Dopamine/D2 Receptor and HD Figure 7. D2-R potentiates striatal neuronal death induced by ExpHtt via ROCK-II activation. A) Immunohistochemical detection of MAP2 protein (red) and nuclei labelling using Hoechst (blue) were performed on striatal neurons transfected for 24 hours with either Htt (top panel) or ExpHtt (bottom panel). The white arrow in the Hoechst panel points to a healthy nucleus (top panel) a fragmented nucleus (bottom planel) in an Htt- and ExpHtt-transfected neuron, respectively. B) Quantification of surviving transfected neurons based on Hoechst labelling. Neurons transfected with Htt (white bars) or ExpHtt (black bars) were treated with quinpirole (10 mM) for 24 hours alone or in the presence of pharmacological inhibition of ROCK (Y27632, 10 mM and Hydroxyfasudil, 10 mM) or ROCK-II siRNA (50 nM). Data are expressed as mean6SEM from 3 independent experiments (100 transfected neurons per condition and per experiment)(***P,0.001, *P,0.05, ns: non significant). doi:10.1371/journal.pone.0008287.g007 Primary Striatal Culture and Drug Treatment Primary striatal neurons were dissected out from 14 days old embryos from pregnant Swiss mice (Janvier, Le Genest Saint Isle, France) as previously described [43]. After dissociation, cell pellets were resuspended in Neurobasal medium supplemented with B27 (Invitrogen), 500 nM L-glutamine, 60 mg/ml Pennicilin-strepto- mycin and 25 mM b-mercapto-ethanol and then plated on poly-L- lysine-coated 24-well (1,336105 cells by well) or 6-well plates (6,3216105 cells by well) at 37uC in a humidified 5% CO2 incubator. Using this preparation, the cell culture contains in majority cells from neuronal and not glial origin. After 7 days in culture, neurons grown in 24 well plates, were transiently transfected with GFP-tagged Htt constructs (provided by HDF Resource Bank, UCLA) encoding the first exon of human Htt containing either 25 (Htt) or 103 (ExpHtt) continuous CAA or CAG repeats. The sequence encoding an enhanced Green Fluorescence Protein (GFP) was inserted in frame at the C- terminus of each construct. Three hours and half after transfection, the medium was removed and replaced by the complete Neurobasal Medium containing dopamine (DA, 100 mM, Calbiochem), quinpirole (Quin, 10 mM RBI). Cells were then replaced at 37uC for the appropriate time. For the pharmacological treatments, Y-27632, (10 mM; Tocris) Hydro- xyfasudil (10 mM; Sigma) or Raclopride (Raclo, 1 mM Sigma) were added 30 min before and during quinpirole treatment. RNA Interference SiRNAs targeted dopamine D1 (D1R), both isoforms of dopamine D2 (D2R) receptors, D2Long and D2Short (D2LR and D2SR, respectively) and ROCK-II were designed to specifically interfere with dopamine D1R, D2R and ROCK-II expression. The target sequences were as follows: D1R, 59- CCUGGAAGAUGCCGAGGAU-39; D2R, 59-CCAGAGAG- GACCCGGUAUA-39 and ROCK-II, 59-CAAUGAAGCUU- CUUAGUAA-39 (Eurogentec). Neurons were transfected at day in vitro 4 (DIV) with 50 nM of siRNA against D1R, D2R and ROCK-II or siRNA control (Cont siRNA #2, Ambion). After 72 hours of silencing, primary neurons were used for transfections experiments and drug treatment. RNA Isolation and RT-PCR Seventy two hours after transfection with D1R or D2R siRNAs, total mRNA was isolated using RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Reverse transcription was per- PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8287