Mouse taste buds use serotonin as a neurotransmitter

Abstract

Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl- and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca2+ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca2+ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.

Figure 1.

Ca²⁺ mobilization in CHO/5-HT_2C cells elicited by serotonin and ATP. Cells were loaded with fura-2, and responses were measured as the ratio of emission to fluorescent excitation at 340-380 nm. A, Concentration-response relationships for 5-HT. Open circles indicate the mean±SEM (31 cells). B, CHO/5-HT_2C cell responses evoked by 3 μm ATP and 3 nm 5-HT. Open columns, Control responses. Filled columns, Effects of mianserin (10 nm), a5-HT_2C antagonist. Mianserin does not affect Ca²⁺ mobilization in response to ATP but reduces responses to 5-HT. Data are from a different experiment from that shown in A. ATP and 5-HT responses were measured in parallel, and responses are normalized to each agonist separately. C, Ca²⁺ mobilization in CHO/5-HT_2C cells is elicited by repeated applications of 5-HT but not by tastants or depolarization with KCl. Sequential responses elicited by 10 nm 5-HT in the presence of 2 mm Ca²⁺ and in nominally 0 mm Ca²⁺ (shaded area labeled “Ca-free”), 50 mm KCl, 100 μm cycloheximide, and 20 mm saccharin and recovery of 5-HT responses after brief application of 10 nm mianserin (shaded area labeled “mianserin”). In this and subsequent figures, fura-2 excitation was extinguished between stimulus episodes to reduce phototoxicity and photobleaching, resulting in discontinuities in the traces. cyx, Cycloheximide; sac, saccharin.

Figure 2.

CHO/5-HT_2C cells are biosensors for detecting serotonin release from isolated taste buds. A, Micrograph of a fixed, isolated taste bud immunostained for serotonin. Two immunopositive taste cells (red) are visible in this plane of focus. ANomarski differential interference contrast image was merged with an immunofluorescent micrograph for this micrograph. B, Micrograph of a fura-2-loaded (green) biosensor cell abutted against an isolated taste bud in a living preparation. A Nomarski differential interference contrast image and a fluorescence microscopy image were merged. C, Ca²⁺ mobilization in a biosensor cell positioned against a taste bud, as in B. The biosensor cell was initially stimulated with 3 nm 5-HT (↓) to verify its sensitivity, followed by 50 mm KCl to depolarize taste cells and 100 μm cycloheximide (cyx). D, A procedure similar to that in C was conducted on a taste bud preparation that had been pretreated with 500 μm 5-hydroxytryptophan to elevate the serotonin concentration in taste cells. Responses evoked by depolarization and taste stimulation are enhanced by this procedure. E, Saccharin (sac), but not aspartame (aspm), evoked serotonin release from taste buds. F, Mianserin (1 nm; shaded area) reversibly reduced the biosensor responses evoked by depolarizing taste buds repeatedly with 50 mm KCl (↓).

Figure 3.

Serotonin release from taste buds depends on Ca²⁺ influx for acid stimulation and KCl depolarization but not for taste stimulation with bitter (cycloheximide) or sweet (saccharin) compounds. A, Sequential biosensor responses from an isolated taste bud after stimulation with 50 mm KCl (↓) or 100 μm cycloheximide (cyx; ↓). The biosensor was tested for sensitivity at the beginning and end of the record with 3 nm 5-HT(↓). During the traces marked“Ca-free,” Ca²⁺ in the bath (2 mm) was exchanged for 3 mm Mg²⁺. B, An experiment similar to that shown in A, with stimulation by 20 mm sodium saccharin (sac; ↓). Note that responses elicited by KCl depolarization, but not by cycloheximide or saccharin, were eliminated by removing Ca²⁺ from the bathing medium. C, Serotonin release stimulated by 8 mm acetic acid, pH 5, was reduced by exchanging Mg²⁺ for Ca²⁺. D, Sequential responses from two different taste buds from a PLCβ2 null mouse. The interruption in the abscissa shows the two recordings. KCl depolarization evoked serotonin release, but neither 20 mm saccharin nor 100 μm cycloheximide stimulated transmitter release. HAc, Acetic acid.