The mechanisms utilized by taste receptor cells (TRCs) to signal the presence of taste stimuli are incompletely understood. Parallel studies on TRCs with molecular and physiological approaches have produced, at times, incongruent data, particularly for sweet and bitter stimuli. As well, there are now previously unrecognized communication pathways among TRCs involving neurotransmitters. Germane questions include the quality sensitivity of individual cells, the roles of second messengers systems and trp channels in transducing responses, and how responses are relayed to the afferent nerve. This proposal continues the specific aims of the previous funding cycle by exploring the physiology of a specific array of bitter/sweet tastants, their underlying signaling pathways, and the interaction of neurotransmitters on these pathways. In the first specific aim, patch clamp and calcium imaging experiments will be performed on dissociated TRCs of rat posterior tongue to explore the multiple sensitivity of individual cells to these stimuli as well as the signaling mechanisms that underlie these responses. These phenotypic groups of cells will be compared to molecular markers T1R/T2R taste receptors, gustducin, and TRPM5 by combining physiology with single cell RT-PCR. In the second specific aim, recently discovered cellular signaling among TRCs by neurotransmitters will be explored. Our previous work demonstrated novel roles for the neurotransmitters norepinephrine (NE) and serotonin (5HT) in the bud and provided evidence for the expression of multiple adrenergic (alpha and beta) and serotonergic (5HT1A and 5HT3) receptor subtypes in TRCs. The paracellular role of the adrenergic system in the taste bud will be examined by phenotyping alpha and beta-expressing TRCs and examining their convergence with sweet/bitter receptors on second messenger systems. Our data suggest that 5HT release not only acts to excite the peripheral nerve (presumably through 5HT3 receptors) but simultaneously act through a paracellular route to inhibit a Subset of 5HT1A expressing TRCs. The phenotype of the 5HT1A-expressing TRCs will be examined. Collectively, these experiments promise a fuller understanding of the pathways for taste information processing within the bud as well as will provide needed complimentary physiological evidence to the recent advances in the molecular biology of TRCS.
