formed using SuperScript III (Invitrogen) followed by cDNA amplification with specific primers (Eurogentec S.A.). The nucleotide sequences of the primers were as follows: D1R, forward 59- TTCTTCCTGGTATGGCTTGG-39, reverse 59-GCTTAGC- CCTCACGTTCTTG-39; D2LR, forward 59-AACTGTACC- CACCCTGAGGA-39, reverse 59-GTTGCTATGTAGACCGTG -39 and D2SR, forward 59-CACCACTCAAGGATGCTGCCCG- 39, reverse 59-GTTGCTATGTAGACCGTG -39. Amplification of Arginin Rich Protein (ARP), used as an housekeeping gene, was performed using the following primers forward 59- GAACGTCGTCTTCGTGTTCA-39, reverse 59- AAAACCTG- GACGAAGGAGGT -39. Immunocytochemistry and Histological Analysis Striatal neurons were fixed in 2% paraformaldehyde in PBS for 40 minutes at room temperature and permeabilized with Methanol/Acetone (1:1 ratio) for 10 min at 4uC. After washing with PBS, cells were preincubated with blocking buffer (10% Normal Goat Serum in PBS) at room temperature for 1 h and then incubated with primary antibody in PBS overnight at 4uC: mouse anti-microtubule-associated protein 2 (1/1000, Sigma), rabbit anti-P-Cofilin (1/500, Cell Signaling,) and mouse anti- Huntingtin (1/1000, Chemicon). Cells were rinsed with PBS and incubated for 2 h at room temperature with the secondary antibody, Cy3-conjugated anti-mouse IgG (1/2000,Jackson Lab- oratories) or Rabbit-FITC (1/200, Sigma). To analyze the nuclei morphology, cells were stained with Hoechst (1/20000 in PBS) for 5 min at room temperature, followed by three PBS washes. Aggregates of ExpHtt were visualized by GFP staining. The cells were mounted under cover slips using a Vectashield medium (Vector Laboratories) and examined with a Leica DM4000B fluorescence microscope (X40). The percentage of transfected neurons containing aggregates of Exp-Htt were determined by counting neurons containing aggregated GFP versus diffuse GFP. Neurons containing condensed or fragmented nuclei were scored as dying cells. The neuritic length was evaluated by tracing the neurites using the software ImageJ (version 1.34 s). The actin cytoskeleton staining was performed by incubating fixed cells with phallo¨ıdine TRITC (1/500) diluted in PBS-2% BSA for 20 min at room temperature. Each experiment was conducted in triplicate, and at least 100 transfected neurons were randomly analyzed in each experiment. Cell Extracts and Western Blot Analysis Samples were prepared from neurons and HEK293 cells homogenized in a boiling lysis buffer containing 1% SDS and References 1. Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, et al. (1985) Neuropathological classification of Huntington’s disease. J Neuropathol Exp Neurol 44: 559–577. 2. Roze E, Saudou F, Caboche J (2008) Pathophysiology of Huntington’s disease: from huntingtin functions to potential treatments. Curr Opin Neurol 21: 497–503. 3. Cass WA (1997) Decreases in evoked overflow of dopamine in rat striatum after neurotoxic doses of methamphetamine. J Pharmacol Exp Ther 280: 105–113. 4. Vonsattel JP, DiFiglia M (1998) Huntington disease. J Neuropathol Exp Neurol 57: 369–384. 5. Bowyer JF, Clausing P, Schmued L, Davies DL, Binienda Z, et al. (1996) Parenterally administered 3-nitropropionic acid and amphetamine can combine to produce damage to terminals and cell bodies in the striatum. Brain Res 712: 221–229. 6. Luo Y, Umegaki H, Wang X, Abe R, Roth GS (1998) Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem 273: 3756–3764. 7. Ricaurte GA, Guillery RW, Seiden LS, Schuster CR, Moore RY (1982) Dopamine nerve terminal degeneration produced by high doses of methylam- phetamine in the rat brain. Brain Res 235: 93–103. Dopamine/D2 Receptor and HD 1 mM sodium orthovanadate and briefly sonicated. Equal amounts of proteins were resolved by SDS-PAGE gels and transfered to PVDF membrane (Amersham). After blocking in 5% nonfat dry milk (for ROCK-I and ROCK-II) or 5% BSA (Bovine Serum Albumin, SIGMA) (for D1R and D2R immunobloting) in TBS for 1 h at room temperature, membranes were incubated overnight at 4uC with primary antibodies, rabbit polyclonal anti- ROCK-I anti-ROCK-II (1/500, Santa Cruz), mouse monoclonal anti-D1R (1/250, Millipore), rabbit polyclonal anti-D2R (1/1000, Millipore) and mouse monoclonal anti-b-tubulin (1/5000,Sigma), and revealed with appropriate anti-rabbit or anti-mouse peroxi- dase-conjugated secondary antibodies (1/5000, Jackson Immu- noResearch Laboratories) and the enhanced chemiluminescent (ECL) detection system (Pierce). Rho Activation Assay Rho activation was analyzed using Rho assay kit (Upstate Biotechnology). After DA (100 mM) and quinpirole (10 mM) treatments for 15 min, cells were lysed in 50 mM Tris-HCl, pH 7,5, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthova- nadate, 50 mM MgCl2, 0,1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate and complete protease inhibitor cocktail (Sigma) for 15 min at 4uC. The Rho active form, Rho-GTP, was specifically immunoprecipitated from these lysates incubating with Rho Assay Reagent (Rhotekin fused to the Rho Binding Domain on agarose beads) for 45 min at 4uC on a rotator. After a short spin, the pellet beads were washed three times with lysis buffer and resuspended in 25 ml of 2X Laemmli buffer. Rho-GTP activated bound to rhotekin-agarose beads was subjected to 15% SDS-PAGE, followed by Western blotting with mouse monoclonal anti-Rho (-A,-B,-C) antibody (1/250, Upstate). Statistical Analysis All data were statistically analyzed using GraphPad Prism (GrahPad Software Inc., San Diego,CA). Two-way ANOVA followed by post hoc Bonferroni’s test was carried out and for all statistical analyses the difference between comparisons was considered to be significant when P,0.05. Author Contributions Conceived and designed the experiments: CD ER JC SB. Performed the experiments: CD BGR EM DC JC SB. Analyzed the data: CD BGR EM NB ER DC JC SB. Contributed reagents/materials/analysis tools: NB JC SB. Wrote the paper: CD JC SB. 8. Cyr M, Beaulieu JM, Laakso A, Sotnikova TD, Yao WD, et al. (2003) Sustained elevation of extracellular dopamine causes motor dysfunction and selective degeneration of striatal GABAergic neurons. Proc Natl Acad Sci U S A 100: 11035–11040. 9. Cyr M, Sotnikova TD, Gainetdinov RR, Caron MG (2006) Dopamine enhances motor and neuropathological consequences of polyglutamine expanded huntingtin. Faseb J 20: 2541–2543. 10. Charvin D, Vanhoutte P, Pages C, Borrelli E, Caboche J (2005) Unraveling a role for dopamine in Huntington’s disease: the dual role of reactive oxygen species and D2 receptor stimulation. Proc Natl Acad Sci U S A 102: 12218–12223. 11. Charvin D, Roze E, Perrin V, Deyts C, Betuing S, et al. (2007) Haloperidol protects striatal neurons from dysfunction induced by mutated huntingtin in vivo. Neurobiol Dis 29: 22–29. 12. Graveland GA, Williams RS, DiFiglia M (1985) Evidence for degenerative and regenerative changes in neostriatal spiny neurons in Huntington’s disease. Science 227: 770–773. 13. Ferrante RJ, Kowall NW, Richardson EP Jr (1991) Proliferative and degenerative changes in striatal spiny neurons in Huntington’s disease: a combined study using the section-Golgi method and calbindin D28k immunocytochemistry. J Neurosci 11: 3877–3887. PLoS ONE | www.plosone.org 10 December 2009 | Volume 4 | Issue 12 | e8287