Overexpression of artemin in the tongue increases expression of TRPV1 and TRPA1 in trigeminal afferents and causes oral sensitivity to capsaicin and mustard oil

Abstract

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, supports a subpopulation of trigeminal sensory neurons through activation of the Ret/GFRalpha3 receptor tyrosine kinase complex. In a previous study we showed that artemin is increased in inflamed skin of wildtype mice and that transgenic overexpression of artemin in skin increases TRPV1 and TRPA1 expression in dorsal root ganglia neurons. In this study we examined how transgenic overexpression of artemin in tongue epithelium affects the anatomy, gene expression and calcium handling properties of trigeminal sensory afferents. At the RNA level, trigeminal ganglia of artemin overexpresser mice (ART-OEs) had an 81% increase in GFRalpha3, a 190% increase in TRPV1 and a 403% increase in TRPA1 compared to wildtype (WT) controls. Myelinated and unmyelinated fibers of the lingual nerve were increased in diameter, as was the density of GFRalpha3 and TRPV1-positive innervation to the dorsal anterior tongue and fungiform papilla. Retrograde labeling of trigeminal afferents by WGA injection into the tip of the tongue showed an increased percentage of GFRalpha3, TRPV1 and isolectin B4 afferents in ART-OE mice. ART-OE afferents had larger calcium transients in response to ligands of TRPV1 (capsaicin) and TRPA1 (mustard oil). Behavioral sensitivity was also exhibited by ART-OE mice to capsaicin and mustard oil, measured using a two-choice drinking test. These results suggest a potential role for artemin-responsive GFRalpha3/TRPV1/TRPA1 sensory afferents in mediating sensitivity associated with tissue injury, chemical sensitivity or disease states such as burning mouth syndrome.

Figure 1. K14-keratin promoter drives increased expression of artemin in the tongue epithelium

Tongue sections from ART-OE (A) and wildtype (B) mice immunolabeled using anti-artemin antibody. Fluorescent signal is localized to basal epithelial cells (arrowheads in A and B) and in the heavily keratinized filiform papillae (arrows). Epi, epithelial layer. Bar in B = 100 µm.

Figure 2. Tongues of ART-OE mice have increased innervation

Wildtype (A, B) and ART-OE (C, D) sections of tongue immunolabeld with antibodies against GFRα3 (A, C) and TRPV1 (B, D). ART-OE tongues are hyperinnervated in epithelial and subepithelial compartments by GFRα3-positive fibers, some of which contain TRPV1. TRPV1-positive fibers in WT tongues were rare (none apparent in B, but see Fig. 3). Arrowheads in A, C and D indicate positive-labeling of afferents in mucosa. Bar in D = 100 µm.

Figure 3. Fungiform papillae are innervated by GFRα3 afferents in WT and ART-OE mice

Sections of tongue from wildtype (A, B) and ART-OE (C, D) mice show representative fungiform papilla (arrows in A and C) immunolabeled with antibodies against GFRα3 (A, C) and TRPV1 (B, D). GFRα3 (not shown) and TRPV1-fiber labeling to fungiform papillae of ART-OEs (D, H) was generally greater compared to wildtype (B, F) structures. Some fibers appeared associated with the single taste bud structure of the papilla made visible using hemotoxylin and eosin staining (E, G). H&E staining of WT (E) and ART-OE (G) sections was done on sections shown in F and H, respectively. Arrows in E and G show taste bud. Bar in D = 50 µm; bar in E = 40µm.

Figure 4. Few geniculate ganglia neurons are immunopositive for GFRα3 or TRPV1

Immunolabeling for GFRα3 (A-C) and TRPV1 (D-F) on sections of trigeminal (TG, A, D) and geniculate (Gen, B, C, E and F) ganglia. Trigeminal ganglia (from ART-OE mice) were labeled in parallel with geniculate ganglia and serve as a positive control for staining. Few if any geniculate neurons were GFRα3-positive in WT (B) and ART-OE (C) mice. TRPV1 reactivity was evident in many neurons of the trigeminal ganglia (D) but few neurons labeled in geniculate ganglia of WT (E) and transgenic (F) mice. Scale bar in D for A, D = 100 µm; bar in F is for B, C, E and F and = 60µm.

Figure 5. The percentage of lingual neurons that express GFRα3, TRPV1 and IB4 is increased in ART-OE mice

A. Representative photomicrographs of WGA-labeled lingual-projecting somata from WT (top) and ART-OE (bottom) mice immunolabeled for TRPV1 (red), IB4 (blue) and TRPV1/IB4 overlap. Neurons in ART-OE ganglia exhibited somal hypertrophy and an increased percentage of neurons expressing TRPV1, IB4 and TRPV1/IB4 (arrows, overlap panel). B. Quantification of the percentage of WGA-positive lingual somata that express GFRα3 and TRPV1, IB4 or CGRP. ART-OE ganglia (red bars) had an increased percentage of WGA-positive neurons that co-expressed GFRα3, TRPV1, IB4, or both TRPV1 and IB4 compared to WT mice (blue bars). No significant change in ART-OE WGA-positive neurons that express CGRP occurred. The mean percentage ± SEM is plotted. Analysis was done with n=4 WT and 4 ART-OE mice. Asterisk indicates p<0.05 vs. WT.

Figure 6. Artemin expression causes hypertrophy of lingual nerve fibers

A. Low magnification electron microscopy image comparing representative lingual nerve from WT (left) and ART-OE (right) mice demonstrates enlarged nerve diameter in ART-OE mice. B. No difference in the percentage of myelinated and unmyelinated axons in the lingual nerve of WT (blue, n=3) and ART-OE (red, n=3) mice was detected. Rightward shift in distribution of axons in diameter (microns) in ART-OE mice indicates hypertrophy of myelinated (C) and unmyelinated (D) axons in transgenic nerves (p<0.002 and p<0.009, two-way ANOVA). Bar in A = 50µm.

Figure 7. ART-OE mice display oral sensitivity to capsaicin and mustard oil

Ten WT (white bars) and 10 ART-OE (black bars) mice of both sex (40 mice total) were tested for oral sensitivity to capsaicin and mustard oil using a two-bottle drinking aversion assay. A. Male and female ART-OE mice drank less capsaicin containing water compared to WT mice (males WT 1.57 ± 0.09 ml; ART-OE 1.00 ± 0.07 ml, p≤0.005: females WT 1.72 ± 0.11 ml; ART-OE 0.98 ± 0.04, p≤0.005). B. Both male and female ART-OE mice drank less mustard oil containing water (males WT 1.47 ± 0.14 ml; ART-OE 1.11 ± 0.06 ml, p≤0.05: females WT 1.68 ± 0.10 ml; ART-OE 1.16 ± 0.06, p≤0.005). C. Same data as panel A but calculated as a percentage of total water consumed (CAP - males WT 29.08 ± 1.37 %; ART-OE 22.16 ± 1.26%, p≤0.005; females WT 28.87 ± 1.90 %; ART-OE 19.74 ± 0.79 %, p≤0.005). D. Calculated as a percentage, female ART-OE mice drank less mustard oil containing water (MO - females: WT 31.15 ± 2.47 %, ART-OE 22.69 ± 1.58 %; p≤0.005). Male ART-OE mice did not drink significantly less mustard oil containing water when calculated as a percentage of total water (see text). E and F. The amount of water (containing 0.5% ethanol) consumed across genotype and sex was unchanged. However, the total amount of water consumed by ART-OE males tested for mustard oil sensitivity was less than WT males (males WT 5.29 ± 0.18 ml, ART-OE 4.52 ± 0.20 ml, p≤0.005). This was not the case for female mice (WT 5.47 ± 0.16 ml; ART-OE 5.19 ± 0.17 ml, p>0.05). Mean ± SEM. Asterisk indicates p<0.05 vs WT.