Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice

Abstract

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

Figure 1

AF-353 is specific for P2X3 in isolated geniculate ganglion cells A, change in fluorescence ratio of a P2X2/P2X3 DKO (solid line) and WT (dashed line) ganglion cell in response to 10 μm ATP and 55 mm KCl. B, summary of WT (circles; n = 23), P2X2/P2X3 DKO (DKO; squares; n = 16), and P2X3KO (triangles, n = 9) in response to ATP and KCl. C, change in fluorescence ratio of a P2X3KO ganglion cell in response to 10 μm ATP, 10 μm ATP with 100 μm AF-353, and 55 mm KCl. D, effect of 10 μm AF-353 (n = 9) or 100 μm AF-353 (n = 7) on ATP response in all P2X3KO ganglion cells. E, effect of 10 μm 5-HT or 10 μm 5-HT plus 100 μm AF-353 on intracellular Ca²⁺ levels in 5-HT3A-GFP expressing neurons. F, effect of 100 μm AF-353 on ATP released from isolated lingual epithelium containing circumvallate taste buds. B, D and E, individual cells are indicated by symbols while the population is represented as box plots. Asterisks indicate significance (P < 0.05 two-way repeated measures ANOVA for (B); P < 0.05 paired Student's t test for (F). 5-HT, serotonin; DKO, double knockout; KO, knockout; WT, wild-type.

Figure 2

Multiple populations of geniculate ganglion cells respond differently to AF-353 A, change in fluorescence ratio of two ganglion cells in response to 10 μm ATP, 10 μm ATP with 10 μm AF-353 and 55 mm KCl. In the cell shown in the upper trace 10 μm AF-353 completely blocks the ATP response whereas it only blocks about 50% of the response in the cell shown in the lower trace. Drug application order was the same between top and bottom traces. B, effect of various concentrations of AF-353 on ganglion cells of WT (circles; n = 19 cells), X2KO (diamonds; n = 7 cells) and X3KO; triangles; n = 7–9). WT cells were separated into two categories according to their response to ATP at 1 μm AF-353. Cells above the mean response were classified as ‘less sensitive’ (closed circles) while cells below the mean response were classified as ‘more sensitive’ (open circles). For WT, individual cells are represented as circles with straight lines connecting individual cells. For X2KO and X3KO symbols indicate means ± SEM. Asterisks indicate significance (P < 0.001 Mann–Whitney test between ‘more sensitive’ and ‘less sensitive’ cells). X2KO, P2X2KO; X3KO, P2X3KO; WT, wild-type.

Figure 3

Expression of P2X2 and P2X3 in geniculate ganglion neurons A–C, geniculate ganglion showing P2X2 (magenta) and P2X3 (green) immunoreactivity. Image is a maximum Z-projection of 12 optical sections through a ∼16 μm tissue section. Scale bar = 100 μm. Brightness and contrast were adjusted linearly to preserve relative expression level information. D, quantification of P2X2 and P2X3 immunofluorescence. D, top, histograms showing the distribution of P2X2 (magenta) and P2X3 (green) immunofluorescence of individual ganglion cells. D, bottom, scatterplot showing the relative intensities of P2X2 and P2X3 immunoreactivity for all ganglion cells. Intensity values were normalized to maximum values for each image analysed. n = 769 cells from five mice. IR, immunoreactivity.

Figure 4

Effect of topical application of AF-353 on chorda tympani nerve responses A, representative integrated chorda tympani nerve response to different tastants before, and 10 and 30 min after the application of AF-353 (1.1 mm) on the tongue. Responses to all tastants were totally abolished after a 10 min treatment with AF-353. Responses start recovering 30 min after a rinse with water, denoting a reversible effect of the antagonist. Taste stimuli were applied for 30 s (bar beneath recording) and rinsed for 50 s with water. B, percentage of neural response remaining after application of AF-353 at various concentrations on the tongue for 10 min. As all qualities were similarly affected, responses to all qualities were averaged (means ± SD) for each concentration of AF-353 applied to the tongue. Increasing the concentration of AF-353 proportionally decreased taste responses to all qualities.

Figure 5

Effect of i.p. injection of AF-353 on chorda tympani nerve responses A, representative integrated chorda tympani nerve responses to different tastants before and 10 min after the i.p. injection of AF-353. A high concentration of AF-353 (43.7 μm) measured in the plasma totally abolished responses to all taste qualities (top traces). A lower concentration of AF-353 (1.3 μm) decreased taste responses by about 50% (bottom traces). Taste stimuli were applied for 30 s (bar beneath recording) and rinsed for 50 s with water. B, percentage of neural response remaining 10 min after injection of AF-353 at various concentrations. As there was no tastant-specific effect of AF-353, all taste responses at each plasma concentration were averaged, with means ± SD plotted. Taste responses decrease with increasing concentration of AF-353 in the plasma. C, percentage of change in baseline amplitude following injection of AF-353. The concentration of AF-353 in the plasma does not significantly influence the basal activity of the nerve.

Figure 6

Effect of i.p. injection of AF-353 on behavioural taste responses to SC-45647 Following 3 days of training, mice were tested in the lickometer after i.p. injection of the solvent propylene glycol on day 4, and propylene glycol + AF-353 on day 5. Only the last complete presentation of each concentration was used for analysis to diminish the effect of thirst on intake. The grey horizontal line represents the expected value from random licking from all concentrations. A, average number of licks to different concentrations of SC-45647 compared to total licks for mice with plasma concentrations of AF-353 greater than 100 μm (means ± SEM, n = 12). B, ratio of number of licks for 300 μm SC-45647 to the total number of licks relative to the concentration of AF-353 in the plasma for all mice tested. Each point represents an individual mouse injected with AF-353; for injections with the vehicle propylene glycol, the lick ratios are represented by the means ± SEM for all mice tested (n = 30). Blue symbols represent behavioural responses to the vehicle, while red represents responses in the presence of AF-353.