We also report for the first time a reduced 5-HT2 receptor function in HD animals. These data demonstrate that the HD mutation disrupts 5-HT2 receptor responses, and could be associated with previously described decreases of 5-HT2A/2C receptors in models of HD [26,27]. However, even though such alterations have been observed in other mouse models of depression [68,69], the reduced 5-HT2 receptor function was displayed by both male and female HD mice and could not explain per se the female-specific depression-like behaviors. Interestingly, variation in levels of 5-HTT expression has been shown to be a source of changes in 5-HT2A receptor function [70]. In conclusion, we have found that the female-specific depression phenotype of the R6/1 model is associated with decreased levels of serotonin and its metabolite across various brain regions, which are likely due to altered 5-HT1A receptor signaling as suggested by the increased 8-OH-DPAT-induced hypothermia displayed only by female HD mice. The identification of this specific molecular abnormality presents as a novel target for the early treatment or prevention of depression in HD. Finally even though male HD animals did not display any impaired emotionality-related behaviors, they shared some aspects of molecular dysfunction with the female HD mice such as a decreased 5-HT2A receptor function when compared to WT animals. Materials and Methods Ethics Statement All experiments were performed in accordance with the guidelines of the HFI Animal Ethics Committee (approval ID, AEC #08-100) and the National Health and Medical Research Council (NHMRC). Animals R6/1 transgenic hemizygote males [71] were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and bred with CBB6 (CBA6C57/B6) F1 females to establish the R6/1 colony at the Howard Florey Institute (HFI). After weaning, animals were grouped housed (4 mice per cage with 2 of each genotype) and maintained on a 12 h light/dark cycle with access to food and water ad libitum. To avoid any possible ‘‘litter effects’’, we ensure that animals from each litter were represented in each experimental condition using an appropriate randomization. All experiments were performed on male and female wild-type (WT) and R6/1 (HD) mice at 8 weeks of age and in accordance with the guidelines of the HFI Animal Ethics Committee (approval ID, AEC #08-100) and the National Health and Medical Research Council (NHMRC). Each animal was only exposed to a unique behavioral test. Drugs The 5-HT2A/C receptor agonist, 1-(2, 5-dimethoxy-4-odophe- nyl)- 2-aminopropane ((6)DOI) and the 5-HT1A receptor agonist, 8-hydroxy-di-n-propylaminotetralin ((6)-8-OH-DPAT) were sup- plied by Sigma (Aldrich, NSW, Australia). Body Temperature Core temperature was measured at ambient temperature of 2361uC in gently restrained mice using a thermocouple probe (ID-Tech-Bioseb, France; 0.71 mm diameter). Forced-Swimming Test (FST) Mice were individually placed into a glass beaker (13 cm diameter) filled with 12 cm deep water (25–26uC) and video recorded for 300 secs. Total immobility time was manually scored by an experienced experimenter blind to treatment and mouse genotype. To study the effects of acute sertraline (Pfizer Inc, CT, USA), mice were injected intraperitoneally (i.p.) with either vehicle (1% Tween-20, 1 ml/100 g body weight) or sertraline (20 mg/kg) 60 mins before FST [27]. A total of 105 animals (48 males and 57 females) were assessed on FST (n = 10–15 mice per group). Tail-Suspension Test (TST) Mice were suspended by the tail to a hook using adhesive tape. The duration of immobility over a 6-min session was manually scored by an experienced experimenter blind to treatment and mouse genotype. To study the effects of acute sertraline (Pfizer Inc, CT, USA), mice were injected intraperitoneally (i.p.) with either vehicle (1% Tween-20, 1 ml/100 g body weight) or sertraline (20 mg/kg) 30 mins before TST [27]. A total of 75 animals (41 males and 34 females) were assessed on TST (n = 8–12 mice per group). Novelty-Suppressed Feeding Test (NSFT) As previously described [27], mice were food deprived for 48- hours prior to testing but allowed 2-hours of feeding after an initial 24-hour period. Water was available ad libitum. During the test, a single food pellet was placed on a piece of filter paper in the centre of the test arena (80680680 cm). Individual mice were then placed into a corner of the arena and the latency to grasp and feed on the food pellet within 5 minute was recorded. Upon the initiation of feeding or reaching the time limit, mice were removed and allowed to feed on a single food pellet of pre-determined weight for 5 minutes (to assess the level of hunger). A total of 59 animals (29 males and 30 females) were assessed on NSFT (n = 12– 17 mice per group). Saccharin-preference test Based on a validated protocol [33], single housed mice were exposed to both 0.1% saccharin and tap water solutions across four 15 h-overnight periods (18:00 h to 9:00 h). To avoid place preference, the position of the 2 bottles was varied every day across the left or right side of the feeding compartment. In order to take into account time for the animals to become accustomed to the testing environment, saccharin preference score (saccharin intake/ total fluid intake) was calculated as the mean of the 2 last measures. A total of 48 animals (22 males and 26 females) were assessed on saccharin preference test (n = 8–14 mice per group). 8-OH-DPAT-induced hypothermia As previously described [39], basal values were determined just before subcutaneous injection of the 5-HT1A receptor agonist 8- OH-DPAT (0.1 and 0.3 mg/kg) or vehicle (0.9% NaCl, 1 ml/ 100 g body weight), and body temperature was measured every 10 min thereafter. The response to 8-OH-DPAT was calculated as the decrease (from baseline) in body temperature during 60-min post injection. A total of 128 animals (58 males and 70 females) were used for 8-OH-DPAT-induced hypothermia (n = 8–14 mice per group). 8-OH-DPAT-induced change of serum corticosterone levels Mice na¨ıve to behavioral testing were killed 60-min after acute subcutaneous injection of the 5-HT1A receptor agonist 8-OH- DPAT (0.3 mg/kg) or vehicle, between 8.30–10.00am. Blood was collected, left to clot at room temperature for 30 mins and centrifuged at 1000 g for 15 mins. Serum was then collected and corticosterone quantified with an Enzyme Immuno Assay PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22133