Acid Stimulation (Sour Taste) Elicits GABA and Serotonin Release from Mouse Taste Cells

Several transmitter candidates including serotonin (5-HT), ATP, and norepinephrine (NE) have been identified in taste buds. Recently, γ-aminobutyric acid (GABA) as well as the associated synthetic enzymes and receptors have also been identified in taste cells. GABA reduces taste-evoked ATP secretion from Receptor cells and is considered to be an inhibitory transmitter in taste buds. However, to date, the identity of GABAergic taste cells and the specific stimulus for GABA release are not well understood. In the present study, we used genetically-engineered Chinese hamster ovary (CHO) cells stably co-expressing GABAB receptors and Gαqo5 proteins to measure GABA release from isolated taste buds. We recorded robust responses from GABA biosensors when they were positioned against taste buds isolated from mouse circumvallate papillae and the buds were depolarized with KCl or a stimulated with an acid (sour) taste. In contrast, a mixture of sweet and bitter taste stimuli did not trigger GABA release. KCl- or acid-evoked GABA secretion from taste buds was Ca2+-dependent; removing Ca2+ from the bathing medium eliminated GABA secretion. Finally, we isolated individual taste cells to identify the origin of GABA secretion. GABA was released only from Presynaptic (Type III) cells and not from Receptor (Type II) cells. Previously, we reported that 5-HT released from Presynaptic cells inhibits taste-evoked ATP secretion. Combined with the recent findings that GABA depresses taste-evoked ATP secretion [1], the present results indicate that GABA and 5-HT are inhibitory transmitters in mouse taste buds and both likely play an important role in modulating taste responses

Glutamate May Be an Efferent Transmitter That Elicits Inhibition in Mouse Taste Buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (∼50%) respond to 100 µM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca2+. In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 µM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors

Intracellular Ca2+ and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells

ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca 2+ and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca 2+ release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca 2+ has a dual role: (1) Ca 2+ opens TRPM5 channels to depolarize receptor cells and (2) Ca 2+ plus membrane depolarization opens ATP-permeable gap junction hemichannels

Autocrine and Paracrine Roles for ATP and Serotonin in Mouse Taste Buds

Receptor (type II) taste bud cells secrete ATP during taste stimulation. In turn, ATP activates adjacent presynaptic (type III) cells to release serotonin (5-hydroxytryptamine, or 5-HT) and norepinephrine (NE). The roles of these neurotransmitters in taste buds have not been fully elucidated. Here we tested whether ATP or 5-HT exert feedback onto receptor (type II) cells during taste stimulation. Our previous studies showed NE does not appear to act on adjacent taste bud cells, or at least on receptor cells. Our data show that 5-HT released from presynaptic (type III) cells provides negative paracrine feedback onto receptor cells by activating 5-HT 1A receptors, inhibiting taste-evoked Ca 2+ mobilization in receptor cells, and reducing ATP secretion. The findings also demonstrate that ATP exerts positive autocrine feedback onto receptor (type II) cells by activating P2Y1 receptors and enhancing ATP secretion. These results begin to sort out how purinergic and aminergic transmitters function within the taste bud to modulate gustatory signaling in these peripheral sensory organs

Norepinephrine is coreleased with serotonin in mouse taste buds

ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT

Presynaptic (Type III) cells in mouse taste buds sense sour (acid) taste

Taste buds contain two types of cells that directly participate in taste transduction – receptor (Type II) cells and presynaptic (Type III) cells. Receptor cells respond to sweet, bitter and umami taste stimulation but until recently the identity of cells that respond directly to sour (acid) tastants has only been inferred from recordings in situ, from behavioural studies, and from immunostaining for putative sour transduction molecules. Using calcium imaging on single isolated taste cells and with biosensor cells to identify neurotransmitter release, we show that presynaptic (Type III) cells specifically respond to acid taste stimulation and release serotonin. By recording responses in cells isolated from taste buds and in taste cells in lingual slices to acetic acid titrated to different acid levels (pH), we also show that the active stimulus for acid taste is the membrane-permeant, uncharged acetic acid moiety (CH3COOH), not free protons (H+). That observation is consistent with the proximate stimulus for acid taste being intracellular acidification, not extracellular protons per se. These findings may also have implications for other sensory receptors that respond to acids, such as nociceptors

Serotonin, released during glutamate stimulation, inhibits taste buds.

<p>ATP biosensors were used to monitor taste-evoked transmitter release from taste buds. <b>A</b>, Traces show responses from a biosensor positioned near an isolated taste bud to measure ATP release elicited by taste stimulation. A sweet-bitter taste mix (↓, taste; 1 mM sucralose, 0.1 mM SC45647, 10 µM cycloheximide, 1 mM denatonium) evoked ATP release (biosensor response) that was inhibited by 100 µM glutamate (↓, taste+glu). Glutamate-evoked inhibition of ATP secretion was fully restored by adding combination of CNQX (30 nM) and DL-APV (15 µM) (present throughout the shaded area) to the bath. <b>B</b>, Summary of data. Open circles show normalized peak biosensor responses triggered by taste, taste+glutamate, taste+glutamate in the presence of CNQX and DL-APV, and finally, a repeat taste stimulus. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030662#pone-0030662-g002" target="_blank">Fig. 2</a>, offset filled symbols show mean ± 95% CI, ***, p<0.001, repeated measures ANOVA, N = 4). <b>C</b>, In another experiment, a sweet-bitter taste mix (↓, taste) evoked ATP release (biosensor response) that was inhibited by 100 µM glutamate (↓, taste+glu). Glutamate-evoked inhibition of ATP secretion was partially reversed by adding WAY100635 (WAY, 10 nM, present throughout the shaded area), a 5-HT_1A antagonist, to the bath. <b>D</b>, Summary of data. Open circles show normalized peak biosensor responses of each experiment triggered by taste, taste+glutamate, and finally taste+glutamate in the presence of WAY100635. Offset closed symbols show mean ± 95% CI. ***, p<0.001, repeated measures ANOVA, N = 5).</p

Glutamate, NMDA, and kainic acid induce serotonin release from isolated taste buds and cells.

<p>Serotonin (5-HT) biosensors were positioned against circumvallate taste buds to measure stimulus-evoked transmitter release. <b>A</b>, Traces show biosensor responses. When the biosensor was not near a taste bud (TB-), the biosensor responded only to 3 nM 5-HT (↓, 5-HT) but not to 100 µM glutamate (↓, glu) or KCl depolarization (not shown), verifying that the biosensor did not respond to stimuli that activate taste buds. In contrast, when the biosensor was positioned against a taste bud (TB+), KCl depolarization (↓, KCl) and glutamate alike (↓, glu) elicited biosensor responses, indicating stimulus-evoked 5-HT release. <b>B</b>, Simultaneous recordings from an isolated Presynaptic cell (top trace, Pre) and a 5-HT biosensor (bottom trace, 5-HT-bio). Stimulating the Presynaptic cell with 30 µM NMDA (↓, NMDA) triggered 5-HT secretion, as evidenced by the robust biosensor response (bottom). <b>C</b>, In another experiment, NMDA (↓, NMDA) (30 µM) triggered 5-HT release from a taste bud. The NMDA-evoked release of 5-HT was reversibly reduced by DL-APV (15 µM, present throughout shaded area). <b>D</b>, Summary of NMDA-evoked 5-HT release before, during and after the presence of DL-APV. Open circles represent normalized peak biosensor responses. Offset closed symbols show mean ± 95% Confidence Interval (95% CI). *, p<0.05, repeated measures ANOVA, N = 5). <b>E</b>, Kainic acid (↓, KA) (3 µM) also induced 5-HT release from a taste bud. KA-induced 5-HT release was reversibly inhibited by CNQX (30 nM, present throughout shaded area). <b>F</b>, Summary of experiments testing CNQX, plotted as in D. ***, p<0.001, repeated measures ANOVA, N = 9).</p

Presynaptic (Type III) taste bud cells respond to glutamate, kainic acid (KA), and NMDA.

<p>Taste cells were isolated from mouse circumvallate papillae and their responses to ionotropic glutamate receptor agonists recorded by Ca²⁺ imaging. <b>A</b>, Representative traces of an identified Presynaptic (Type III) taste cell depolarized by KCl (50 mM) (↓, KCl) followed by stimulation with glutamate (100 µM) (↓, glu) <b>B</b>, Another Presynaptic cell responded to KCl depolarization (↓, KCl) and 100 µM KA (↓, KA). <b>C</b>, A different Presynaptic cell responded to KCl (↓, KCl) and to 100 µM NMDA (↓, NMDA). <b>D</b>, In another Presynaptic cell, responses were evoked by KCl depolarization (↓, KCl), KA (↓, KA), and NMDA (↓, NMDA) alike. Note, as shown in all records in this figure, KCl stimulation typically elicited more robust responses than did glutamate, KA, or NMDA. <b>E</b>, Venn diagrams representing the relative proportions of Receptor and Presynaptic taste cells that responded to glutamate, as well as the overlap of responses of glutamate-sensitive Presynaptic cells to NMDA and/or KA.</p

Stimulating Presynaptic (Type III) taste cells evokes GABA release.

<p>GAD-GFP mice were used to isolate and identify Presynaptic taste cells. <b>A,</b> Traces show concurrent Ca²⁺ recordings from an isolated Presynaptic cell (upper traces, Pre) and an apposed GABA biosensor (lower traces, GABA-bio). Stimulating the Presynaptic cell with 50 mM KCl (↓, KCl) evoked a transient Ca²⁺ response and the biosensor reported that GABA was released (lower trace). CGP55845 (10 µM, “CGP”, present throughout shaded area) reversibly blocked the biosensor responses without affecting Presynaptic cell responses. <b>B,</b> From another experiment, KCl and ATP (10 µM) evoked Ca²⁺ responses in a Presynaptic cell (upper traces) but only KCl depolarization triggered GABA release (lower traces). <b>C,</b> Another experiment, 50 mM KCl and 10 mM acetic acid (HAc, pH 5.0) evoked a transient Ca²⁺ elevation in the Presynaptic cell (upper traces) and triggered GABA release (lower traces). <b>D,</b> Summary of data from identified Presynaptic cells. KCl depolarization (N = 4) and sour tastant (HAc, N = 4) stimulate Presynaptic cells to release GABA. CGP55845 (CGP) inhibits KCl-evoked biosensor responses. Neither tastants (sweet/bitter taste mixture, N = 3) nor ATP (N = 4) stimulates Presynaptic cells to release GABA. ns, not significant. ***, <i>p</i><0.001, , Student <i>t</i>-test.</p