The Contribution of SERCA to Taste Perception Figure 2. Expression of Serca3 protein in taste buds. Immunofluorescence images of mouse circumvallate (CV), foliate (Fo), fungiform (FF), and soft palate (SP) sections stained with the anti-Serca3 antibody show that immunoreactivity was detected in a subset of taste bud cells from all four major taste fields. Scale bars: 50 mm in CV and Fo, 25 mm in FF and SP. doi:10.1371/journal.pone.0023165.g002 displayed mutually exclusive patterns (Fig. 3, lower panels), indicating that Serca3 is not expressed in the presynaptic cells. Taken together, these results show the specific expression of Serca3 in TRCs, suggesting a possible role for Serca3 in sweet/ umami/bitter taste sensation. Normal morphology and expression of taste-related proteins in Serca3 KO taste tissues To characterize the possible function of Serca3 in taste bud structure and physiology, we first examined the expression of several taste-related proteins and taste bud morphology in Serca3 KO mice [47]. The lingual epithelium and taste buds of the mutant mice appeared grossly normal. Immunostaining of the mutant taste buds with anti-Serca3 antibody detected no specific signals, verifying the lack of Serca3 protein expression in the KO mice. Immunohistochemical studies showed no obvious abnor- malities in expression of the taste-signaling components T1R3, a- gustducin, PLCb2, Trpm5, and neural cell adhesion molecule (NCAM) in the mutant taste buds (Fig. 4). Increased behavioral responses to bitter taste in Serca3 KO mice To characterize behavioral responses of the Serca3 KO mice to tastants, we performed 48-h two-bottle preference tests with compounds representative of five primary taste qualities: sweet (caloric sucrose and the noncaloric artificial sweetener sodium saccharin), umami (monosodium L-glutamate or MSG), bitter (denatonium and quinine hydrochloride), sour (citric acid), and salty (NaCl). Data analysis indicated that there were no statistically significant differences between the wild-type (WT) and KO mice in the preference scores to sweet and umami taste stimuli, although the KO mice tended to display higher preference scores for the compounds at lower concentrations than did WT mice (Fig. 5A– C, Table S4). Interestingly, our data showed that mice deficient for Serca3 displayed significantly increased aversive responses to the bitter substances compared with WT mice (Fig. 5D and E, Table S4). We found no significant difference in the preference to ionic taste stimuli (sour and salty tastes) between the WT and KO mice (Fig. 5F and G, Table S4). We also carried out a brief-access procedure, which was designed to minimize possible postingestive effects. The results also showed that there were no significant differences between the responses of Serca3 KO and WT controls (n = 7 for each genotype) for the sweet and umami tastants (Fig. 6, Table S4), but there was a trend towards higher preference in KO mice for these palatable stimuli (Fig. 6A and B), as we observed in two-bottle preference tests (Fig. 5A–C). The behavioral responses of Serca3 KO mice to sour (citric acid) and salty (NaCl) taste substances were indistinguishable from those of the control WT mice (Fig. 6E and F). In contrast, KO mice displayed significantly increased avoidance of the bitter-tasting compound, denatonium at 0.1 mM, the lowest concentration we tested (Fig. 6C). For higher concentrations of denatonium (0.3 and 1 mM) and for another bitter-tasting substance, quinine (0.05, 0.25, and 1 mM), KO mice also seemed to exhibit increased avoidance responses, although the increases were statistically insignificant (Fig. 6C and D, Table S4). PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23165