Evidence for a role of glutamate as an efferent transmitter in taste buds

Abstract

Background: Glutamate has been proposed as a transmitter in the peripheral taste system in addition to its well-documented role as an umami taste stimulus. Evidence for a role as a transmitter includes the presence of ionotropic glutamate receptors in nerve fibers and taste cells, as well as the expression of the glutamate transporter GLAST in Type I taste cells. However, the source and targets of glutamate in lingual tissue are unclear. In the present study, we used molecular, physiological and immunohistochemical methods to investigate the origin of glutamate as well as the targeted receptors in taste buds.

Results: Using molecular and immunohistochemical techniques, we show that the vesicular transporters for glutamate, VGLUT 1 and 2, but not VGLUT3, are expressed in the nerve fibers surrounding taste buds but likely not in taste cells themselves. Further, we show that P2X2, a specific marker for gustatory but not trigeminal fibers, co-localizes with VGLUT2, suggesting the VGLUT-expressing nerve fibers are of gustatory origin. Calcium imaging indicates that GAD67-GFP Type III taste cells, but not T1R3-GFP Type II cells, respond to glutamate at concentrations expected for a glutamate transmitter, and further, that these responses are partially blocked by NBQX, a specific AMPA/Kainate receptor antagonist. RT-PCR and immunohistochemistry confirm the presence of the Kainate receptor GluR7 in Type III taste cells, suggesting it may be a target of glutamate released from gustatory nerve fibers.

Conclusions: Taken together, the results suggest that glutamate may be released from gustatory nerve fibers using a vesicular mechanism to modulate Type III taste cells via GluR7.

Figure 1

Gel image of PCR products for VGLUTs 1, 2 and 3 in pooled taste buds. PCR products for VGLUT 1, 2, and 3 were not observed in pooled taste buds from fungiform (FF) and circumvallate (CV) papillae as well as in non taste epithelium (NT). Brain (BR) was used as a control. Bands are for the expected size for VGLUT1 (398 bp), VGLUT2 (360 bp) and VGLUT3 (523 bp). Left lane indicates a 100-bp ladder.

Figure 2

VGLUT1 immunoreactivity in TRPM5-GFP mice. The figures illustrate the expression of GFP under the control of the TRPM5 promoter (green), VGLUT1 (red) in circumvallate papillae (CV; A-C), fungiform papillae (FF; D-F). In circumvallate taste buds, VGLUT1 is not expressed in taste cells but only in nerve fibers surrounding these taste cells. Each figure represents merged images from a Z-series. The Photoshop digital filters Dust & Scratches and Unsharp Mask were used in some images to eliminate pixel noise in this and subsequent figures containing confocal images.

Figure 3

VGLUT2 immunoreactivity in TRPM5-GFP and GAD67-GFP mice. Laser scanning confocal images illustrate the expression of GFP under the control of the TRPM5 promoter (green), VGLUT2 (red) and P2X2 (blue) in circumvallate papillae (CV; A-D), fungiform papillae (FF; E-H). Panels I-K represent VGLUT2 immunoreactivity in GAD67-GFP mice. In fungiform and circumvallate taste buds, VGLUT2 is not expressed in taste cells but only in nerve fibers surrounding these taste cells. Moreover, the VGLUT2-positive fibers are co-localized with P2X2 fibers, a marker for gustatory fibers. Each figure represents merged images from a Z-series.

Figure 4

Gel image of PCR products for VGLUT1 and 2 in geniculate and trigeminal ganglia. The geniculate ganglion (GG) and the trigeminal ganglion (TG) both expressed VGLUT1 and 2.

Figure 5

VGLUT1 and 2 immunoreactivity in geniculate ganglion neurons. A-C: Laser scanning photomicrographs of VGLUT2 (red) and P2X2 (blue). D-F: VGLUT1 (green), VGLUT2 (red). Note that all P2X2-expressing neurons also express VGLUT2 and that VGLUT2 completely co-localizes with VGLUT1.

Figure 6

Calcium responses to glutamate in Type II and Type III taste cells. A: In T1R3-GFP cells, only high concentrations of glutamate (100 mM) elicit a calcium response. B: In GAD67-GFP cells, glutamate at 10 mM or 1 mM may elicit a calcium response. Type III cells possess voltage-gated calcium channels and respond to high concentrations of KCl (55 mM). C: In GAD67-GFP cells, NBQX, a specific antagonist of AMPA/Kainate receptors partially inhibited the response to 1 mM glutamate. D: Average effect of NBQX on responses to glutamate (One way repeated measures ANOVA with a Tukey post test; **: P < 0.005; *: P < 0.05, n = 9 cells).

Figure 7

Gel image of PCR products for Kainate receptors in taste tissues. PCR products for GluR7, KA1 and KA2 were observed in pooled taste buds from fungiform (FF) and circumvallate (CV) papillae but not in non taste epithelium (NT). Brain (BR) was used as a control. Bands are for the expected size for GluR5 (549 bp), GluR6 (498 bp), GluR7 (441 bp), KA1 (487 bp) and KA2 (210 bp). Left lane indicates a 100-bp ladder.

Figure 8

GluR7 immunoreactivity in taste tissues of TRM5-GFP and GAD67-GFP mice. The expression of GluR7 is shown in circumvallate (CV) papillae in mice expressing GFP (green) under the control of the TRPM5 promoter (A-D) or under the GAD67 promoter (E-H). The GluR7 is co-localized with a subset of GAD67-GFP cells but not with TRPM5-GFP cells suggesting that GluR7 is expressed in a subset of Type III cells. The overlay represents a maximum intensity projection obtained from a z series stack of images, so that the labelled taste cells are not in the same layer.