The cellular code for mammalian thermosensation

Abstract

Mammalian somatosenory neurons respond to thermal stimuli and allow animals to reliably discriminate hot from cold and to select their preferred environments. Previously, we generated mice that are completely insensitive to temperatures from noxious cold to painful heat (-5 to 55°C) by ablating several different classes of nociceptor early in development. In the present study, we have adopted a selective ablation strategy in adult mice to study this phenotype and have demonstrated that separate populations of molecularly defined neurons respond to hot and cold. TRPV1-expressing neurons are responsible for all behavioral responses to temperatures between 40 and 50°C, whereas TRPM8 neurons are required for cold aversion. We also show that more extreme cold and heat activate additional populations of nociceptors, including cells expressing Mrgprd. Therefore, although eliminating Mrgprd neurons alone does not affect behavioral responses to temperature, when combined with ablation of TRPV1 or TRPM8 cells, it significantly decreases responses to extreme heat and cold, respectively. Ablation of TRPM8 neurons distorts responses to preferred temperatures, suggesting that the pleasant thermal sensation of warmth may in fact just reflect reduced aversive input from TRPM8 and TRPV1 neurons. As predicted by this hypothesis, mice lacking both classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50°C. Our results provide a simple cellular basis for mammalian thermosensation whereby two molecularly defined classes of sensory neurons detect and encode both attractive and aversive cues.

Figure 1.

Ablation of TRPV1- and TRPM8-expressing cells. a, To determine the cells required for thermosensation, we generated mice that carried BAC transgenes containing an insertion of GFP fused via a foot and mouth disease virus 2A-peptide to DTR at the initiation codon of the TRPV1 or TRPM8 genes. These mice were treated with DT to eliminate sensory neurons in adult animals. b, In situ hybridization of sections through DRG demonstrates that ablation of TRPV1- and TRPM8-expressing neurons in DT-treated TRPV1-DTR and TRPM8-DTR mice are selective for the targeted cells.

Figure 2.

Specific ablation of sensory neurons is achieved by DT treatment of DTR-transgenic mice. a, DT treatment of TRPV1-DTR and TPRM8-DTR mice generates animals with specific loss of TRPV1- and TRPM8-expressing neurons. In situ hybridization (ISH) of sections through DRG were probed with molecular markers as indicated. Lumbar DRG from several different mutant mice were analyzed by ISH for expression of TRPV1, TRPA1, TRPM8, and Mrgprd. Serial sections were stained for NeuN-positive neurons to provide a total neuron count. Data represent the percentage of total neurons that were positive by ISH and are shown as means ± SEM (n = 3); significance was assessed using Student's t test. **p < 0.01. b, c, Capsaicin-elicited eye wipes (b) and icilin-induced wet dog shakes (c) were used to assess loss of TRPV1 and TRPM8 function, respectively. b, TRPV1-DTR and TRPV1^−/−, but not TRPM8-DTR mice, lose eye-wipe responses. **p < 0.01, Student's t test. c, In contrast, responses to icilin are lost in TRPM8-DTR and TRPM8^−/− mice, but not TRPV1 mice.**p < 0.01, Student's t test. Data represent means ± SEM (n ≥ 6 animals).

Figure 3.

Ablation of TRPV1- and TRPM8-expressing sensory neurons does not alter the numbers of spinal cord interneurons. In situ hybridization was performed with probes to different neuropeptides on sections through the dorsal horn of the spinal cord of control and mutant animals as indicated. No significant differences between genotypes were observed for numbers of positive neurons.

Figure 4.

Elimination of TRPV1- and TRPM8-expressing neurons impair avoidance to heat and cold temperatures, respectively. Two-plate preference tests were used to assess thermal responses of TRPV1-DTR and TRPM8-DTR mice. In these experiments, the fixed plate was set at 30°C. a, TRPV1-DTR mice (red squares) show no behavioral responses to hot temperatures (45–50°C; p < 0.01, Student's t test) that are strongly aversive to control mice (TRPV1-DTR nontransgenic DT-treated littermates; open circles). In contrast, TRPV1^−/− animals (pink squares) exhibit responses that are indistinguishable from controls. b, TRPM8-DTR mice (dark blue squares) have greatly attenuated responses to cold compared with controls (TRPM8-DTR nontransgenic DT-treated littermates; open circles), but retain strong aversion to heat. Significant differences between genotypes were observed from 0–25°C and at 35°C (p < 0.01, Student's t test). TRPM8^−/− mutants (pale blue squares) are also less sensitive to cool temperatures (5–20°C) than controls, but are significantly more responsive to cold (0–10°C) than TRPM8-DTR mice (^#p < 0.05, Student's t test). Time at test temperature represents the fraction of time mice spent on the test plate versus the fixed plate; data are means ± SEM (n ≥ 6 animals).

Figure 5.

TRPA1-expressing neurons do not play a detectable role in cold reflex behavioral responses. a, In situ hybridization of sections through DRG was used to examine TRPA1 expression. After ablation of TRPV1 neurons, all TRPA1 expression was lost. b, A cold plantar assay was used to assess responses of various mutant mice to rapid cooling. In this assay, TRPM8^−/− mutants (pale blue bar) are less sensitive than controls (*p < 0.05, Student's t test), but are significantly more responsive than TRPM8-DTR mice (**p < 0.01, Student's t test). In contrast, loss of TRPA1 input (elimination of TRPV1 cells) had no effect on paw withdrawal (TRPV1-DTR; red bar) even in mice in which TRPM8 cold-responsive cells were also killed (TRPV1-DTR/TRPM8-DTR; hashed bar). Data represent means ± SEM (n ≥ 6 animals).

Figure 6.

Mrgprd neurons contribute to the detection of noxious cold. a, Representative in situ hybridization of sections through DRG from TRPM8-DTR/Mrgprd-DTR mice illustrates selective loss of TRPM8- and Mrgprd-expressing cells. b, In cold plantar tests, TRPM8-DTR mice (blue bars) have significantly longer withdrawal latencies than controls (**p < 0.01, Student's t test). In contrast, behavioral responses of Mrgprd-DTR mice (gray bars) were indistinguishable from controls (n.s., Student's t test). Combined loss of TRPM8 and Mrgprd neurons (dark gray bars) causes a significantly greater cold deficit than ablation of TRPM8 cells alone (*p < 0.05, Student's t test). Elimination of TRPV1 cells in addition to the TRPM8 and Mrgprd cells (cross-hatched bar) had no additional effect on paw withdrawal latency. As expected, TRPV1-DTA mutants (mice with an extensive loss of TRPV1-lineage neurons, black bar) exhibit significantly reduced withdrawal latency compared with controls and other genotypes (**p < 0.01, Student's t test). Data represent means ± SEM (n ≥ 6 animals).

Figure 7.

Mrgprd neurons contribute to the detection of painful heat. a, Representative in situ hybridization of sections through DRG from TRPV1-DTR/Mrgprd-DTR mice illustrates selective loss of TRPV1- and Mrgprd-expressing cells. b, A standard hot plate test that measures latency to show escape reactions to 55°C was used to examine responses to noxious heat; significant differences between genotypes were assessed using Student's t test. Mrgprd-DTR mice (pale gray bar) display withdrawal responses indistinguishable from controls (open bar), whereas TRPV1-DTR mice (red bars) exhibit reduced response to noxious heat (*p < 0.05). Additional ablation of TRPM8 cells (hatched bar) had no significant effect on response latency. In contrast, double mutants in which both the TRPV1 and Mrgprd cells were eliminated (dark gray bar) and TRPV1-DTA mice that extensively lack both of these classes of nociceptor (black bar) were essentially unresponsive within the cutoff for this assay. Data represent means ± SEM (n ≥ 6 animals).

Figure 8.

TRPV1 and TRPM8 neurons code for hot, cold, and warm stimuli. a, In two-plate choice assays with the fixed plate set at 25°C, TRPV1-DTR mice (red squares) exhibit marked preference for test temperatures ≥ 40°C, whereas their control littermates (open circles) display profound avoidance to 45–50°C. In contrast, TRPM8-DTR animals (blue squares) display no preference for warm and show increasing avoidance as temperature rises >35°C. b, When the fixed plate was set at 45°C, TRPM8-DTR mice (blue squares) preferred all colder temperatures, whereas TRPV1-DTR animals (red squares) were significantly more averse to cold than controls (red squares) over the 10–20°C temperature range (p < 0.01, Student's t test). c, d, Mice with combined loss of TRPV1- and TRPM8-expressing cells (TRPV1-DTR/TRPM8-DTR, purple squares) were also tested using the two-plate preference assay with the fixed plate set at 25°C (c) or 45°C (d). The double mutant animals exhibited no temperature preference across the full range of temperatures. Time at test temperature represents the fraction of time mice spent on the test plate versus the control plate. Data represent means ± SEM (n ≥ 6 animals).