(Cayman Chemical Company, Ann Arbor, MI, USA). A total of 46 animals (23 males and 23 females) were used for 8-OH-DPAT- induced corticosterone release (n = 5–7 mice per group). DOI-induced Head-Twitches Mice were administered with vehicle or the 5-HT2A/C receptor agonist (6)DOI (0.3 and 1 mg/kg, i.p.) and immediately placed inside an observation area. The number of head twitches was counted for a 15-min period starting 15 min after drug administration. A total of 69 animals (32 males and 37 females) were used for DOI-induced head-twitches (n = 8–10 mice per group). Real-time PCR for quantification of mRNA expression Mice na¨ıve to behavioral testing were killed for dissection of raphe. Total RNA was isolated using Qiagen RNeasy extraction kits (Qiagen, NSW, Australia) and stored at 280uC. RNA concentration and quality were determined using Nanodrop spectrophotometer. cDNA was reverse transcribed from 1 mg of total RNA per sample using Applied Biosciences Reverse Transcription kits (PE Applied Biosystems, Foster City, CA, USA) with random hexamers. The reverse transcription PCR conditions were 25uC—10 min, 48uC—30 min and 95uC— 5 min. cDNA products were stored at 220uC. Quantitative real- time PCR was performed using SYBR Green PCR master mix (Sigma-Aldrich) on a PE ABI Prism 7500 Light Cycler system (PE Applied Biosystems). All primer pairs were optimized for working concentrations before use and primer sequences are as follows. Serotonin 1A receptor (5-HT1AR) F: CCC CAA CGA GTG CAC CAT, R: GCG CCG AAA GTG GAG TAG AT; Serotonin transporter (5-HTT) F: CTT CAG CCC CGG ATG GTT, R: GTG GAC TCA TCA AAA AAC TGC AAA; TPH2 F: GAC AAC GTG CCG CAA CTG R: TGA AGC CAG ATC GCT CTT TCA; Cyclophilin F: CCC ACC GTG TTC TTC GAC A, R: CCA GTG CTC AGA GCT CGA AA. Cyclophilin was used as the endogenous housekeeping gene as it is not altered in the R6/1 mouse line at this age (Zajac, unpublished data). Real-time PCR conditions were 50uC—2 min, 95uC—10 min, followed by 40 cycles of 95uC—15 s and 60uC— 1 min. All reactions were performed on five-eight individual subjects per group, each in triplicate. Melt curve analyses were performed to ensure that only one reaction product was obtained. A total of 23 animals (12 males and 11 females) were used for gene expression analyses (n = 5–6 mice per group). Whole tissue measurements of 5-HT and its metabolite Mice na¨ıve to behavioral testing were killed for brain dissection. Tissue levels of endogenous 5-HT and its metabolite 5- References 1. Duff K, Paulsen JS, Beglinger LJ, Langbehn DR, Stout JC (2007) Psychiatric symptoms in Huntington’s disease before diagnosis: the predict-HD study. Biol Psychiatry 62: 1341–1346. 2. Julien CL, Thompson JC, Wild S, Yardumian P, Snowden JS, et al. 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Dissected brain structures were homogenized in 5– 10 volumes (v/w) of ice-cold 0.1 M HClO4 containing 1.34 mM disodium EDTA and 0.05% Na2S2O5. Homogenates were centrifuged at 30,000 g for 20 min at 4uC. After neutralization with 2 M KH2PO4/K2HPO4, pH 7.4, containing 0.01 mg per mL ascorbate oxidase (Boehringer Mannheim, Meylan, France), supernatants were further centrifuged at 30,000 g for 20 min. Aliquots (10 mL) of clear supernatants were injected into a high performance liquid chromatography (HPLC) column (Ultrasphere IP, Beckman, Gagny, France; 2560.46 cm, C18 reversed phase, particle size 5 mm) protected with a Brownlee pre-column (3 cm, 5 mm). The mobile phase for the elution (at a flow rate of 1 mL per min) consisted of (in mM): KH2PO4, 70; triethylamine, 3.1; disodium EDTA, 0.1; octane sulphonate, 1.05; methanol, 16%, adjusted to pH 3.02 with solide citric acid. The electrochemical detection system (ESA 5011, Bedford, MA, USA) comprises an analytical cell with dual coulometric monitoring electrodes (+50 and +350 mV). The generated signals were integrated by a computing integrator (Millenium 32, Waters, Saint Quentin Fallavier, France). A total of 27 animals (14 males and 13 females) were used for HPLC analyses (n = 6–8 mice per group). Statistical Analysis Analyses of variance (ANOVAs) were used to examine main effects and/or interactions. A 2-way ANOVA was used to analyze the effects of sex and genotype on saccharin preference, NSFT as well as measurement of gene expression (qPCR) and serotonin levels (HPLC). For the experiments involving pharmacological treatments, the effects of sex, genotype and treatment(s) were analyzed by a 3-way ANOVA. To determine specific group differences in case of significant main effects (or interaction), the ANOVAs were followed by Fisher’s LSD or Bonferroni post-hoc tests. 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