antibody specificity. We verified the reproducibility of the results by performing immunostaining with at least 40 taste tissue sections from five each of WT and Serca3 KO mice, with total 20 sections from at least 2 mice from each Trpm5-GFP and Gad1-GFP transgenic line, and with 30 taste tissue sections from three rats for Serca2 immunostaining experiments. Taste solutions Taste solutions were prepared in deionized water using reagent- grade chemicals purchased from Sigma-Aldrich. All taste stimuli were presented at room temperature. Two-bottle taste preference tests Sex- and age-matched Serca3 KO and WT control (FVB/NJ) mice (9–10 for each group, except 5 KO mice for NaCl tests) were used for two-bottle taste preference tests as previously described [19,59]. The animals were 7–15 weeks old at the start of each experiment. The mice were given access for 48 h to two bottles, one containing deionized water and the other a tastant solution, and to food ad libitum. After 24 h, intake of each fluid was recorded and the bottle positions were switched to minimize any positional effect. After another 24 h, fluid intake was again recorded. Mice were then given two bottles of deionized water for at least 4 days between different tastant trials. Each tastant was presented in an ascending concentration series: sweet, 5, 10, 20, 40, and 80 mM sucrose (caloric) and 0.1, 0.3, 1, 3, and 10 mM sodium saccharin (noncaloric artificial sweetener); umami, 0.1, 0.3, 1, 3, and 10 mM monosodium L-glutamate (MSG); bitter, 0.01, 0.03, 0.1, 0.3, 1, and 3 mM denatonium benzoate and 0.01, 0.03, 0.1, 0.3, and 1 mM quinine hydrochloride; sour: 0.1, 0.5, 1, and 2.5 mM citric acid; salty: 37.5, 75, 150, and 300 mM NaCl. Brief-access tests Sex- and age-matched Serca3 KO and WT control (FVB/NJ) mice (7 for each group) were used for these tests. The animals were 5– 7 weeks old at the onset of each experiment. The licking rate of individual mouse presented with a series of different tastants was measured using a Davis MS-160 gustometer (DiLog Instruments, Tallahassee, FL) as previously described [60]. Test stimuli consisted of serial concentrations of: sodium saccharin (0.1, 1, and 10 mM); MSG (1, 3, 10 mM) in the presence of 100 mM amiloride and 1 mM inosine monophosphate (IMP); denatonium benzoate (0.1, 0.3, and 1 mM); quinine hydrochloride (0.05, 0.25, and 1 mM); citric acid (1, 3, and 10 mM); and NaCl (0.1, 0.3, and 0.5 M). In order to measure responses to attractive stimuli (saccharin and MSG), mice were fed 1 g food and 1.5 ml water over the 24 h before testing and were tested every second day. For aversive stimuli (denatonium, quinine, citric acid, and NaCl), mice were placed on a 22-h restricted water- access schedule and were tested daily. To ensure adequate sampling, a maximum of five different solutions (including a water control) were presented during a test. Stimulus presentation order was randomized within blocks. The duration of each trial (5 s) was regulated by a computer-controlled shutter that allowed access to the sipper tube with a 10 s inter-presentation interval. A water rinse presentation (1 s) was interspersed between the test trials for the normally avoided stimuli to help control for potential carry-over effects as previously described [61]. Data analysis Data from the two-bottle preference tests are presented as percent preference scores, which were calculated using average daily fluid intakes of individual mice: [solution intake/total fluid (solution+water) intake]6100. A preference score of 50% indicates The Contribution of SERCA to Taste Perception neither preference nor avoidance of the solution compared with water; a score .50% suggests preference for the solution, and ,50% avoidance of the solution, compared with water. For brief- access tests, the average number of licks per trial for each stimulus concentration was divided by the average number of water licks per trial, yielding a tastant/water lick ratio. Preference scores (two-bottle tests) and tastant/water lick ratio (brief-access tests) were analyzed using a mixed-model analysis of variance (ANOVA), and genotype (between-subjects) and tastant concentration (within-subjects) effects were evaluated. If there was a significant effect, then post hoc pairwise comparisons of means at each test solution concentration were conducted with Student t- tests between genotype groups (two-tailed unpaired distribution, except for NaCl tests in two-bottle tests, for which a two-sample equal variance was used). All statistical analyses were performed using Microsoft Excel software and the software package Statcel (OMS, Tokyo, Japan). Statistical rejection was set at the 0.05 level for all data analysis. Gustatory nerve recordings The procedures for the mouse glossopharyngeal (GL) nerve recordings were described previously [62]. Briefly, Serca3 KO and WT control (FVB/NJ) mice (6–8 for each group) were anesthetized with an intraperitoneal injection of sodium pentobarbital (40–50 mg/ kg of mouse body weight) and maintained at a surgical level of anesthesia with additional injection of sodium pentobarbital. The trachea of each animal was cannulated, and the mouse was then fixed in the supine position with a head holder. The right GL nerve was exposed by removal of the digastric muscle and posterior horn of the hyoid bone and then dissected free from underlying tissues and cut near its entrance to the posterior lacerated foramen. For whole-nerve recording, the entire nerve was placed on a platinum wire recording electrode. An indifferent electrode was positioned nearby in the wound. Neural responses induced by chemical stimulations of the tongue were fed into an amplifier (Grass Instruments, West Warwick, RI), monitored on an oscilloscope and audiomonitor. Whole nerve responses were integrated with a time constant of 1.0 sec and recorded on a computer for later analysis using PowerLab system (AD Instruments, Colorado Springs, CO). The solutions used to stimulate the posterior part of the tongue were as follows: sucrose, 0.5 M; mono potassium glutamate (MPG), 0.1 M; denatonium, 0.1, 0.3, 1, and 3 mM; quinine, 0.1, 0.3, 1, and 3 mM; HCl, 10 mM; and NaCl, 0.1 M. The compounds were applied to the tongue for 1 min, followed by rinse with deionized water. In the data analysis for whole-nerve response to each stimulus, the magnitude of the integrated response at 10, 15, 20, 25, 30, 35, 40, 45, and 50 s after stimulus onset was measured and averaged. Relative response magnitudes (averaged) for each test stimulus were normalized to the response magnitude to 0.1 M NH4Cl. Genotype differences in responses to compounds tested at single concentrations were assessed using t-tests. Responses to compounds tested at multiple concentrations were analyzed using a mixed-model ANOVA assessing effects of genotype (between-subjects factor) and concen- tration (within-subjects factor). If significant effects were detected using ANOVA, then planned comparison or post hoc t-tests were used to compare strain means for specific concentrations. For all tests, P,0.05 was considered significant. Supporting Information Table S1 Primers for cell type-specific marker genes used in cell typing qPCR. (DOC) PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23165