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Neuroscience 141 (2006) 1171–1180

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LIGODENDROCYTES EXPRESS P2Y12 METABOTROPIC RECEPTOR

N ADULT RAT BRAIN

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. AMADIO,a G. TRAMINI,a A. MARTORANA,b

. T. VISCOMI,a G. SANCESARIO,b G. BERNARDIa,b

ND C. VOLONTÉa,c*

Santa Lucia Foundation/CNR, Via del Fosso di Fiorano 64, 00143ome, Italy

University of Rome Tor Vergata, Facoltà di Medicina, Dipartimento dieuroscienze, Rome, Italy

Institute of Neurobiology and Molecular Medicine, CNR, Via del Fossoi Fiorano 64, 00143 Rome, Italy

bstract—In the CNS, nucleotide receptors termed P2 recep-ors are identified on neurons and glial cells, mediating neu-on–neuron, glia–glia and glia–neuron communication. In theresent work, we qualify in vivo in the adult rat CNS theellular/subcellular distribution of P2Y12 receptor protein inerebral cortex, white matter and subcortical nuclei (striatumnd substantia nigra), by means of immunofluorescence-onfocal, electron microscopy and Western blot analysis.2Y12 receptor immunoreactivity colocalizes neither witharkers such as neuronal nuclei, neurofilament light chain,

albindin and tyrosine hydroxylase, nor with glial fibrillarycidic protein and isolectin B4, but with myelin basic proteinnd the oligodendrocyte marker RIP, in both cell bodies androcesses, indicating therefore oligodendrocyte localization.lectron microscopy identifies P2Y12 receptors in both theerikaryon and under the plasmalemma of oligodendrocyteell bodies and radiating processes, until the paranodal re-ion of fibers. By Western blot analysis, P2Y12 receptorhows a specific band of 42–44 kDa, matching the molecularass predicted from amino acid sequencing. Since in plate-

ets P2Y12 receptor is known to regulate adhesion/activationnd thrombus growth/stability, from our results we couldpeculate by analogy that, in oligodendrocytes, P2Y12 recep-or signaling might contribute to the migration and adhesionf the glial processes to axons to be myelinated. © 2006

BRO. Published by Elsevier Ltd. All rights reserved.

ey words: cerebral cortex, myelin basic protein, purinergiceceptors, subcortical nuclei.

n the CNS, a large array of nucleotide receptors (P2eceptors) responding to ADP and ATP is increasinglydentified on neurons and glial cells, and found to be in-olved in neuron–neuron, glia–glia and glia–neuron com-unication (Kirischuk et al., 1995; Fields and Stevens,000; Cotrina et al., 2000; James and Butt, 2002; Amadio

Correspondence to: C. Volonté, Santa Lucia Foundation/CNR Via delosso di Fiorano 64, 00143 Rome, Italy. Tel: �3906-501703084; fax:3906-501703321.-mail address: cinzia.volonte@inmm.cnr.it (C. Volonté).bbreviations: EM, electron microscopy; GFAP, glial fibrillary acidicrotein; IB4, isolectin B4; MBP, myelin basic protein; NeuN, neuronaluclei; NFL, neurofilament light chain protein; PB, phosphate buffer;

PBS, phosphate-buffered saline; PBT, phosphate-buffered saline con-

aining 0.5% Triton X-100; TH, tyrosine hydroxylase.

306-4522/06$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.05.058

1171

t al., 2002; Volonté et al., 2003; Cavaliere et al., 2004a,b).2 receptors are classically divided into ionotropic ATP-ated ion channels (P2X), mediating rapid and selectiveermeability to Na�, K� and Ca2� cations (Bean, 1992;ubyak and el-Moatassim, 1993; Abbracchio et al., 2003),nd into metabotropic G-protein-coupled receptors (P2Y),

nducing slower responses and involving second-messen-er systems (von Kugelgen, 2006). Molecular cloning tech-iques have then identified seven distinct P2X (P2X1–7)nd eight different P2Y (P2Y1,2,4,6,11,12,13,14) receptors.urthermore, P2Y receptors can be pharmacologicallyubdivided into adenine nucleotide-preferring receptors,ainly responding to ADP and ATP (P2Y1,11,12,13 recep-

ors); into uracyl nucleotide-preferring receptors, reactingo either UTP or UDP (human P2Y4, P2Y6 receptors); intoeceptors of mixed selectivity (P2Y2, rodent P2Y4 recep-ors); and finally into receptors responding to sugar-nucle-tides UDP-glucose and UDP-galactose (P2Y14 receptor)Ralevic and Burnstock, 1998; Communi et al., 2001; Ab-racchio et al., 2003). It is nevertheless unclear to whatxtent such a big heterogeneity is limited to molecular andharmacological properties, or whether it is further accom-lished also at cellular and functional level. For instance,icroglial cells widely express several P2X and P2Y re-

eptors, likely involved in the management of inflammatoryeurological diseases characterized by microglia responseBianco et al., 2005). Nevertheless, particularly the P2Y12

eceptor is modulated in microglia cells during develop-ent and recovery after facial nerve axotomy, suggesting

hat chemotaxis of microglia to axotomized motor neuronsould promote regeneration through this specific subtypeSasaki et al., 2003). Using northern blot analysis andn situ hybridization, it was moreover demonstrated thathe P2Y12 receptor is widely expressed in the brain (exceptippocampal and cortical neurons), showing a patternhroughout white and gray matter consistent with astrocytexpression (Hollopeter et al., 2001). However, colocaliza-ion has not been found between P2Y12 and GFAP-posi-ive astrocytes in the rat brain cortex and nucleus accum-ens, despite a strong signal for P2Y12 receptor mRNAFranke et al., 2004). Finally, a scattered distribution of the2Y12 receptor in rat white matter, presumed to occur inligodendrocyte progenitors cells, was also observedLaitinen et al., 2001) and this was confirmed by furthertudies that reported the simultaneous expression, Ca2�

ignaling and function of several P2X and P2Y subtypes inligodendrocyte progenitor cells in vitro (Agresti et al.,005a,b). Nevertheless, it is still unclear whether the

2Y12 receptor is commonly present in the adult rat brain

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S. Amadio et al. / Neuroscience 141 (2006) 1171–11801172

r its expression is restricted only during development orarticular functional and pathological conditions.

In the present work, we therefore qualify by means ofmmunofluorescence-confocal, electron microscopy (EM)nd Western blot techniques, the cellular and subcellularistribution of the P2Y12 receptor protein in selected areasf the adult rat CNS in vivo.

EXPERIMENTAL PROCEDURES

istological procedures

he experimental protocol used in this study was approved by thetalian Ministry of Health and was in agreement with the guidelinesf the European Communities Council Directive of November 24,986 (86/609/EEC) for the care and use of laboratory animals. Allfforts were made to minimize the number of animals used andheir suffering. Wistar rats (n�6) (body weight 200–250 g; Harlan,dine, Italy) were deeply anesthetized by i.p. injections of sodiumentobarbital (60 mg/kg) and transcardially perfused with 250 mlf saline (0.9% NaCl) followed by 250 ml of 4% paraformaldehyde

n phosphate buffer (PB, 0.1 M pH 7.4). Each brain was immedi-tely removed, post-fixed in the same fixative for 2 h and thenransferred to 30% sucrose in PB at 4 °C until it sank.

ouble immunofluorescence

ransverse sections (40 �m thick) were cut on a freezing mic-otome and were processed for double immunofluorescence stud-es. Non-specific binding sites were blocked with 10% normalonkey serum in 0.3% Triton X-100 in phosphate-buffered salinePBS) for 30 min at room temperature. The sections were incu-ated in a mixture of primary antisera for 24 h in 0.3% Triton X-100

n PBS. Rabbit anti-P2Y12 (1:300, Alomone, Jerusalem, Israel)as used in combination with either mouse anti-NeuN (neuronaluclei, 1:100, Chemicon International, Inc., Temecula, CA, USA),ouse anti-calbindin-D-28K (1:200, Sigma, Milan, Italy), mouse

nti-TH (tyrosine hydroxylase, 1:500, Sigma), goat anti-NFL (neuro-lament-light chain protein, 1:100, Santa Cruz, Milan, Italy),ouse anti-MBP (myelin basic protein, 1:200, Chemicon Interna-

ional), mouse anti-RIP (anti-oligodendrocytes, 1:200, Chemiconnternational), mouse anti-GFAP (glial fibrillary acidic protein,:400, Sigma) or biotinylated IB4 (isolectin B4 from Griffonia sim-licifolia seeds, 1:200, Sigma). The secondary antibodies used forouble labeling were Cy3-conjugated donkey anti-rabbit IgG (1:00, Jackson Immunoresearch, West Baltimore Pike, PA, USA,ed immunofluorescence), Cy2-conjugated donkey anti-mousegG1:100, Jackson Immunoresearch, green immunofluorescence) ory2-conjugated donkey anti-goat IgG (1:100, Jackson Immunore-earch, green immunofluorescence). Cy2-conjugated streptavidin1:100, Jackson Immunoresearch, green immunofluorescence)as used for IB4 histofluorescence. The sections were washed inBS three times for 5 min each and then incubated for 3 h in aolution containing a mixture of the secondary antibodies in 1%ormal donkey serum in PBS. After rinsing, the sections wereounted on slide glasses, allowed to air dry and coverslipped withel/mount™ anti-fading medium (Biomeda, Foster City, CA, USA).

riple immunofluorescence

fter double immunofluorescence, the sections were mounted onlide glasses, and allowed to air dry. A rectangle was then drawnround the sections with a PAP pen. To allow the use of a secondouse antibody in the same immunolabeling protocol, the unla-eled monoclonal anti-TH (mouse IgG1 isotype) was labeled withenon technology (Molecular Probes, Eugene, OR, USA). Briefly,

he antibody (1:500) was incubated with Zenon Alexa Fluor 647 r

ouse IgG1 labeling reagent (molar ratio 6:1), which contains auorophore-labeled (Ex/Em 650/668) anti-mouse Fab fragment.he labeled Fab fragments bind to the Fc portion of the mousenti-TH IgG1 antibody and excess Fab fragments are neutralizedy the addition of a nonspecific IgG (Zenon blocking reagent-ouse IgG). The addition of non-specific IgG prevents cross-

abeling of the Fab fragment, in experiments where multiple pri-ary antibodies of the same type are present. After rehydration inBS, the sections were incubated with the staining solution inhosphate-buffered saline containing 0.5% Triton X-100 (PBT) inhumidified chamber for 2 h at room temperature. The sectionsere washed twice in PBT and for 5 min in PBS at room temper-ture. Sections were then fixed in 4% paraformaldehyde in PB for5 min at room temperature, to avoid the dissociation of the Zenonab fragment from the primary antibody, washed three times withBS, allowed to air dry and coverslipped with gel/mount anti-

ading medium.

nti-P2Y12 receptor specificity

he polyclonal antiserum used in this study was raised against2Y12 highly purified peptide corresponding to a specific epitopeot present in any other know protein: residues 125–142 of human2Y12 (2nd intracellular loop). The specificity of the P2Y12 signalas furthermore assessed by incubating the brain slices or theestern blots either in the absence of the primary antiserum, or in

he presence of the primary antiserum together with the neutral-zing P2Y12 immunogenic peptide (�g protein ratio 1:1 betweeneptide and antiserum).

onfocal microscopy

ouble and triple label immunofluorescence was analyzed byeans of a confocal laser scanning microscope (CLSM) (LSM10, Zeiss, Arese Milan, Italy) equipped with an argon laser emit-ing at 488 nm, a helium/neon laser emitting at 543 nm and aelium/neon laser emitting at 633 nm. Representative brain re-ions, in particular including the frontal cortex, corpus callosum,he caudate-putamen, and the midbrain with the cerebral pedunclesnd substantia nigra, were examined in five to 10 coronal sectionsrom at least six animals. Levels of immunofluorescence for eachntibody were assessed in each region by visual inspection andssigned with one of three degrees: strong, moderate or negligible.

M

nimals (n�4) were deeply anesthetized with 3.5% chloral hy-rate in saline solution. The animals were then transcardiallyerfused with saline solution followed by 200 ml of fixative (3%araformaldehyde with 0.1–0.5% glutaraldehyde in 0.1 M PB, pH.4). The brain was removed from the skull and coronal sections70 �m) of the basal ganglia including the striatum, the globusallidus, the entopeduncular nucleus, the substantia nigra wereut on vibrating microtome and collected in PBS 0.1 M pH 7.4. Theections from those animals perfused with high concentrations oflutaraldehyde (0.5%) were treated with sodium borohydrateSigma) 0.1% in PBS 0.01 M, pH 7.4. Sections containing cortex,triatum, corpus callosum and substantia nigra were chosen fromour animals, and were immunostained by the ABC method andre-blocked by incubating the sections in 2% normal goat serum

n PBS 0.1 M, for 1 h. Then sections were incubated with primaryntibody against the P2Y12 (1:500 rabbit anti-P2Y12) diluted in.1 M PBS and supplemented with goat serum 1% for 48 h at 4 °C.he sections were washed several times in 0.1 M PBS, pH 7.4 and

ncubated in biotinylated goat anti-rabbit IgG (1:100; Vectastainlite, Vector Laboratories, Peterborough, UK) for 2 h, at room

emperature. The sections were then washed (PBS) and incu-ated in the ABC complex (1:100; Vector Laboratories) for 1 h at

oom temperature. After washing, the reaction product was re-

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S. Amadio et al. / Neuroscience 141 (2006) 1171–1180 1173

ealed by incubating the section in 0.05% 3,3=-diaminobenzidineDAB) (Sigma) in the presence of 0.01% H2O2. The reaction wastopped by several washes in Tris–HCl buffer followed by PBS.ections were post-fixed in osmium tetroxide (1% in PB 0.1 M, pH.4) for 10 min, dehydrated in ascending series of dilution ofthanol (with the presence of 1% uranyl acetate in 70% ethanol)ollowed by propylene oxide (Aldrich, Milan, Italy) and then em-edded overnight in resin (Durcupan ACM, Fluka, Gillingham,orset, UK), mounted on glass slides and then cured at 60 °C for8 h. The areas of interest were examined in the light microscope,hen ultrathin sections were obtained with an ultramicrotomeReichert-Jung Ultracut E, Leica, Nussloch, Germany), and col-ected on 400-mesh copper, counterstained with lead citrate andxamined using a Zeiss EM900 electron microscope. The speci-city of the pre-embedding immunolabeling was demonstrated byhe absence of labeling for the respective antigen when the pri-ary antibody was omitted.

The ultrastructural analysis was performed exclusively on theost superficial portions of the tissue in contact with the embed-ing plastic, to minimize artificial differences in labeling attributedo potential differences in the penetration reagents. Regions usedor this analysis were chosen on the basis of P2Y12-immunoreac-ivity and the morphological integrity of the tissue. The labeledrofiles were examined in 32 ultrathin sections from three sepa-ate rats. Four sections from the striatum and four from the sub-tantia nigra were selected. All immunoreactive processes wereounted in randomly sampled electron micrographs taken at mag-ifications of 7000–30,000�. Micrographs were examined to de-ermine the relative frequencies with which the immunoreactiveroducts were present within neuronal somata, dendrites, axons,r glia, as well as the synaptic or apposing relationships betweenhe profiles.

The classification of neuronal elements was made accordingo the description by Peters et al. (1991). Neuronal somata weredentified by the characteristics of the nucleus, Golgi apparatusnd endoplasmic reticulum; dendrites were distinguished from notyelinated axons by their larger diameter and/or the abundancef uniformly distributed microtubules and synaptic inputs fromxon terminals. Neuronal profiles were classified as not myelin-ted axons if they were 0.1–0.25 �m in cross-sectional diameternd contained microtubules and/or small vesicles. Axon terminalsere defined as elements 0.25 �m or larger in diameter containingumerous small synaptic vesicles. Synapses formed by axonerminals were defined as asymmetric when their post-synapticensity was thicker that the pre-synaptic one, and as symmetrichen both membranes showed equal electron density. Two struc-

ures were considered opposed when the two plasma membranesere parallel and not separated by glial processes, but no mem-rane specialization was visible.

solation of cerebral areas and protein extraction

istar rats (n�2) were deeply anesthetized by i.p. injections ofodium pentobarbital (60 mg/kg) and, after decapitation, brainsere removed. Each brain was transversally cut on a vibratome

300 �m). By a dissection microscope, the specific cerebral areasere isolated and homogenized in Ripa buffer (1% Nonidet P-40,.5% sodium deoxycholate, 0.1% SDS in PBS containing pro-ease inhibitors). After a short sonication, the homogenates werencubated on ice for 1 h and centrifuged at 14,000 r.p.m. for 10 mint 4 °C. Protein quantification was performed in the supernatantsy Bradford colorimetric assay (Biorad, Milan, Italy).

estern blot analysis

qual amount of cell lysate (100 �g of protein from each cerebralrea) and 25 �g of total protein from highly enriched primaryligodendrocytes in culture (a kind gift from Dr. Cristina Agresti

nd Dr. Francesca Aloisi) was separated by electrophoresis on

pi

0%–12% SDS-PAGE and transferred to nitrocellulose mem-ranes Hybond-C extra (Amersham Biosciences, Cologno Monz-

ig. 1. Immunolocalization of P2Y12 receptor within rat brain. Adult trans-erse rat brain sections were processed for double immunofluorescencetudies. Rabbit anti-P2Y12 receptor (red Cy3 immunofluorescence) wassed in the cortex in combination with mouse anti-NeuN (A), in thetriatum with mouse anti-calbindin-D-28K (B) and in the substantia nigraith mouse anti-TH (green Cy2 immunofluorescence) (C). Confocal im-ges illustrate a strong P2Y12 receptor signal throughout the brain, whichoes not colocalize with neuronal markers. The inset (a) shows a mag-ification of P2Y12 protein immunoreactivity in the cortex: a small roundishell (arrow), resembling oligodendrocyte somata, is juxtaposed to a neu-onal body. The inset (b) shows P2Y12 receptor immunoreactivity in theresence of the neutralizing P2Y12 receptor immunogenic peptide. The

nset (c) shows P2Y12 receptor immunoreactivity in the absence of the

rimary antiserum. Scale bar�20 �m in A; 50 �m in B and C; 5 �m in the

nset (a); 10 �m in the insets (b) and (c).

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S. Amadio et al. / Neuroscience 141 (2006) 1171–11801174

se, Italy). The filters were pre-wetted in 5% non-fat milk in TBS-T10 mM Tris pH 8, 150 mM NaCl, 0.1% Tween 20) and hybridizedvernight with P2Y12 antiserum (Alomone, 1:200). The antiserumas immunodetected with an anti-rabbit HRP conjugated antibody

1:2000) and developed by ECL chemiluminescence (Amershamiosciences), using Kodak Image Station (KDS IS440CF).

RESULTS

onfocal analysis

n the present work we studied the topographic cellular andubcellular distribution of the P2Y12 receptor protein inelected areas of the adult rat brain, in vivo. We show byeans of immunofluorescence and confocal analysis that

mmunoreactivity for P2Y12 protein is detectable in long,hick and parallel nerve fibers forming a large- and a close-esh network respectively on the superficial (Fig. 1A) andeep layers (data not shown) of the cortex. P2Y12 receptor

mmunoreactivity confers instead a patchy appearance tohe striatum, being localized mainly in the white matterhile sparing the projecting GABA-ergic neurons enriched

ig. 2. P2Y12 receptor is not present on microglia, astrocytes andespectively in cortex and striatum, shows that the microglial marker IBmmunofluorescence) do not colocalize with P2Y12 receptor (red Cy3

icroglial cell. In C (cortex) and D (striatum), we performed doubl

mmunofluorescence) antisera. In C arrows and arrowheads represent respectivn the inset; 20 �m in B; 5 �m in C; 1 �m in D.

n the gray matter (Fig. 1B). In midbrain, a strong P2Y12

eceptor immunofluorescence is widespread throughouthe cerebral peduncles, the substantia nigra pars reticulatand compacta containing TH-positive dopaminergic neu-ons (Fig. 1C). Moreover, P2Y12 receptor immunoreactivityeither colocalizes with the general neuronal marker NeuN

n cerebral cortex (Fig. 1A), nor with the specific neuronalarker calbindin in striatum (Fig. 1B), nor with TH in sub-

tantia nigra (Fig. 1C). Therefore, the pattern of P2Y12

eceptor distribution appears different in the gray matter,ith respect to the white matter of the upper corticospinal

ract descending through the basal ganglia to the internalapsule and cerebral peduncles (Fig. 1).

We next evaluated the nature of the P2Y12 receptormmunoreactivity. By NeuN immunofluorescence, we de-ect distinct neuronal cells (inset) dispersed longitudinallylong the P2Y12 receptor-positive fibers (Fig. 1A). We thenrobed sections containing both cortex and striatum witharkers for microglial cells using IB4 (Fig. 2A, cortex, andata not shown, striatum), and astrocytes using GFAP

al processes. In A and B double immunofluorescence, performedy2 immunofluorescence) and the astroglial marker GFAP (green Cy2

uorescence). The inset in Fig. 2A shows a higher magnification of ag with P2Y12 (red Cy3 immunofluorescence) and NFL (green Cy2

neuron4 (green Cimmunofle stainin

ely longitudinal or transversal fibers. Scale bar�10 �m in A and 5 �m

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S. Amadio et al. / Neuroscience 141 (2006) 1171–1180 1175

data not shown, cortex, and Fig. 2B, striatum). We findhat immunoreactivity for P2Y12 receptor is always absentrom both microglia and astrocytes. The P2Y12 receptor-ositive fibers observed in the cortex were then labeledith NFL antiserum, specific marker for neuronal pro-esses (Fig. 2C). The P2Y12 receptor immunofluores-ence (in red) appears juxtaposed to fragments of neuro-lament-positive longitudinally and transversally orientedeuronal fibers. The merged field provides only apparentolocalization between the two signals which, dependingn the plane of sectioning, show yellow stripes (arrow) orots (arrowhead) surrounded by red structures (Fig. 2C).his is confirmed also for the white matter of striatum (Fig.D) and substantia nigra (data not shown): a higher mag-ification indeed confirms that the P2Y12 receptor staining

s shaped as a circular crown (red) clearly surrounding thenternal NFL fiber (green). Due to the close vicinity of thesetructures, although the merged field indicates concentricircles providing overlapping signals in yellow, the red andreen immunoreactivities are instead separated, beingriginated from different structures (Fig. 2D).

We then tested two different markers for oligodendro-ytes: MBP staining cell bodies and myelin sheaths, andIP detecting oligodendroglia somata and multiple pro-esses of varying thickness which end in close proximity toyelin sheaths, but not myelin present in the distal portionf axons (Friedman et al., 1989). In the cortex (Fig. 3A, E)nd white matter of striatum (Fig. 3C), we observe a strongolocalization between P2Y12 receptor and MBP, identify-

ng both longitudinal (arrows) and transversal thick fibersFig. 3A, arrowhead). At higher magnification (Fig. 3E), wean clearly distinguish P2Y12-MBP positive fibers with seg-ental structures and breaks on the myelin sheaths, spi-

aling continuous along the length of the axon, likely iden-ified as internodes (arrows) and nodes of Ranvier. More-ver, we observe a few sparse and small P2Y12-MBPositive structures (3–4 �m diameter) with a few radiatinghort processes, resembling oligodendrocyte somatahich are adjacent to neuronal cell bodies (Fig. 3A inset,A inset a).

Whereas in cortex RIP immunoreactivity is confirma-ory on the cellular localization of P2Y12 receptor in oligo-endrocyte somata (Fig. 3B, arrows), the fibers distin-uished by RIP are generally thinner than those identifiedy MBP immunoreactivity, and colocalize only in part with2Y12 protein, in both cortex and striatum (Fig. 3B, D).his because RIP does not recognize distal myelinheaths, and/or because only a subset of the RIP fibersight carry the P2Y12 protein.

Finally, in the substantia nigra and cerebral peduncles,he P2Y12 receptor immunoreactivity is completely over-apping with MBP (Fig. 4). Whereas in the pars reticulatand cerebral peduncles the P2Y12-MBP signal assumes aascicular compact morphology (Fig. 4A) such as in stria-um, in the pars compacta at higher magnification we canistinctly observe that P2Y12 receptor-positive cells arecattered among neurons and emerge contiguous to TH-

abeled neuronal somata (Fig. 4B). p

In all the brain areas tested, immunoreactivity for2Y receptor is lost by incubating the slices either in the

ig. 3. Colocalization of P2Y12 receptor with MBP and RIP. Confocalmages show that P2Y12 immunoreactivity (red Cy3 immunofluores-ence) in the cortex (A, E) and white matter of striatum (C), colocalizesith MBP (green Cy2 immunofluorescence). In A arrows and arrow-eads represent, respectively, longitudinal or transversal fibers. Ehows a higher magnification of a P2Y12-MBP (red Cy3-green Cy2

mmunofluorescence) positive fiber with visible internodal segmentsarrows). In the inset in A, the arrowheads show P2Y12-MBP positiveligodendrocyte somata adjacent to neuronal cell bodies (nb). B (cor-

ex) and D (striatum) represent double immunoreactivity between rab-it anti-P2Y12 (red Cy3 immunofluorescence) and mouse anti-RIPgreen Cy2 immunofluorescence). The arrows in B indicate P2Y12-RIPositive oligodendrocyte somata. Scale bar�10 �m in A, B, C and D;�m in E and the inset in A.

12

resence of the neutralizing P2Y12 receptor immunogenic

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S. Amadio et al. / Neuroscience 141 (2006) 1171–11801176

eptide (Fig. 1B inset b) or in the absence of the primaryntiserum (Fig. 1B, inset c).

M

M shows features that are for many aspects confirmatoryf the confocal analysis. Specific P2Y12 receptor immuno-

abeling appears like an electron-dense amorphous reac-ion product. In all structures observed (n�402), P2Y12

eceptor is confined to plasmalemma and cytoplasm ofany cells (n�71/402). The EM study shows that in coro-al sections of striatum, substantia nigra and cortex (Fig.A), the P2Y12 receptor–immunolabeled cells share manyorphological features. In general, the P2Y12 receptor

ignal is associated with groups of myelinated fibers or, infew cases, with neurons. The P2Y12 receptor–immuno-

abeled cells show a spherical shape with round, largeucleus and a few slender processes radiating from theell body. The nucleus is in eccentric position (N), withhromatin organized in dense clumps. The cytoplasm ap-ears dense and with numerous microtubules. Organelles

ig. 4. Triple immunofluorescence with P2Y12-MBP-TH antisera. Adummunofluorescence studies. Rabbit anti-P2Y12 (red Cy3 immunofluoence) and mouse anti-MBP (green Cy2 immunofluorescence) andositive cells (yellow) in substantia nigra pars compacta, which are inteB). Scale bar�50 �m in A; 10 �m in B.

ike mitochondria, Golgi apparatus, and cisternae of gran- (

lar endoplasmic reticulum are also observed (Fig. 5A).he processes show abundant microtubules and interme-iate filaments. These cells are accordingly identified asligodendrocytes. Specific P2Y12 receptor immunolabeling

s localized not only in the cytoplasm but also under thelasmalemma (Fig. 5B, arrows) of many of these cells.hen observed in the cytoplasm, it is localized mainly in

he granular endoplasmic reticulum cisternae (Fig. 5A,rrows). When observed in coronal sections from the cor-us callosum, P2Y12-immunolabeling is also observedlong the extensions of the radiating processes (Fig. 5C,rrows), just in proximity of myelin sheaths. Moreover,hen observed in longitudinal sections, specific P2Y12-

mmunolabeling was observed in paranodal regions ofome myelinated axons (Fig. 5D, arrows). In this region thentibody stained diffusely the oligodendroglial cytoplasmf the paranodal region, where it was also observed underhe plasmalemma. Other structures identified as neurons,strocytes and microglia are devoid of any specific labeling

rse rat brain sections from substantia nigra were processed for triple) was used in combination with mouse anti-TH (blue immunofluores-

by confocal microscope (A). The magnification shows P2Y12-MBPamong neurons and are contiguous to TH neuronal somata (arrows)

lt transverescencevisualizedrspersed

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S. Amadio et al. / Neuroscience 141 (2006) 1171–1180 1177

estern blotting

e next verified the presence of the P2Y12 receptor pro-ein in the same cerebral areas also by Western blot anal-sis (Fig. 6). We screened total protein extracted fromdult rat cortex, striatum and ventral midbrain including theubstantia nigra (100 �g/sample), and from highly en-iched oligodendrocyte primary cultures (25 �g) (Agrestit al., 1996). Although expressed within different levels inhe different brain regions, the P2Y12 receptor is alwaysecognized as a specific protein band of 42–44 kDa, cor-esponding to the molecular mass predicted from aminocid sequencing (Fig. 6). This protein signal is furthermorebolished when the nitrocellulose membrane is incubated

n the presence of the P2Y12 receptor neutralizing immu-ogenic peptide. The same 42–44 kDa protein band islso observed in highly enriched oligodendrocyte primaryultures from rat forebrain, therefore confirming the local-zation of the P2Y12 on oligodendrocytes in the CNS (Fig.). We did not detect any P2Y12 receptor signal by per-orming immunoreactions in the absence of the primary

ig. 5. P2Y12 receptor immunoreactivity: EM study. P2Y12-immunoreuclei like the striatum, and substantia nigra (A). These cells were ide

n eccentric position (N), with a few slender processes radiating from thnd also under the plasmalemma (B, arrows). When observed in corobserved along the extension of the radiating processes (C, arrows), inbserved in paranodal regions of myelinated axons (D). Scale bar�1

ntiserum (data not shown). i

DISCUSSION

TP released by neurons in an activity dependent mannerFields and Stevens, 2000), and by astrocytes and micro-lia via membrane-transporters, vesicles and ATP-perme-ble channels (Ballerini et al., 2002; Coco and others,003), is known to influence oligodendrocyte functions. For

nstance, ATP- and ADP-mediated activation of metabo-ropic P2 receptors induces migration and inhibits prolifer-tion of oligodendrocyte progenitor cells in vitro (Agresti etl., 2005a,b), increases cytoplasmic Ca2� concentration inature oligodendrocytes in culture (Kirischuk et al., 1995;e and McCarthy, 1994) or in corpus callosum and opticerve (Bernstein et al., 1996; James and Butt, 2001).oreover, the development of Schwann cells and oligo-endrocytes is mediated respectively by activation of neu-onal P2 receptors in the PNS (Stevens and Fields, 2000)r P1 receptors in the CNS (Stevens et al., 2002). Ourresent results therefore confirm and extend these studies,ualifying by various means (immunofluorescence-confo-al analysis, EM, Western blotting) the distribution in vivo

as observed sharing several cellular features in cortex, in subcorticaloligodendrocytes, having a spherical shape with round large nucleusy (A). Specific immunolabeling was localized in cytoplasm (A, arrows)ns taken from the corpus callosum, P2Y12-immunolabeling was also

y of the myelin sheaths. In longitudinal sections, specific labeling wasA; 0.8 �m in B; 0.5 �m in C and D.

activity wntified ase cell bodnal sectio

n selected areas of the adult rat CNS of the metabotropic

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S. Amadio et al. / Neuroscience 141 (2006) 1171–11801178

2Y12 receptor at the interface between axons andligodendrocytes.

In detail, we show here that in cerebral cortex, striatumnd substantia nigra the metabotropic P2Y12 receptor isbsent from neuronal cell bodies (negative double stainingith NeuN in cortex, calbindin in striatum, TH in substantiaigra) and from dendrites and axons (negative doubletaining with NFL in cortex, striatum and substantia nigra).t is moreover absent from astrocytes, confirming previousndings (Franke et al., 2004), and from microglia that,owever, expresses P2Y12 receptor mRNA (Sasaki et al.,003). Throughout the corticospinal tract, the P2Y12 recep-or protein is instead exclusively localized in oligodendro-ytes, therefore suggesting high conserved tissue-homo-eneity and phenotype-specificity. Like constitutive MBPHartman et al., 1982), the P2Y12 receptor immunolabelings strong and uniform within the oligodendrocyte somata,long their radiating processes, up to the myelin sheaths.n contrast, the P2Y12 receptor fluorescence overlaps onlyn part with the RIP signal, known to be absent on myelinn the distal portion of axon (Friedman et al., 1989). Nev-rtheless, not all the proximal RIP-positive fibers possesshe P2Y12 receptor, therefore indicating discontinuity ofxpression and/or selectivity for specific fiber subsets.hus, the analysis of the cellular distribution of the P2Y12

eceptor protein in oligodendrocytes can already provide arst insight on its predicted biological role. In particular, theerikaryal localization of the P2Y12 receptor might be in-icative of a process of receptor protein synthesis,hereas the presence under the plasmalemma can sug-est for instance involvement in receptor-mediated signal-

ng and Ca2� fluxes from intracellular stores, or organiza-

ig. 6. The presence of P2Y12 receptor in rat brain is confirmed byestern blot analysis. Equal amount (100 �g/sample) of total protein

rom adult rat cortex, striatum and ventral midbrain including theubstantia nigra, was separated by SDS-PAGE and transferred byestern blotting. Filters were stained with Ponceau-S and then immu-

ostained with rabbit anti-P2Y12 antiserum. Total protein (25 �g) fromighly enriched oligodendrocyte primary cultures was run on a parallelel and subjected to Western blotting. Incubations with the P2Y12

eceptor antiserum were performed in the absence or presence (neu-ralizing peptide) of the immunogenic peptide. The results are repre-entative of two independent experiments.

ion of membrane constituents for the ensheathment of r

xons. In addition, since the P2Y12 receptor is already wellnown to regulate platelet adhesion/activation and throm-us growth and stability (André et al., 2003), or suggestedo mediate microglia chemotaxis (Nasu-Tada et al., 2005),y analogy we could also speculate that the P2Y12 recep-or signaling in oligodendrocytes might contribute to theigration and adhesion of the glial processes to the axons

o be myelinated. Previous results on both P2 receptor-ependent modulation of Ca2� transients and oligoden-rocyte migration do not exclude our hypothesis (Kirischukt al., 1995; James and Butt, 2001; Agresti et al., 2005a,b).

Throughout the entire cortical layers and basal ganglia,e also observed that the P2Y12 receptor signal is fre-uently juxtaposed to NFL immunoreactivity, thus suggest-

ng a functional neuron–oligodendrocyte crosstalk, androving the association of the P2Y12 receptor not only withhe large spectrum of neurons present in these areasMountcastle, 1997), but especially with longitudinally andransversally oriented intracortical fibers interconnectingortical areas with each other and with subcortical struc-ures. Since there are no actual borders among corticalayers, and neurons cross layer boundaries with their den-rites and axons trees all over, it is conceivable that thebiquitous presence of the P2Y12 receptor on cortical oli-odendrocytes brings up a new marker on the unusualtructural consistency of the cortex, pointing to a broad role ofhis receptor in all the cortex circuitry. This can be extendedo all the upper corticospinal tract, where we always locatehe P2Y12 receptor protein at the interface between neuronalodies, axons and oligodendrocytes, therefore reinforcinghe possibility of a tight structure/function interplay be-ween neurons and oligodendrocytes mediated, perhaps inart, by the P2Y12 receptor. In all these areas, wherever ayelinated axon is running, either single, in fascicles or in

racts, there is a positive P2Y12 receptor immunoreactivity.his could be very well extended to the corpus callosumnd deep layers of the entorhinal cortex, where the P2Y12

eceptor antiserum is described by the manufacturer toabel fibers also connecting these structures.

Since neurons of the cortex and basal ganglia useeveral neurotransmitters which are also active on oligo-endrocytes (Verkhratsky and Steinhauser, 2000), amonghich are GABA, glutamate, dopamine (Umemiya andaymond, 1997; Cheronis et al., 1979; Gerfen, 2000; Cen-

onze et al., 2001), and ATP itself (Soliven, 2001), weoreover do not exclude that in these cerebral areas the2Y12 receptor could be indirectly involved in diseasesharacterized by abnormalities in the production and re-

ease of these neurotransmitters, or in disorders that di-ectly affect oligodendrocyte integrity and ability to pro-uce/maintain myelin sheaths. All these dysfunctions inact target not only neurons, but also the oligodendrocyteounterpart (Schapira, 2005) and confirm the ability ofligodendrocytes to operate in lesioned areas of the CNSFranklin, 2002). Thus, under acute and chronic neurode-enerative/inflammatory diseases, ATP released in aber-ant amount with other neurotransmitters might have aual function: to exacerbate cortex and basal ganglia neu-

onal cell loss, through P2 receptors different from the

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S. Amadio et al. / Neuroscience 141 (2006) 1171–1180 1179

2Y12 subtype (Cavaliere et al., 2002; Ryu et al., 2002;madio et al., 2002; Melani et al., 2005, 2006); to affectligodendrocyte functioning instead involved in the integ-ity protection of the CNS (Stevens et al., 2002), perhapshrough a P2Y12 receptor-dependent mechanism.

CONCLUSION

n conclusion, our study suggests the P2Y12 receptor as aovel building block in the structural consistency of thepper corticospinal tract and in the potential functions re-

ated to neuron-oligodendrocyte crosstalk.

cknowledgments—The research presented was supported byofinanziamento MIUR “Purinoceptors and Neuroprotection.” Weeeply thank Dr. Cristina Agresti and Dr. Francesca Aloisi forroviding cell extracts from highly enriched primary oligodendro-yte cultures.

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(Accepted 25 May 2006)(Available online 10 July 2006)