Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Abstract

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 Ca(2+). 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.

Figure 1. Presynaptic (Type III) taste bud cells respond to glutamate, kainic acid (KA), and NMDA.

Taste cells were isolated from mouse circumvallate papillae and their responses to ionotropic glutamate receptor agonists recorded by Ca²⁺ imaging. A, 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, Another Presynaptic cell responded to KCl depolarization (↓, KCl) and 100 µM KA (↓, KA). C, A different Presynaptic cell responded to KCl (↓, KCl) and to 100 µM NMDA (↓, NMDA). D, 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. E, 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.

Figure 2. Glutamate, NMDA, and kainic acid induce serotonin release from isolated taste buds and cells.

Serotonin (5-HT) biosensors were positioned against circumvallate taste buds to measure stimulus-evoked transmitter release. A, 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, 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). C, 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). D, 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). E, 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). F, Summary of experiments testing CNQX, plotted as in D. ***, p<0.001, repeated measures ANOVA, N = 9).

Figure 3. Serotonin, released during glutamate stimulation, inhibits taste buds.

ATP biosensors were used to monitor taste-evoked transmitter release from taste buds. A, 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, 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 Fig. 2, offset filled symbols show mean ± 95% CI, ***, p<0.001, repeated measures ANOVA, N = 4). C, 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. D, 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).

Figure 4. Diagram showing postulated mechanism for glutamate as an efferent transmitter in taste buds.

Glutamate-stimulated release of serotonin might explain interpapillary inhibition that has been reported by others –. Two taste buds are depicted. Taste stimulation of the taste bud on the right activates a sensory afferent fiber that propagates signals centrally (small arrows to right at bottom) as well as laterally (interpapillary) to an adjacent taste bud via afferent branches(small arrows to left at bottom). Glutamate, released from an afferent axon branch (red arrow, left), activates NMDA and KA receptors on Presynaptic (Type III) taste cells. Glutamatergic stimulation of Presynaptic cells triggers these cells to secrete 5-HT, which inhibits ATP release from Receptor (Type II) cells (blue symbol) .