Recent studies have identified G protein coupled receptors (GPCRs) that respond to umami, bitter and sweet taste stimuli. Downstream signaling pathways for these GPCRs are beginning to be understood through powerful combinations of biochemical and genetic analyses. With the advances, have come significant discrepancies between physiological/behavioral analyses and molecular studies, especially for umami taste. The mechanisms underlying sour taste are far less understood, and many candidate transducer channels remain as candidates. How taste cells process taste signals and transmit information to sensory afferent fibers is virtually unknown. A critical discrepancy exists between physiological evidence that taste cells respond to multiple taste qualities, and molecular evidence that taste cells appear to express GPCRs for only one quality. The present application addresses these key open questions using newly developed methods to examine the gene expression profile of functionally defined taste cells. We hypothesize that signals from receptor cells converge onto a separate class of output cells within taste buds; only output cells form synapses with sensory afferent fibers. Critical tests of this hypothesis may resolve the current controversy on the breadth of tuning of taste cells. For umami and acid tastes, we will carry out functional imaging on isolated taste cells, using criteria derived from detailed studies in the slice preparation. Such functionally defined taste cells will then be subjected to single-cell RT-PCR and/or differential library screening to identify molecules associated with the functional phenotype. To test our hypothesis on output cells, We will employ mice in which functional cell lineages for cells (a) that express PLCb2 or (b) that synthesize biogenic amines are transgenically labeled with Green Fluorescent Protein (GFP) or b-galactosidase. Functional in situ imaging of taste cells from these mice will allow us to test whether there is a separate category of taste bud output cells, akin to ganglion cells in the retina. Differential library screening will then allow us to begin defining the functional relationship between receptor and output cells. ? ?
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