoreactivities(Fig. 4AeC), as well as the microgliosis evaluated by Iba-1 stainingin the striatum (see inserts in panel A of Fig. 4, showing that 6-OHDA treatment led to more intense Iba-1-stained elementswith fewer and thicker processes that are typical of activatedmicroglia, a profile which was no longer evident upon BBG treat-ment). In the SN, there was also a tendency for an increasedimmunoreactivity of GFAP and CD11b, but this did not reach sta-tistical significance, which precluded analyzing a protective effectof BBG (Fig. 4DeE).

3.6. BBG prevents the 6-OHDA-induced damage of SH-SY5Ydopaminergic cells

The observation that BBG simultaneously abrogated the 6-OHDA-induced gliosis but also the 6-OHDA-induced dopami-nergic neurotoxicity, raises the hypothesis that BBG might play adouble role to control PD-related toxicity, by simultaneously con-trolling the loss of dopaminergic cells and neuroinflammation.Whereas the P2X7R-mediated control of neuroinflammation is wellestablished (Di Virgilio, 2007), the direct control of neuro-degeneration by neuronal P2X7R has seldom been documented(Ortega et al., 2011). Thus, we directly tested if BBG affectedneuronal viability in a cellular model constituted by dopaminergic-like cells free of glial cells.

SH-SY5Y cells differentiated with retinoic acid (10 mM for 7days) acquire a dopaminergic phenotype (all identifiable cells areimmuno-positive for TH; Fig. 5A) and are endowed with P2X7R(Fig. 5B). In accordance with their dopaminergic phenotype,differentiated SH-SY5Y cells are sensitive to 6-OHDA; thus expo-sure to 6-OHDA (30 mM) for 24 h led to a decreased MTT reduction(i.e., indicative of cell dysfunction; Fig. 5D) and an increase oflactate dehydrogenase (LDH) release to the medium (i.e., indicativeof cell damage; Fig. 5E). Whereas the presence of 100 nM BBGprevented the cell damage (LDH release) caused by 6-OHDA, itfailed to affect the impact of 6-OHDA on the dysfunction (MTTreduction) of SH-SY5Y cells (Fig. 5DeE); this is probably due to thefact that BBG per se (i.e. in the absence of 6-OHDA) already affectedthe function of SH-SY5Y cells (Fig. 5D), whereas it did not signifi-cantly affect their viability (Fig. 5E).

3.7. Control by BBG on 6-OHDA-induced dysfunction of striatalsynaptosomes

Since the initial dopaminergic damage in PD occurs in dopami-nergic nerve terminals before evolving into an overt dopaminergicneuronal damage (Scotcher et al.,1991; Berendse et al., 2001; Bézardet al., 2001; Forno et al., 1994), we next tested the hypothesis thatP2X7R might be particular efficient to control the dysfunction ofdopaminergic nerve terminals. We first showed that P2X7R werepresent in dopaminergic nerve terminals from the striatum, asdocumented by the co-localization of the immunoreactivity ofP2X7R andof twomarkers of dopaminergic nerve terminals, namelyTH (Fig. 6A) and dopamine transporters (DAT, Fig. 6B) in rat striatalsynaptosomes. Indeed, P2X7R immunoreacticity was identified in6.4�1.3% (n¼4) of TH-immunopositive striatal nerve terminals andin 5.3�1.9% (n¼ 4) of DAT-immunopositive striatal nerve terminals.Albeitweused an antibodyagainst P2X7Rpreviously validated to beselective based on correlative functional criteria (e.g. Alloisio et al.,2008) and on the use of shRNA (Díaz-Hernández et al., 2008), theuseof P2X7Rantibodies todetect P2X7R innerve terminals is alwaysprone to criticism and requires a functional confirmation (Andersonand Nedergaard, 2006). Thus, we next tested the ability of BBG tocontrol the 6-OHDA-induceddysfunction of striatal nerve terminals.The exposure of rat striatal synaptosomes for 2 h to 30 mM of 6-OHDA led to a 6.7 � 0.8% decrease (n ¼ 4, p < 0.05) of Alamar Bluereduction (Fig. 6C); this synaptic dysfunction was abrogated(p< 0.05) by pre-treatment with 100 nM BBG, which was devoid ofeffect per se (p > 0.05, n ¼ 3).

4. Discussion

The present study provides combined behavioral, morpho-logical and neurochemical evidence supporting the contentionthat P2X7 receptor (P2X7R) antagonism attenuates the dysfunc-tion and damage characteristic of a rat model of Parkinson’s

Table 1Effects of the P2X7 receptor antagonist, Brillant Blue G (BBG, 45 mg/kg) on the decrease of monoamine levels (ng/mg tissue) in the rat striatum and mesencephalon upon 6-OHDA administration.

Group Sham Sham þ BBG 6-OHDA 6-OHDA þ BBG

Striatum DA 1332.0 � 349.3 1294 � 311.9 44.9 � 21.7* 2214.0 � 349.3##

DOPAC 471.4 � 99.9 464.6 � 92.57 56.2 � 16.8* 391.7 � 58.4##

Mesencephalon DA 298.5 � 142.8 129.3 � 67.16 14.2 � 4.1* 286.9 � 94.4##

DOPAC 53.0 � 34.6 45.21 � 29.52 6.5 � 3.0* 125.8 � 31.8##

DA: dopamine; DOPAC: 3,4 dihydroxyphenylacetic acid. Data are mean � SEM of 6 rats/group. *p < 0.05 vs. Sham, ##p < 0.05 vs. 6-OHDA, using a using an one-way ANOVAfollowed by Tukey’s post-hoc test.

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disease (PD). Thus, the selective P2X7R, Brilliant Blue G (BBG),attenuated abnormal behavioral responses such as the asym-metric rotational behavior characteristic of an unilaterally dopa-minergic denervated rats, as well as the deficits in aversive andhabit-related working memory that were triggered by 6-hydroxydopamine (6-OHDA) exposure, used as a PD model. Inaccordance with this ability of P2X7R blockade to prevent dopa-minergic function, we directly documented that BBG attenuatedthe 6-OHDA-induced depletion of the levels of dopamine in theareas mostly afflicted in PD, namely the striatum and the nigra.This was further confirmed by the analysis of histochemical fea-tures characteristic of PD, such as the loss of the dopaminergicmarker (tyrosine hydroxylase) and the exacerbation of markers ofastrogliosis (GFAP immunoreactivity) and of microgliosis (CD11bimmunoreactivity), all of which were attenuated by BBG in 6-OHDA-exposed rats. Additional in vitro studies also concurredthis neuroprotective profile of BBG and actually revealed a doubleability of BBG to prevent the 6-OHDA-induced synaptotoxicity aswell as neurotoxicity. The concurring results from in vivo andin vitro models pertinent to PD largely eliminates the limitation ofnot knowing how the brain concentration of BBG upon peripheraladministration relates to the in vitro described potency of BBG(see Díaz-Hernández et al., 2012). Also, the similar impact of twoselective but chemically different P2X7R antagonists on 6-OHDA-induced rotational behavior further argues for the involvement ofP2X7R in the effects of BBG.

Fig. 3. P2X7 receptor blockade attenuates the loss of a dopaminergic marker (tyrosine hyhydroxydopamine (6-OHDA). (A, B) Representative photomicrographs of TH-immunostainor the SN (B, 5� objective) from sham-injected rats (left pictures), 6-OHDA-injected ratsBrilliant Blue G (BBG, 45 mg/kg, ip). (C, D) Bar graphs displaying the semi-quantitative analyscoronal sections (50 mm thick and 300 mm apart) that were representative of the ipsilateral##p < 0.05 vs. 6-OHDA, using a one way ANOVA followed by Bonferroni post hoc test.

These results indicate the key participation of P2X7R in differentprocesses contributing to dysfunction and damage of the brainregions affected in this 6-OHDA model of PD. This is in agreementwith the concept that ATP is an extracellular signal indicative ofbrain damage (Di Virgilio, 2000). This is supported by the directobservation that different noxious brain stimuli increase theextracellular levels of ATP (Lutz and Kabler, 1997; Juranyi et al.,1999; Davalos et al., 2005; Gourine et al., 2005; Melani et al.,2005) and by the combined genetic and pharmacological studiesthat converge to indicate a neuroprotective role associated with theblockade of P2X7R in animal models of different brain insults (seeIntroduction). We now provide evidence to join to this list theability of P2X7R blockade to alleviate the cardinal behavioral(contralateral rotations), morphological and neurochemical fea-tures of PD.

The exploration of the cellular processes affected by the P2X7Rantagonist BBG revealed several combined features controlled byP2X7R in this animal model of PD. Thus, we found that BBG pre-vented the dysfunction of striatal nerve terminals caused by 6-OHDA. This is in agreement with the previously reported localiza-tion of P2X7R in nerve terminals (Deuchars et al., 2001; Lundy et al.,2002; Cavaliere et al., 2004; Alloisio et al., 2008) and the role ofP2X7R to control the maturation of nerve terminals (Díaz-Hernández et al., 2008) and the release of neurotransmitters(Wirkner et al., 2005; Marcoli et al., 2008). We now extended theseobservations to show that P2X7R were specifically located in

droxylase e TH) in the striatum and substantia nigra (SN) of rats challenged with 6-ing in coronal brain sections (50 mm thick) containing the striatum (A, 1� objective)(central pictures) or 6-OHDA-injected rats treated with the P2X7 receptor antagonistes of TH immunoreactivity in the striatum (C) and SN (D). Analyses were made in serialstriatum or SN. Data are mean � SEM of n ¼ 5 per group. *p < 0.05 vs. sham-operated,

Fig. 4. P2X7 receptor blockade attenuates the astrogliosis and microgliosis in the striatum of rats challenged with 6-hydroxydopamine (6-OHDA). (A, D) Representative photo-micrographs showing the super-imposed immunohistochemical detection of the astrocytic marker GFAP (green) and of the microglia marker CD11b (red) in coronal brain sections(50 mm thick) containing the striatum (A, 20� objective) or the substantia nigra (B, 20� objective) from sham-injected rats (left pictures), 6-OHDA-injected rats (central pictures) or6-OHDA-injected rats treated with the P2X7 receptor antagonist Brilliant Blue G (BBG, 45 mg/kg, ip). The inserted pictures in (A) are Iba-1-stained microglia in each experimentalcondition, which are representative of 3 experiments. (B, C, E, F) Bar graphs displaying the semi-quantitative analyses of GFAP (B, E) and CD11b immunoreactivity (C, F). Analyseswere made in serial coronal sections (50 mm thick and 300 mm apart) that were representative of the striatum (B, C) or substantia nigra (E, F). Data are mean � SEM of n ¼ 5 pergroup. *p < 0.05 vs. sham-operated, ##p < 0.05 vs. 6-OHDA-challenged, using a one way ANOVA followed by Bonferroni post hoc test. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of this article.)

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152148

Fig. 5. P2X7 receptor blockade attenuates the damage of dopamine-differentiated SH-SY5Y cells challenged with 6-hydroxydopamine (6-OHDA). (AeC) Confocal microscopyphotographs showing that the differentiated SH-SY5Y cells were labeled with dopaminergic marker tyrosine hydroxylase (TH) detected immunohistochemically in (A) and werealso endowed with also P2X7R immunoreactivity (B), as can be appreciated in the merged image (C). There was no staining when the primary antibodies were omitted in the assay(not shown). (D) Bar graph showing that the P2X7 receptor antagonist Brilliant Blue G (BBG, 100 nM) did not modify the loss of functionality of SH-SY5Y cells caused by exposure to6-OHDA (30 mM), as gaged by the ability of SH-SY5Y cells to reduce MTT, probably because BBG already caused per se a reduction of the functionality of SH-SY5Y cells. (E) Bar graphshowing that the P2X7 receptor antagonist Brilliant Blue G (BBG, 100 nM) attenuated the damage of SH-SY5Y cells caused by exposure to 6-OHDA (30 mM), as gaged by the release ofthe cytoplasmatic enzyme lactate dehydrogenase (LDH), with all values express as percentage of control (100%, as indicated by the dashed line). Data are mean � SEM of n ¼ 5 pergroup. *p < 0.05 vs. control (no added drugs), ##p < 0.05 vs. 6-OHDA-treated, using a one way ANOVA followed by Bonferroni post hoc test.

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152 149

dopaminergic nerve terminals of the rat striatum, which is inagreement with the ability of P2R to control the release of dopa-mine (Trendelenburg and Bültmann, 2000; Krügel et al., 2003) aswell as dopaminergic functions (Kittner et al., 2001, 2004).

Fig. 6. P2X7 receptor blockade attenuates the dysfunction of striatal synaptosomes challengea percentage of the striatal synaptosomes that are immuno-positive for the dopaminergic mwere also endowed with also P2X7R immunoreactivity (in red), as indicated by the arrowsshown). (C) Bar graph showing that the P2X7 receptor antagonist Brilliant Blue G (BBG, 100(30 mM), as gaged by the decreased reduction of Alamar blue. Data are mean � SEM of n ¼ 4a one way ANOVA followed by Bonferroni post hoc test. (For interpretation of the reference

Additionally, we provide the first direct demonstration that P2X7Rdirectly control the metabolic viability of nerve terminals, asconcluded from the ability of BBG to control the loss of metaboliccapacity of purified nerve terminals exposed to 6-OHDA.

d with 6-hydroxydopamine (6-OHDA). (A, B) Superimposed photographs showing thatarkers (in green) tyrosine hydroxylase (TH in A) or dopamine transporters (DAT in B). There was no staining when the primary antibodies were omitted in the assay (notnM) decreased the loss of functionality of striatal synaptosomes exposed to 6-OHDA

per group. *p < 0.05 vs. control (no added drugs), ##p < 0.05 vs. 6-OHDA-treated, usings to color in this figure legend, the reader is referred to the web version of this article.)

M.R.S. Carmo et al. / Neuropharmacology 81 (2014) 142e152150

Additionally, we also found that BBG attenuated the astrogliosisand microgliosis caused by exposure to 6-OHDA. This is agreementwith the ascribed localization of P2X7R to astrocytes and tomicroglia and with the ability of P2X7R to control the function ofthese glia cells (Bianco et al., 2006; Ferrari et al., 2006; Monif et al.,2009; Gandelman et al., 2010), namely in the substantia nigra(Marcellino et al., 2010). This may be a crucial contributing factorfor the ability of P2X7R to control neurotoxicity, since P2X7R areup-regulated in glial cells upon noxious stimuli (Franke et al., 2004)and several studies have proposed that the P2X7R-mediatedneurotoxicity is mostly due to the control of the noxious impactof glial cells on neuronal viability in conditions of brain damage(Cavaliere et al., 2005; Melani et al., 2006; Skaper et al., 2006; Choiet al., 2007; Matute et al., 2007; Gandelman et al., 2010; Lee et al.,2011). However, the use of differentiated dopaminergic cells in vitroalso allowed us to highlight a direct ability of BBG to preventneuronal damage through putative neuronal P2X7R. In fact, we firstconfirmed that the differentiation of human neuroblastoma SH-SY5Y cells with retinoic acid yields cells with a dopaminergicphenotype (Lopes et al., 2010) and that these cells are endowedwith P2X7R, as previously shown by others (Larsson et al., 2002;Orellano et al., 2010). Furthermore, BBG afforded a direct protec-tion of 6-OHDA-induced neurotoxicity, which supports a directneuroprotective effect of P2X7R, as observed by others using PC12cells (Hracskó et al., 2011) and different neurons in culture (Junet al., 2007; Ortega et al., 2011; Nishida et al., 2012; Anccasi et al.,2013).

Overall, the reported results indicate a potentially triple mech-anism operated by BBG to control 6-OHDA-induced dysfunctionand damage characteristic of PD, namely: 1) the initial synapto-toxicity occurring in dopaminergic nerve terminals of the striatum,2) the gliotic-like features that are associated with the evolution ofneurodegenerative disorders (Marchetti and Abbracchio, 2005;McGeer and McGeer, 2008) and 3) the direct control of theviability of dopaminergic neurons in the substantia nigra, which arethe histological hallmark of PD (Bernheimer et al., 1973; Bézardet al., 2001). This triple potential actions of P2X7R prompt thecontention that P2X7R might affect the different processes associ-ated with the different phases of PD, namely the initiation (striataldopaminergic synaptotoxicity), the propagation (astrogliosis andmicrogliosis) and the execution (nigra dopaminergic cell loss),which contribute to understand the robust ability of the P2X7Rantagonist BBG to prevent the neurochemical features (preserva-tion of striatal and nigra levels of dopamine) and the behavioralfeatures characteristic of the tested 6-OHDA model of PD, namelythe asymmetric rotational. However, it is worth noting that BBGwas more effective to control the 6-OHDA-induced morphologicalmodifications in the striatum than in the substantia nigra. Thissuggests that P2X7R might be more effective to control the initialphases (striatal synaptotoxicity) rather than the later phases (nigradopaminergic cell loss) of PD-related neurodegeneration. This isconsistent with the observation that, in an aggressive model of PD(using anMPTP dosage that resulted in a 50%mortality after 72 h), asingle dose of BBG was ineffective to prevent the decrease ofdopamine levels (Hracskó et al., 2011). Therefore, the present studymainly argues for the possibility that P2X7R might be novelmodulatory drugs to be exploited as novel aiding therapy tomanage early PD.

Acknowledgments

This study was supported by FCT (PTDC/SAU-FCF/100423/08;PEST-C/SAU/LA0001/2011), by UC-Brazil, by the Brazilian NationalResearch Council (CNPq), by the Coordination of Improvement ofHigher Education Personnel (CAPES), by the National Institute of

Sciences and Technology (INCT-IBISAB) and by CAPESeFCT. Wewish to warmly thank Luísa Cortes (Microscopy Unit e CNC),Francisco Queiróz and Fabio Klant for their help.

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