Using biosensors to detect the release of serotonin from taste buds during taste stimulation

Abstract

CHO cells transfected with high-affinity 5HT receptors were used to detect and identify the release of serotonin from taste buds. Taste cells release 5HT when depolarized or when stimulated with bitter, sweet, or sour tastants. Sour- and depolarization-evoked release of 5HT from taste buds is triggered by Ca2+ influx from the extracellular fluid. In contrast, bitter- and sweet-evoked release of 5HT is triggered by Ca2+ derived from intracellular stores.

Fig. 1. Serotonin elevates Ca²⁺ in CHO cells transfected with 5HT2c receptors

Concentration-response plot for 5HT. Responses were measured as Fura 2 emission ratios, F340/F380, and normalized to the ratios at 1 μM 5HT. EC50 ~ 9 nM. Points show mean ± s.e.m. (N = 31 cells from 2 experiments), sigmoidal non-linear regression curve fitted with Prism v4 (GraphPad). Adapted from Huang et al., 2005.

Fig. 2. Biosensor cells detect serotonin released from isolated taste buds

A. a fixed, isolated taste bud immunostained for serotonin. At least one immunopositive taste cell (arrow) is visible. The lingual epithelium was incubated with 500 μM hydroxytryptophan (5HTP) prior to isolating this taste bud (see later). B. a Fura 2-loaded biosensor cell abutted against an isolated taste bud in a living preparation. In both A and B, Nomarski differential interference contrast and fluorescence microscopy images were merged. C. cartoon showing placement of a biosensor onto an isolated taste bud in a configuration used for the experiments described in this report, as in B. D. Ca²⁺ elevation in a 5HT biosensor cell positioned against a taste bud as in B, C. Bath-applied 3 nM 5HT (↓) was used initially to verify the sensitivity of the biosensor, followed by 50 mM KCl to depolarize taste cells and 100 μM cycloheximide (cyx). The lingual epithelium had been incubated with 500 μM 5HTP to elevate 5HT in taste cells. Responses evoked by depolarization and taste stimulation are enhanced by this procedure. All subsequent records were recorded from taste buds isolated from epithelium incubated in 5HTP. E. saccharin, but not aspartame, elicts 5HT release from taste buds. Biosensor responses were elicited by perfusing KCl, 2 mM and 20 mM saccharin (sac), but neither 1 mM nor 10 mM aspartame (aspm) were effective stimuli. F. Mianserin, a 5HT2c receptor antagonist, reversibly reduces 5HT biosensor responses. Responses were evoked by depolarizing taste buds with 50 mM KCl (↓). 1 nM mianserin was present during the time indicated by the shaded region. Mianserin also reversibly blocked responses evoked by saccharin, cycloheximide, and acetic acid (data not shown). G. 5HT release from taste buds depends on Ca²⁺ influx for KCl depolarization but not for taste stimulation with a bitter (cycloheximide) compound. Sequential biosensor responses from an isolated taste bud stimulated with 50 mM KCl (↓) or 100 μM cycloheximide (cyx, ↓). The 5HT biosensor was calibrated at the beginning and end of the recording with 3 nM serotonin (5HT, ↓). During the shaded region, Ca²⁺ in the bath (2 mM) was exchanged for 8 mM Mg²⁺. Adapted from Huang et al., 2005.