A sensory-labeled line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia

Abstract

Many primary sensory neurons are polymodal, responding to multiple stimulus modalities (chemical, thermal, or mechanical), yet each modality is recognized differently. Although polymodality implies that stimulus encoding occurs in higher centers, such as the spinal cord or brain, recent sensory neuron ablation studies find that behavioral responses to different modalities require distinct subpopulations, suggesting the existence of modality-specific labeled lines at the level of the sensory afferent. Here we provide evidence that neurons expressing TRPM8, a cold- and menthol-gated channel required for normal cold responses in mammals, represents a labeled line solely for cold sensation. We examined the behavioral significance of conditionally ablating TRPM8-expressing neurons in adult mice, finding that, like animals lacking TRPM8 channels (Trpm8(-/-)), animals depleted of TRPM8 neurons ("ablated") are insensitive to cool to painfully cold temperatures. Ablated animals showed little aversion to noxious cold and did not distinguish between cold and a preferred warm temperature, a phenotype more profound than that of Trpm8(-/-) mice which exhibit only partial cold-avoidance and -preference behaviors. In addition to acute responses, cold pain associated with inflammation and nerve injury was significantly attenuated in ablated and Trpm8(-/-) mice. Moreover, cooling-induced analgesia after nerve injury was abolished in both genotypes. Last, heat, mechanical, and proprioceptive behaviors were normal in ablated mice, demonstrating that TRPM8 neurons are dispensable for other somatosensory modalities. Together, these data show that, although some limited cold sensitivity remains in Trpm8(-/-) mice, TRPM8 neurons are required for the breadth of behavioral responses evoked by cold temperatures.

Figure 1.

Development of the Trpm8^DTR mouse line. A, A complementary DNA transgene for the simian DTR, a GFP fusion protein, was inserted into the trpm8 BAC clone at sequences corresponding to the second coding exon. B, Transgene expression (GFP-expression) in TG and DRG neurons. C, DTR-expressing central projections (green) were localized to the outermost lamina of the dorsal horn of the lumbar spinal cord and did not overlap with those binding to IB₄ (red). D, Immunolabeling revealed no transgene expression in non-peptidergic neurons that bind IB₄.

Figure 2.

Selective ablation of TRPM8-expressing sensory neurons. A, Schematic of the TRPM8 neuron ablation protocol. Adult mice, both Trpm8^DTR (DTR) and control littermates (WT), were given two intraperitoneal injections of 50 μg/kg DTx, which selectively ablated only targeted neurons (green cells). B, One week after injection of a single dose of DTx, the expression of TRPM8 transcripts was reduced to 12.6 ± 3.8% of control and then to 0.18 ± 0.04% if two injections were performed (n = 5, ^#p < 0.0001), as measured by qPCR. GFP-like immunoreactivity was abolished in Trpm8^DTR mice injected with DTx (D) compared with vehicle-injected Trpm8^DTR mice (C) or control Trpm8^GFP mice (E) subjected to the same DTx injection protocol as in D. F, Comparative analysis of RNA transcripts isolated from control and ablated DRG was performed by microarray and plotted as the fold reduction (log10) in expression between the two conditions. Dashed line indicates the 1.5-fold reduction threshold for significant reduction in expression.

Figure 3.

Ablation of TRPM8 afferents abolishes acute cold behaviors. A, Evaporative cooling induced by application of a droplet of acetone to the hindpaw evokes stereotyped behaviors from wild-type, un-injected Trpm8^DTR, and control mice as determined on a 5-point score scale (see Materials and Methods; Knowlton et al., 2011). The response scores of Trpm8^−/− and ablated mice were significantly reduced from control to 1.1 ± 0.1 and 1.2 ± 0.1 (n = 12, ***p < 0.001), respectively, and were not different from one another (p > 0.05). B, Intraperitoneal icilin injections (50 mg/kg body weight) induced robust wet-dog shakes in control but were absent in both Trpm8^−/− and ablated mice (*p < 0.05 each compared with control). The latency to nocifensive flinching (C) and licking/wringing (D) behaviors of the forepaw when mice were placed on a 0°C metal plate was recorded. The average latency to flinch and licks in control mice was 6.2 ± 2.1 and 21.3 ± 2.5 s, respectively (n = 6), and similar to that observed in wild-type and Trpm8^DTR mice (p > 0.05). Latencies of Trpm8^−/− and ablated mice were significantly higher compared with control at 58.0 ± 7.9 s (n = 10) and 69.8 ± 15.0 s (n = 10) for flinching, respectively, and 72.9 ± 15.4 and 98.5 ± 17.1 s for licking, respectively (*p < 0.05, **p < 0.01). Dashed line marks the standard 60 s cutoff time point.

Figure 4.

Mouse mobility on the two-temperature choice assay is altered in absence of TRPM8 channels and neurons. A, The MouseChaser tracking software was developed to analyze mouse movement on the two-temperature choice assay. Screen capture from software after annotation of video with position of mouse. The red square is an estimate of the mouse's position with the yellow diamond marking the center of which the x and y coordinates are logged over the course of the video. B, Movements of wild-type mice in the two-temperature choice chamber, with the control surface held constant at 30°C and the test at temperatures from 10 to 50°C, were monitored over a 5 min period. Pseudocolored exploration plots show qualitatively equal activity on both surfaces set to 30°C, with reduced movement to and on the test surface as it was either heated or cooled. C, Comparative analysis of mobility from noxious cold (5°C) to noxious heat (45°C) for control, Trpm8^−/−, and ablated mice. D, Avoidance of the test surface was measured as the number of times the animal crossed between surfaces, with control mice showing decreased mobility as the temperature deviated from 30°C. Both Trpm8^−/− and ablated mice avoided noxious heat but, unlike controls, did not avoid 20 versus 30°C (**p < 0.01, n = 6–12). Trpm8^−/− mice did avoid cold as temperatures dropped below 20°C but not to the same extent as control mice (*p < 0.05). However, ablated mice showed no avoidance behaviors until test plate temperatures of 5°C. E, Preference for warmth was determined by quantifying the time spent on the 30°C surface, with all genotypes showing strong preference for warmth over painful heat (p < 0.001 compared with test at 30°C). Control and Trpm8^−/− mice showed warm preference at temperatures ≤15°C (p < 0.001 compared with test at 30°C), whereas ablated animals showed no preference between 10 and 20°C (p > 0.05 compared with test at 30°C) and moderate preference at 5°C (p < 0.01 compared with test at 30°C). Preference behaviors for ablated mice were different from control and Trpm8^−/− animals at all temperatures below 20°C (**p < 0.01, ***p < 0.001).

Figure 5.

Heat, mechanical, and proprioceptive behaviors do not require TRPM8 neurons. Latencies to hindpaw flinching on a hotplate were the same at (A) 48°C and (B) 52°C in all genotypes tested (p > 0.05, n = 6–12). C, Mildly noxious mechanical stimulation of the hindpaw with the electronic von Frey apparatus yielded similar paw-withdrawal thresholds in all genotypes (n = 6–12, p > 0.05). D, Dynamic stroke of the hindpaw with a puffed-up cotton swab yielded similar response frequencies in both control and ablated mice (n = 8–11, p > 0.05). E, The average maximum grip strengths were similar across all genotypes, as was the latency to drop from an accelerating rotarod (F) (n = 6–27, p > 0.05).

Figure 6.

TRPM8 neuron ablation selectively diminishes inflammation- and nerve injury-induced cold allodynia. A, Inflammation significantly increased ipsilateral, but not contralateral, response scores to evaporative cooling in control mice by 2 d after CFA injection (n = 10). Both Trpm8^−/− (n = 6) and ablated mice (n = 10) showed significantly lower sensitization compared with controls (***p < 0.001) but were significantly increased over baseline and the contralateral side (p < 0.01). There were no significant differences in sensitization between Trpm8^−/− and ablated mice (p > 0.05). B, After induction of irritation to the sciatic nerve (CCI), control mice showed an increase in ipsilateral scores, whereas contralateral scores remained unchanged (n = 8). Both Trpm8^−/− and ablated mice (n = 8 each) showed significantly lower sensitization compared with controls and were significantly increased over baseline and the contralateral side (p < 0.01). Unlike CFA, ablated mice showed a significant increase in nocifensive behaviors compared with control mice (*p < 0.05, **p < 0.01, ***p < 0.001). In both the CFA (C) and CCI (D) models, the significant (p < 0.001) decrease in latencies to hindpaw lift from a radiant heat source were identical between all genotypes tested (p > 0.05). Similarly, mechanical thresholds were increased on the ipsilateral side (p < 0.001) but were indistinguishable (p > 0.05) between all genotypes before and after injury in both pain models (E, F).

Figure 7.

Mice lacking TRPM8 or neurons are deficient in cooling-induced analgesia. A, In a neuropathic pain model (CCI), control mice exhibited increased sensitivity to mechanical stimuli that was temporally alleviated by cooling the affected paw (17°C for 5 min). Before cooling [baseline (BL)], ipsilateral thresholds were significantly lowered from that recorded contralateral (p < 0.001). After cooling, ipsilateral thresholds were significantly increased over baseline (3.8 ± 0.3 g at baseline compared with 7.1 ± 0.4 g at 5 min after cooling; ***p < 0.001) and indistinguishable from contralateral thresholds (7.7 ± 0.5 g at baseline compared with 7.3 ± 0.3 g at 5 min after cooling, ^#p > 0.05). Trpm8^−/− (B) and ablated (C) mice showed decreased ipsilateral mechanical thresholds in the CCI model (p < 0.001) similar to controls and were unaffected by cooling.

Figure 8.

Model for TRPM8 neuronal function. Our data suggest the presence of TRPM8 neurons that mediate innocuous cool (green) and likely provide inhibitory input, either through spinal inhibitory interneurons (yellow) or directly to nociceptors (pink) to induce cooling analgesia. Cold nociceptors (blue) likely also express TRPM8 and provide input on noxious cold temperatures that drive thermal-avoidance behaviors.