Introduction 1 2 3 4 11 12 15 2,4,6 1 1 3 7 1,2,4,6,12 2 1 16 2 17 18 4,6 19 20 21 22 23 24 25 12 26 7 27 Our work thus aimed to provide the complete topographical analysis of known P2X and P2Y subtypes that are present in rat striatum and SN in vivo, and to investigate the dynamic presence of P2 proteins after the induction of experimental parkinsonism by dopamine-denervation achieved by using the unilateral 6-hydroxydopamine (6-OHDA) rat model. By upgrading the current map of P2 receptors expressed in the brain, our study discloses the potential impact of these receptors in the normal and lesioned nigro-striatal circuit. Materials and methods Histological procedures Wistar rats (Harlan, Udine, Italy) were anesthetized by i.p. injections of sodium pentobarbital (60 mg/kg), and transcardially perfused with saline (0.9 % NaCl) followed by 4% paraformaldehyde, in phosphate buffer (PB, 0.1 M pH 7.4). Each brain was immediately removed, post-fixed in the same fixative for 2 h, and then transferred to 30% sucrose in PB at 4°C, until it sank. The experimental protocol used in this study was approved by the Italian Ministry of Health and was in agreement with the guidelines of the European Communities Council Directive of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering. Double immunofluorescence Transverse sections (40-μm thick) were cut on a freezing microtome and were processed for double immunofluorescence studies. Non-specific binding sites were blocked with 10% normal donkey serum in 0.3% Triton X-100, in phosphate buffered saline (PBS) for 30 min at room temperature. The sections were incubated in a mixture of primary antisera for 24–48 h in 0.3% Triton X-100 in PBS. Rabbit anti-P2r (1:300, Alomone, Jerusalem, Israel) was used in combination with either mouse anti-calbindin-D-28K (1:200, Sigma, Mi, Italy), mouse anti-tyrosine hydroxylase (TH, 1:500, Sigma), mouse anti-parvalbumin (1:200, Chemicon International, Temecula, CA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:400, Sigma), mouse anti-myelin basic protein (MBP, 1:200, Chemicon International), mouse anti-neurofilament H non-phosphorylated (SMI 32, 1:500, Sternberger Monoclonals, Lutherville, MD, USA), mouse anti-neurofilament H and M non-phosphorylated (SMI 33, 1:500, Sternberger Monoclonals), mouse anti-neurofilament 160 (NF160, 1:500, Sigma) or goat anti-neurofilament-L protein (NF-L, 1:100, Santa Cruz, Mi, Italy). The secondary antibodies used for double labeling were Cy3-conjugated donkey anti-rabbit IgG (1:100, red immunofluorescence, Jackson Immunoresearch, West Baltimore Pike, PA, USA), Cy2-conjugated donkey anti-mouse IgG (1:100, green immunofluorescence, Jackson Immunoresearch) or Cy2-conjugated donkey anti-goat IgG (1:100, green immunofluorescence, Jackson Immunoresearch).  The sections were washed in PBS three times for 5 min each, and then incubated for 3 h in a solution containing a mixture of the secondary antibodies in 1% normal donkey serum in PBS. After rinsing, the sections were mounted on slide glasses, allowed to air dry and coverslipped with gel/mount anti-fading medium (Biomeda, Foster City, CA, USA). Confocal microscopy Double- or triple-label immunofluorescence was analyzed by means of a confocal laser scanning microscope (CLSM) (LSM 510, Zeiss, Arese, Mi, Italy) equipped with argon laser emitting at 488 nm, helium/neon laser emitting at 543 nm, and helium/neon laser emitting at 633 nm. Specificity of the antibodies was positively proved by performing confocal analysis in the absence of the primary antibodies, but in the presence of either anti-rabbit or anti-mouse secondary antibodies. Specificity was further confirmed for the P2r antiserum by performing immunoreactions in the simultaneous presence of the P2r neutralizing immunogenic peptides. Isolation of cerebral areas and protein extraction Wistar rats were anesthetized by i.p. injections of sodium pentobarbital (60 mg/kg) and, after decapitation, brains were removed. Each brain was transversally cut on a vibratome (300 μm). The specific cerebral areas were isolated with the aid of a dissection microscope and homogenized in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS containing protease inhibitors). After short sonication, the homogenates were incubated on ice for 1 h and centrifuged at 14,000 r.p.m. for 10 min at 4°C. Protein quantification was performed in the supernatants by Bradford colorimetric assay (Biorad, Milan, Italy). Western blot analysis 1,2,4 5 4/14 6 2 Anti-P2r specificity The polyclonal P2r antisera used in this study were raised against P2r highly purified peptides (identity confirmed by mass spectrography and amino acid analysis, as indicated in the certificate of analysis provided by the manufacturer), corresponding to specific epitopes not present in any other known protein. The specificity of the P2r signals was moreover assessed by incubating Western blots either in the absence of the primary antiserum, or in the presence of the primary antiserum together with the neutralizing P2r immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum). 6-OHDA lesion and Nissl staining 28 29 n Results P2X and P2Y receptor proteins in rat striatum 1 Fig. 1 1,2,4 4 Panel A 1 1 a 1 b 1 Panel B 2 2 e f Panel C 4 4 g i h i Panel D 4 4 n q o 1,2,4 c A d B m C 4 r D Scale bars A a b B e f C h i l g D n o  1 1 A a 1 1 A 1 A b 1 1 1 1 1 1 A c 2 1 B 1 B e f 2 2 1 B d 3 1 Table 1 Map of P2 receptor proteins in striatum and substantia nigra   Striatum Substantia nigra 1 +++ +++ 2 +++ +++ 3 ++ ++ 4 +++ +++ 5 + +++ 6 + ++ 7 ++ ++ 1 + ++ 2 +++ +++ 4 +++ +++ 6 + +++ 11 – – 12 +++ +++ 13 – – 14 – +++ Materials and methods 4 1 C g i 4 h l 4 4 1 C m 5,6,7 1 1 2 1 4 1 D n q o 4 r 30 31 6 1 11,13,14 1 12 1 26 P2X and P2Y receptor proteins in substantia nigra 2 2,5 2 A B 1,4 1 6,14 2 C D 4 1 3,6 1 1 2 12 1 2 12 26 2,5 a A b B 2 6,14 c C d D 2 11,13 1 Fig. 2 2,5 6,14 2,5 a A b B 6,14 c C d D Scale bars  6-Hydroxydopamine modulates the expression of selected P2 receptors in striatum and substantia nigra 3 A a b Fig. 3 Panel A arrows asterisks a b Panel B 1 Panel C 4 c Scale bars  A B a b C c  1,4 2 12 2 n Table 2 Map of P2 receptor modulation after dopamine denervation   Ipsilateral Striatum  Ipsilateral SN 1 = ↑GABA 2 ↓GABA ↓TH 3 ↓GABA ↓TH, ↑GABA 4 ↓GABA ↓TH, ↑GABA 5 = ↓TH 6 = ↓TH, ↑GABA 7 = = 1 = ↓TH 2 = = 4 ↓GABA ↑GFAP 6 = ↓TH 11 = = 12 = = 13 = = 14 = ↓TH TH GABA GFAP 2 1 3 B 3,4,6 2 4 3 C Discussion 32 33 34 1–7 1,2,4,6,11–14 11 13 5,6 1,6,14 3 6,7 1 1,4 2 2–5 1,4,6,14 2–4 4 2,4 7 27 12 26 35 36 3 37 30 31 38 21 39 41 42 43 44 45 46 1,3,4,6 4 47 48 43 44 49 26 50 In summary, the importance of our work is twofold. We first provide the complete topographical analysis of all known P2X and P2Y receptor subtypes expressed in vivo at their protein levels in rat striatum and SN, which, when considered alongside functional studies, supports a key role for extracellular ATP as a cotransmitter/neuromodulator in these brain areas. Then, we prove that dopamine denervation in the 6-OHDA animal model of Parkinson’s disease generates a significant rearrangement of P2 receptor proteins in these nuclei, therefore disclosing the participation of P2 receptors in the lesioned nigro-striatal circuit. While requiring further investigation, our findings indicate a potential but noteworthy pharmacological and therapeutic novel outcome for Parkinson’s disease.