PMC:1181811 / 25695-31076 JSONTXT

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Concentrations of each solution were made fresh daily using distilled water, and all taste stimuli were presented at room temperature. All animal protocols were approved by the UTHSC Institutional Animal care and Use Committee.\n\nBrief-access tests\nAll behavioral tests were conducted in the commercially available Davis MS-160 gustometer (DiLog Instruments, Inc., Tallahassee FL). Testing procedures were similar to those described earlier [29,37]. Briefly, after 24 hours of water deprivation, naïve mice are given a single 20-minute trial consisting of access to a single bottle of distilled water (sipper tube training). On day 2, mice could initiate up to sixteen 5 s trials with a single lick to one of four bottles containing distilled water (trial training). Testing occurred in sessions 3 and 4, with one test session per day per mouse. Trials were 5 s in length with an inter-trial interval of 10 s, and mice had up to 120 s to initiate a trial; if a trial was not initiated during this interval, the shutter closed for 10 s and the next trial was presented. Mice were tested with 2 concentrations each of 4 different taste stimuli [1 and 3 mM QHCl, 1 and 3 mM DB, 3 and 10 mM PROP (unpublished data), and 0.01 and 0.1 M sucrose]. Stimulus trials were presented in 3 blocks of 8 trials, for a total of 24 possible trials per test session. Each block consisted of each concentration of stimulus plus four presentations of distilled water in random order. Individual mice were also tested in random order.\nThe dependent measure for each computed for each mouse was the lick ratio (average number of licks to stimulusx /average number of licks to water) where x is a given concentration of stimulus and the average number of licks to water is derived from the water trials during both test sessions. Lick ratio data for each stimulus were compiled for all individual mice, and means were prepared for each strain. B6 vs. D2 comparisons (Fig. 1A) were made using main effects ANOVA. Lick ratios for individual mice to sucrose were generally ~1.0 (data not shown), indicating that either concentration of this compound was licked at a similar rate to water by these thirsty animals. This stimulus was intended as a \"neutral\" stimulus, albeit one that has different sensory properties than water, and therefore not analyzed further. This was done to encourage sampling on \"non-water\" trials, as there is some evidence that mice detect distilled vs. adulterated water in brief-access taste tests based on olfactory clues; there is no evidence that mice can detect or distinguish among concentrations of a particular stimulus [37].\n\nQTL mapping\nLinkage analysis was conducted on BXD mice using freely available software (Map Manager QTX [38]), and BXD genotype data shared by Robert W. Williams, University of Tennessee Health Science Center [45]. Simple interval mapping was conducted. This method evaluates the association between trait values (lick ratios) and expected genotype of a hypothetical quantitative trait locus (QTL) at multiple analysis points between each pair of adjacent marker loci. The significance of each potential association is measured by the likelihood ratio statistic (LRS; e.g. [46]). Permutation analysis (x2000) was used to determine genome-wide significance criteria for LRS scores. Significance was set at p \u003c 0.05 and suggestive refers to p \u003c 0.63. Additional markers used to refine the QTL on chromosome 6 were identified from the Jackson Laboratories online resources for the BXD RI strains [40].\n\nIdentification of T2R alleles\nOligonucleotides were based on published mTas2r B6 or 129/SvJ cDNA sequences or on the public B6 genome. Entire coding regions plus ~50 kb of flanking sequence of each single-exon Tas2r was amplified from D2 or BXD RI genomic DNA (Jackson Laboratories, Bar Harbor, ME) by polymerase chain reaction (PCR) using a high-fidelity polymerase TaqPro Complete (Denville Scientific, South Plainfield, NJ). PCR products were subcloned into pGemT-Easy (Promega, Madison, WI) and sequenced at the University of Maryland School of Medicine Biopolymer Core. The sequences of D2 products were compared to B6 sequences available in Genbank (see Additional file 2), and polymorphisms identified. When possible, unique restriction sites were identified that differentiated B6 and D2 alleles, and the corresponding restriction endonucleases were used in diagnostic digests of Tas2r cDNAs amplified from genomic DNA of each BXD/Ty RI strain. For Tas2r120, the absence of a PCR product was considered diagnostic of the D2 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