Function, Innervation, and Neurotransmitter Signaling in Mice Lacking Type-II Taste Cells

IHC confirms lack of GNAT3- and PLCβ2-expressing Type II cells in Skn-1a^-/- mice. A, Lingual sections from Skn-1a^-/- and WT littermates were labeled with antibodies against SNAP25 (magenta) and GNAT3 (green) or PLCβ2 (green). In Skn-1a^-/-, there was no detectable levels of Type II cell markers GNAT3 or PLCβ2. B, Semi-quantitative IHC reveals increased numbers of Type III cells in circumvallate, fungiform, and soft palate taste buds. Each dot represents a single taste bud. Box plots are summaries of all datapoints. Acquisition settings were equalized between WT and KO for all channels. Images are maximal z-projections of 12- to 16-μm image stacks. CV = circumvallate, FF = fungiform; *p < 0.05 by Kruskal–Wallis test followed by Dunn test for multiple comparisons.

Skn-1a^-/- mice have suppressed responses to Type II-mediated taste modalities. A, Chorda tympani nerve activity of Skn-1a^-/- and WT littermates was monitored in response to lingually applied taste solutions (100 mM NH₄Cl, 500 mM sucrose, 10 mM quinine-HCl, 100 mM mono-sodium glutamate, 100 mM mono-sodium glutamate plus 0.5 mM inosine monophosphate, 100 mM NaCl, 10 mM HCl, and 10 mM citric acid). Integrated nerve activity over 30 s of stimulation was normalized to baseline; N = 6 mice for each genotype. B, Baseline normalized chorda tympani activity of Skn-1a^-/- mice in response to NaCl (30, 100, and 300 mM) with pre- and concurrent application of 100 μM amiloride. No significant differences were detected between WT and Skn-1a^-/-. Bar charts indicate sample mean, error bars represent SEM, and superimposed dot plot represents each individual animal. ns = no significance; *p < 0.05 by two-way repeated measures ANOVA with Holm–Sidak post hoc test.

No change in the number of 5-HT_3A-GFP geniculate ganglion neurons in Skn-1a^-/- mice. Skn-1a^-/- mice were crossed to 5-HT_3A-GFP mice. A, B, Geniculate ganglia of Skn-1a^-/-/5-HT_3A-GFP and WT littermates (still expressing 5-HT_3A-GFP) were labeled with antibodies against GFP (green), P2X3 (magenta), and SNAP25 (blue). The number of labeled cells was counted for each label. C, Percent of total cells showing GFP, P2X3, or SNAP25 immunoreactivity. KO of Skn-1a had no effect. D, Percent of total cells showing each combination of labels. KO of Skn-1a had no effect. Data are a summary of from three WT and four KO mice. Bar chart depicts sample mean and error bars show SEM.

KO of Skn-1a does not affect responsiveness of geniculate ganglion neurons to ATP or 5-HT. Skn-1a^-/- mice were crossed to 5-HT_3A-GFP mice. A, B, Raw calcium imaging traces of isolated, individual GFP+ (green) or GFP- (black) geniculate ganglion neurons in response to 10 μM ATP, 10 μM 5-HT, or 55 mM KCl. C, D, Summary of responses to exogenous stimuli; N = 7 GFP+, 4 GFP- from 3 Skn-1a^-/-/5-HT_3A-GFP, and 6 GFP+ and 6 GFP- from 3 Skn-1aWT/5-HT_3A-GFP. Bar charts indicate sample mean, error bars represent SEM, and superimposed dot plot represents each individual cell; *p <0.05 by Two-way ANOVA with Holm–Sidak post hoc test.

No change in the innervation density of taste buds in Skn-1a^-/- mice. A, B, Lingual slices of Skn-1a^-/- and WT littermates were labeled with antibodies against SNAP25 and P2X3. C. P2X3 immunoreactivity density was calculated for each taste bud. Dots represent individual taste buds and the box plot demonstrates distribution of data. CV, circumvallate; Fol, foliate; FF, fungiform; SP, soft palate. Data are from six WT and five Skn-1a^-/- mice. No significant interactions were found by Kruskal–Wallis test.

5-HT_3A-GFP innervation in Skn-1a^-/- mice. 5-HT_3A-GFP mice were crossed with Skn-1a^-/-. Skn-1a^-/- mice that had the GFP transgene and WT littermates that also had the GFP transgene were used for experiments. A, B, Maximal z-projections of fungiform taste buds from WT and KO mice labeled with antibodies against GFP (green) and P2X3 (magenta). A’, B’, Same image as A, B but after prepressing which included Otsu thresholding. C, Analysis was performed on each optical plan of individual taste buds to quantify the relative immunoreactivity of P2X3 and GFP; *p < 0.05 by Kruskal–Wallis test followed by Dunn test for multiple comparisons. Three to four mice from each genotype were used, and 228 taste buds were quantified.

Purinergic receptor antagonism eliminates residual chorda tympani responses to lingually applied taste solutions. The chorda tympani response to taste solutions was measured in Skn-1a^-/- before and after application of 1 mM AF353. Data are representative integrated responses to each stimulus. The y-axis scale is an order of magnitude smaller on the responses after AF353. Responses are representative of four animals.