Odontoblast TRPC5 channels signal cold pain in teeth

Abstract

Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system.

Fig. 1. TRPC5 is essential for inflammatory tooth pain.

Percent change in sucrose consumption relative to baseline (dashed line) after DPI. DPI enhanced the consumption of 5% room temperature sucrose water to 293 ± 46% above baseline (^###P = 0.00001). Lack of TRPC5 (^##P = 0.005; 171 ± 21%), but not TRPM8 (P = 0.9; 299 ± 34%) or TRPA1 (P = 0.1; 225 ± 43%), reverts glucose consumption after DPI to baseline. The reduction in sucrose consumption in TRPC5^−/− (n.s. P = 0.5), but not TRPA1^−/− (^##P = 0.006) or TRPM8^−/− (^##P = 0.004), was not different from the respective controls without DPI. n.s., not significant.

Fig. 2. Extracellular recordings from mouse tooth nociceptors in a novel jaw-nerve preparation.

(Top) Illustration of mouse head with jaws and their innervation. The mandible-inferior alveolar nerve preparation is derived from the lower jaw and transferred to an organ bath consisting of an external solution and a mini-tube that is connected to a temperature-controlled application system. Connection of the application system’s heating coil to a heating/cooling thermostat board permits rapid exchange of solution temperature in the mini-tube, where the preparation is exposed to chemical compounds and cold temperatures. Both the external and internal bath are supplied with oxygenized extracellular solution. Because of the short length of the inferior alveolar nerve, suction electrodes from glass capillaries are applied and the amplifier is used in a single-ended configuration. The external bath is required to prevent cold block in the nerve when the teeth in the mini-tube are exposed to cold. Action potentials from the inferior alveolar nerve are recorded in gap-free mode with Spike 2 (Materials and Methods).

Fig. 3. Tooth nociceptor cold responses are much larger than skin cold nociceptor responses.

(A) Schematic illustration of extracellular recordings from jaw-nerve as compared to skin-nerve preparations.(B to D) Comparison of the (B) cold response magnitude, (C) peak frequency, and (D) threshold temperature of C57BL/6J teeth (n = 45) and skin (n = 59) nociceptors. Statistical significance was identified by a two-sided Student’s t test: ***P = 2 × 10⁻⁸, ***P = 3 × 10⁻¹¹, and *P = 0.05. Skin nociceptor cold responses are from (6, 50) and refer to the same background strain at equivalent stimulus conditions.

Fig. 4. TRPC5 and TRPA1 are sufficient as cold sensors in healthy teeth.

(A) Percent cold-sensitive tooth nociceptors blocked by HC-030031 (n = 9 of 9 fibers) and ML204/HC-070 (n = 6 of 13 fibers) and respective fraction of block (means ± SEM). (B) C57BL/6J wt tooth nociceptor recording with temperature (top) and instantaneous frequency pattern (I.F.P.; bottom) blocked by HC-070 and HC-030031. Circles represent action potentials. (C) Tooth cold responses in TRPA1^−/− (n = 10 of 138, P = 1.0), TRPC5^−/− (n = 8 of 217, ^‡P = 0.04), and TRPC5/A1-DKO (n = 5 of 177, ^‡P = 0.02), chi-square tests versus C57BL/6J (n = 45 of 570). (D) Typical cold response of a TRPC5/A1-DKO tooth nociceptor with temperature (top) and instantaneous frequency pattern (I.F.P.; bottom). As in (B), circles represent action potentials, and horizontal bars and arrows indicate applications and respective time intervals. (E to G) Teeth nociceptor cold response characteristics according to genotype, (E) cold response magnitude [P = 0.2 between groups by one-way analysis of variance (ANOVA)], (F) peak firing frequency (P = 0.01), and (G) temperature threshold (P = 0.06). TRPC5/A1-DKO were statistically different from C57BL/6J wt (^#P = 0.04 and ^#P = 0.02) and TRPC5^−/− (^#P = 0.05, ^##P = 0.001, and ^##P = 0.007). TRPC5^−/− were statistically different from TRPA1^−/− (^#P = 0.03) and C57BL/6J (^#P = 0.05) in least significant difference post hoc tests. (H to J) Histograms in bins of 2 s (H) and respective box plots of (I) dynamic and (J) constant cold responses. Significant differences between groups by one-way ANOVA existed for dynamic (10 to 42 s of the histogram; P = 0.01) but not constant cold (44 to 80 s of the histogram; P = 0.6). TRPC5/A1-DKO (n = 5) and TRPA1^−/− (n = 10) were different from C57BL/6J (^#P = 0.02 and ^#P = 0.03, n = 45) and TRPC5^−/− (^#P = 0.01 and ^##P = 0.02, n = 8). Histograms present data as means ± SEM, and asterisks refer to two-sided Student’s t test comparison of respective knockouts with the C57BL/6J background strain (3 × 10⁻⁶ < *P < 0.002). Lines represent medians, squares represent the mean, boxes represent the interquartile range (IQR), and whiskers represent 2.2-fold of IQR after exclusion of the >2.2 IQR outliers identified by crosses.

Fig. 5. Most cold-sensitive DPANs use TRPM8, but TRPA1 and TRPC5 are also functional cold transducers.

(A) Characterization and quantification of cold-sensitive cultured DPANs (C57BL/6J: n = 23 of 136) based on Ca²⁺ transients measured with Fura-2 AM and sensitivity to TRP channel modulators menthol (me, n = 7), carvacrol (crv, n = 3), both (n = 10, descending diagonal stripes), neither (n = 4, ascending diagonal stripes), and riluzole (rlz, n = 1). Cold-sensitive DPANs insensitive to menthol and carvacrol (ascending diagonal stripes) are unchanged in TRPC5^−/− (n = 1 of 14 in 93) versus C57BL/6J (3 of 23 in 136; n.s. P = 0.86), but increased in TRPM8A1-DKO (n = 23 in 457; ^‡‡P = 0.002 and ^‡‡‡P = 0.00003). In TRPC5A1-DKO, cold-sensitive cells (n = 20 of 100) were mostly menthol sensitive (n = 15; crv+ n = 10; both, n = 7 and none, n = 2). TRPC5M8-DKO cold-sensitive cells (n = 5 of 103) were mostly sensitive to carvacrol (n = 3; me+ n = 2; both, n = 1 and none, n = 1). Inset: Photomicrograph of cultured mouse TG neurons with red DiI retro-labeled DPANs. (B and C) Ca²⁺ transient traces from (B) C57BL/6J representative of four types of cold-sensitive neurons and (C) TRPM8A1-DKO (red), TRPC5A1-DKO (green), and TRPC5M8-DKO (blue). Bottom: Temperature stimulator command. (D) TRPM8/A1-DKO control (cntrl) cold responses (n = 23) were smaller than in C57BL/6J (n = 23; ^###P = 0.0007) but not in TRPC5^−/− neurons (n = 13; P = 0.2). ML204 (circles = treated neurons) blocked most cold responses in TRPM8/A1-DKO (≥50% block = filled circles, n = 12; *P = 0.01) and some in C57BL/6J neurons (n = 15; *P = 0.02), but not in TRPC5^−/− neurons (n = 13; n.s. P = 0.2). n.s., not significant. Lines are medians, squares mean, boxes IQR, whiskers 2.2-fold IQR after exclusion of >2.2 IQR outliers identified by crosses.

Fig. 6. Functional TRPC5 in trigeminal neurons.

(A) Relative expression of TRP channel genes and nociceptor-specific subtypes of voltage-gated sodium channel genes in mouse DPANs given as transcripts per million (TPM). Data are presented as means ± SEM. Dots represent each replicate. Replicates with value 0 are not represented in the logarithmic scale. TRPC5 was not among the transcripts (see table S1). (B) Red DPANs (arrowheads) in TRPC5 reporter mouse ganglion (TG) multiphoton stacks of maxillo-mandibular regions. A total of 176 DPANs had 11 TRPC5⁺ neurons (seven TGs, five mice). (C) Typical doubly rectifying current-voltage relation observed in a cultured TRPC5⁺ neuron of a reporter mouse (n = 13). TRPC5 current is small and is only identified as Englerin-sensitized current subtracted from the baseline current in the absence of chloride in the solution.

Fig. 7. TRPC5 channels are located in the odontoblast layer.

(A) TRPC5 reporter mouse molar tooth whole mount with densely packed TRPC5⁺ odontoblasts at the pulp-dentin boundary. (B and C) In tight association with sensory nerves. Green, TRPC5; red, βIII-tubulin; circle indicates area shown in (D) and (E) from a subsequent section. cp, coronal pulp; rp, radicular pulp. (E) Oblique section through the predentinal radicular pulp to visualize the tight association of TRPC5⁺ odontoblast processes with their sensory nerves.

Fig. 8. TRPC5 is expressed in normal human teeth and increases with pulpitis.

(A) Whole-mount panography of a human tooth; regions evaluated for TRPC5 expression are indicated; dotted line, level used in root measurement. (B) TRPC5⁺ nerve fibers in the radicular pulp double labeled with TRPM8. (C) TRPC5⁺ nerve fibers double labeled with PGP9.5 within the inner third of the dentinal tubules (type IV) ~40 μm above the odontoblast-predentinal border. (D) TRPC5⁺ branched fibers (type II/III) in the predentin/dentin layer. (E) Quantification of TRPC5⁺ and TRPM8⁺ fiber types (four teeth). TRPC5/PGP9.5 and TRPM8/PGP9.5 for type I: 13/51 and 7/22; type II/III: 41/70 and 0/35; type IV: 26/105 and 6/49. (F) Hematoxylin and eosin (H&E)–stained human tooth whole mount with degenerated dentin (caries) and pulpitis. (G) Abundant TRPC5⁺ and decreased TRPM8⁺ (arrows) nerve fibers in the pulpitic tooth root. (H) TRPC5⁺ nerve fibers (type IV) in degenerating dentin (inset, H&E). (I) Increased TRPC5⁺ (190 of 660 versus 394 of 607 fibers; **P = 0.002), decreased TRPM8⁺ (197 of 660 versus 67 of 607; **P = 0.005), and similar proportions of colabeled fibers (black) in normal versus pulpitic tooth roots [132 ± 78 (n = 4) versus 86 ± 21 (n = 7; P = 0.2)] and in the tooth pulp [TRPC5⁺: 15 of 51 versus 81 of 161; *P = 0.01; TRPM8⁺: 622 of 161 versus 9 of 22; **P = 0.008; colabeled fibers (black): 12 ± 3 (n = 4) versus 18 ± 8 (n = 9); P = 0.3]. Error bars, SEM.