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Mammalian somatosenory neurons respond to thermal stimuli allowing animals to reliably discriminate hot from cold and select their preferred environments. We previously 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. Here we have adopted a selective ablation strategy in adult mice to dissect this phenotype and thereby 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, while 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. Thus, 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. Notably, 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 form TRPM8 and TRPV1-neurons. As predicted by this hypothesis, mice lacking both these classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50 °C. Taken together these 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.
Thermosensation provides valuable information about the environment and triggers strong emotional and behavioral responses over a wide temperature range. For example, noxious heat and cold are highly unpleasant sensations that trigger powerful escape reactions in most animals. However, mammalian thermosensation does far more than simply trigger reflex withdrawal from painful heat or cold: for instance, humans reliably distinguish unpleasantly hot and cold stimuli, while intermediate temperatures (e.g. cool or warm) can be very pleasant and drive attractive rather than aversive responses.
The ability of mammals to sense the temperature in their environment relies primarily on specialized somatosensory neurons in the trigeminal and dorsal root ganglia (DRG) that project axonal processes to the outer layers of the skin (Woolf and Ma, 2007). How are thermal cues encoded in these neurons at the periphery?
A major advance in the understanding of mammalian temperature sensation came with the characterization of TRPV1 as a high temperature and capsaicin gated-channel that is expressed in heat sensitive DRG neurons (Caterina et al., 1997). Subsequent identification of TRPM8 as a low temperature sensor (McKemy et al., 2002, Peier et al., 2002) as well as several other TRP-channels that appear to be stimulated by either heating or cooling led to the proposal that multiple TRP-channels act in concert as differentially tuned “molecular thermometers” to orchestrate appropriate responses over a wide range of temperatures (Jordt et al., 2003, Patapoutian et al., 2003).
In keeping with its role as a putative cold temperature sensor, knockout of TRPM8 results in a large selective deficit in an animal’s ability to detect cool (15 – 25 °C) temperatures (Bautista et al., 2007, Colburn et al., 2007, Dhaka et al., 2007). However, knockout of other thermosensory TRP-channels, provides more modest support for this model (Kwan et al., 2006, Vriens et al., 2011) and the limited thermosensitive defect of TRPV1-KO mice has led to questions about the role of TRPV1 in vivo (Woodbury et al., 2004, Park et al., 2011). Nonetheless, agonist dependent ablation (Karai et al., 2004, Mishra and Hoon, 2010) or silencing (Cavanaugh et al., 2009) of TRPV1-neurons, resulted in significant deficits in sensing hot, suggesting that TRPV1 marks heat sensitive neurons. Unfortunately, resiniferatoxin-mediated ablation of TRPV1-cells is never complete (Mishra and Hoon, 2010) and the capsaicin induced lesions (Cavanaugh et al., 2009) are largely undefined reducing the strength of conclusions that can be drawn from this type of approach.
Recently, we generated mice that were completely insensitive to a very broad range of thermal stimuli (from −5 °C to 55 °C) (Mishra et al., 2011). In these animals, TRPV1 mediated expression of diphtheria toxin (DTA) resulted in ablation of several different molecular classes of nociceptor including the TRPV1-, TRPM8- and Mrgpr-neurons because TRPV1-DTA is expressed in common precursor cells (Mishra et al., 2011). Here we used a different molecular genetic approach to selectively eliminate each of these neural classes in adult mice and thereby delineated a simple logic for detection of temperature spanning the entire physiologically relevant range.
All procedures followed the NIH Guidelines for the care and use of laboratory animals, and were approved by the National Institute of Dental and Craniofacial Research Animal Care and Use Committee. TRPV1-DTR and TRPM8-DTR mice are BAC transgenics; BACs were engineered by recombineering (Lee et al., 2001) using RP23-181P10 and RP24-78N24 respectively to generate transgenic constructs (Figure 1a). As expected before DT treatment transgenically expressed GFP was readily detected in the appropriate number of somatosensory neurons. However, although we searched extensively outside peripheral ganglia in the brain and other organs that have been reported to express TRPV1 (Cavanaugh et al., 2011) we found no expression of GFP even using sensitive antibody staining techniques. Thus these BAC-transgenes selectively target DTR to the appropriate class of somatosensory neuron (see Figure 1 for additional characterization). The other strains have been described previously: Mrgprd-DTR (Cavanaugh et al., 2009) (a generous gift from David Anderson), TRPV1-DTA (Mishra et al., 2011), TRPM8 −/− (Bautista et al., 2007) and TRPV1 −/− (Caterina et al., 2000).
Animals were intercrossed to generate experimental animals (of either sex) as described in the text; the TRPV1-DTR and TRPM8-DTR were hemizygous and Mrgprd-DTR heterozygous in all experiments. For diphtheria toxin mediated cell ablation we began administering DT in adult mice (> 5 weeks old); behavioral testing began at least 1 week and no more than 2 months after DT administration using mice weighing 20–30g (2–4 month old). Over this 2 month time period, repeat experiments established that phenotypes were completely stable. Control mice for each experimental series were DT treated non-transgenic littermates and were co-housed with DTR-siblings. We tested mice in behavioral assays before recording its ear-tag number, thus experimenters were blind to the genotype of individual mice. No significant differences in behavior between different sets of controls were noted and for simplicity data was combined from the TRPV1-DTR and TRPM8-DTR littermate controls unless otherwise specified.
We experimentally established the effectiveness of DT mediated ablation in the different lines and assessed the minimal dosage required to achieve > 95 % elimination of target cells. For TRPM8-DTR animals, this required daily injection of DT (100ng) for 7 days; for TRPV1-DTR and Mrgprd-DTR animals, cell elimination was achieved by regular injection of 200ng DT (administered for 5 days, followed by 2 days off, for 3 weeks); double and triple mutants all received this higher DT dose. We used the TRPV1-DTR line to check that DT indeed ablates cells from the ganglia by counting neurons in serial sections through the entire L4 ganglion of control (8660 ±667 NeuN positive neurons) and DT treated TRPV1-DTR mice (5760 ±467 NeuN positive neurons; significant difference between groups; P< 0.01, Student’s t test, means ±s.e.m. (n=4). As expected approximately 33 % of total neurons are lost, consistent with selective loss of TRPV1-expressing neurons (Caterina et al., 2000). Core body temperature (Table 1) was measured using a rectal probe (Fine Science Tools); measurement was performed on 3 separate days.
Eye wipe assays were performed to investigate the afferent functions of the ophthalmic branch of the trigeminal nerve. Capsaicin induced eye-wipes were counted for 1 minute following delivery of 50 μl of solution (50 μM capsaicin), PBS elicited eye wipes were subtracted from those measured for capsaicin. Wet-dog shakes were induced by i.p. injection of 50 mg/Kg icilin (Sigma) and numbers of whole body shakes were counted over 30 minutes as described (Dhaka et al., 2007).
A two choice temperature assay was employed to determine thermal preference; mice were placed in an apparatus that had a fixed reference plate set at 25, 30, or 45 °C and a test-plate whose temperature was adjusted between 0 °C and 50 °C (T2CT, Bioseb, France). Each animal was tested twice for each set of parameters; the first assay was initiated by placement of the mouse on the plate set at the test-temperature, in the second assay the initial placement was reversed so that the mouse was placed on the fixed-plate. Mouse position was tracked over 5 minutes using an automated tracking system (Bioseb, France). Only assays in which mice sampled both plates within the first minute were scored.
Thermal reflex response assays were used to assess acute temperature sensitivity. For hot responses, mice were placed on a plate at 55 °C and latency to display withdrawal of hind limbs was measured; cut-off to prevent injury was set at 30 seconds. A cold plantar assay (Brenner et al., 2012) was used to assess sensitivity to low temperatures; for this assay, animals were habituated (> 15 min) in individual chambers with a 1/16 inch glass plate floor and a dry ice pellet was applied below the hind-paw. The time for paw-withdrawal was measured; for each reflex-response assay, animals were tested more than 3 times.
In situ hybridization (ISH) was performed at high stringency (washed 30 min, 0.2x SSC, 70 °C) as described previously (Hoon et al., 1999). ISH of molecular markers were performed on tissue from >5 transgenic and control mice. Serial sections from >10 sections per mouse were hybridized and numbers of cells counted in order to quantize numbers of neurons. Immunohistochemistry was performed with chicken anti-GFP (1:1000 Abcam), and rabbit anti NeuN (1:800 Abcam) and developed with donkey anti-chicken Alexa488 or donkey anti-rabbit Alexa 488 (Jackson Immunolabs) respectively. Images were collected using a Microphot FX microscope (Nikon) and images were processed with Adobe Photoshop.
Previously, our attempt to ablate the thermoreceptor neurons expressing TRPV1 with constitutively expressed DTA was frustrated because TRPV1 is expressed in the precursors of several functionally distinct classes of nociceptor (Mishra et al., 2011). Here we adopted a different strategy that combined molecular genetic targeting and toxin injection to selectively kill defined classes of these somatosensory neurons in adult mice. This approach makes use of the fact that mice are normally insensitive to diphtheria toxin (DT) because they do not express a receptor that mediates cellular uptake of the active toxin. This means that cell-specific DT-sensitivity can be achieved by directed expression of the human diphtheria toxin receptor (DTR) (Saito et al., 2001). Thus to target putative thermoreceptive neurons, we engineered BAC-transgenic mice that express DTR the under the control of TRPV1- and TRPM8-regulatory sequences (see Figure 1a) and used DT injection in adult mice to selective ablate these classes of somatosensory neurons. In situ hybridization studies (Figure 1b and Figure 2a) indicated that TRPV1-neurons are effectively and selectively killed by DT-injection in mice expressing the TRPV1-DTR transgene and that TRPM8-neurons (but not other classes of nociceptor) are eliminated in TRPM8-DTR mice. For simplicity, we refer to the toxin treated animals as TRPV1-DTR and TRPM8-DTR mice respectively.
Given that approximately one third of somatosensory neurons express TRPV1, we expected that ablation of this class of somatosensory neuron should result in a dramatic reduction in the total neural count. Indeed, we found toxin treatment reduced the number of NeuN positive neurons by about 30 % in the DRG of TRPV1-DTR mice (see Methods for details). Analysis of interneurons in the spinal cord (Figure 3) revealed no noticeable differences between mutant and control animals indicating that TRPV1-cell ablation destroys peripheral input without grossly affecting the somatosensory circuitry. We also noted that core body temperature of TRPV1-DTR mice remained indistinguishable from that of controls (Table 1). Finally, we assayed capsaicin induced eye-wipes as a high sensitivity behavioral assay for TRPV1 function and observed no response in TRPV1-DTR animals (Figure 2b), while responses to the TRPM8-agonist icilin remained indistinguishable from those of toxin treated wild-type controls (Figure 2c). Thus TRPV1-DTR animals have a highly selective and essentially complete loss of TRPV1-expressing neurons.
TRPM8 is expressed in a much smaller subset of neurons than TRPV1 (see Figure 1b). Thus no significant differences in the number of DRG-neurons were detected in TRPM8-DTR mice. However, as expected, behavioral assays showed that these animals are completely unresponsive to the potent TRPM8-agonist, icilin (Figure 2c) while they retain responses to capsaicin that were indistinguishable from controls (Figure 2b). Like the TRPV1-DTR mice, TRPM8-DTRs exhibited completely normal expression of markers of spinal cord interneurons (Figure 3) and core body temperature (Table 1).
We next used a two-plate choice assay to assess temperature preference in TRPV1- and TRPM8-DTR animals. Controls included toxin treated littermates not carrying the transgenes as well as TRPV1−/− and TRPM8−/− mice. In essence, the assay allows us to set the temperature of one plate at a permissive temperature (fixed-plate) and to study the response of individual mice to changes in temperature of the other plate (test-plate) over a wide and physiologically relevant range (from 0 – 50 °C). When the fixed-plate was set at 30 °C, control mice showed a clear preference for the test-plate at 35 °C but exhibited increasingly strong avoidance of the test-plate when the temperature was raised or lowered outside the 25 – 40 °C range (Figure 4, open circles). Notably, TRPV1−/− mice behaved exactly like control animals in this assay (Figure 4a, pink circles). In contrast, DT-mediated ablation of TRPV1-neurons completely eliminated aversion to elevated temperatures (40 – 50 °C), but did not affect avoidance of cold (Figure 4a, red squares) highlighting the role of TRPV1-cells (but not TRPV1-itself) as selectively tuned hot sensors. The opposite phenotype was observed with TRPM8-DTR animals, which exhibited normal aversion to hot temperatures but a striking loss of response to cool and cold (Figure 4b, blue squares). Our data [and previous studies (Bautista et al., 2007, Colburn et al., 2007, Dhaka et al., 2007)] showed a qualitatively similar phenotype for TRPM8 −/− mice, with less aversion to cool (15 – 25 °C) temperatures than control mice (Figure 4b, pale blue circles, P < 0.05, Students t-test). However, ablation of TRPM8-cells resulted in a significantly stronger cold (0 – 15 °C) deficit than knockout of the TRPM8 gene (Figure 4b). Taken together our results confirm the hypothesis that TRPV1 and TRPM8-neurons provide selective, aversive input in response to heat and cold respectively and demonstrate that each of these classes of neurons utilize unknown thermosensors in addition to either TRPV1 or TRPM8.
One prominent, but controversial, candidate cold-sensor is the TRP-channel, TRPA1 (Story et al., 2003, Jordt et al., 2004, Bautista et al., 2006, Kwan et al., 2006, Caspani and Heppenstall, 2009, Karashima et al., 2009, Knowlton et al., 2010). Here, we demonstrate that TRPA1 is not expressed in the cold sensing TRPM8 cells but rather is exclusively found in TRPV1 expressing cells (Figure 2) confirming previous reports (Story et al., 2003, Mishra and Hoon, 2010). Interestingly, we noted that TRPM8-DTR mice exhibited modest but appreciable aversion to 0 °C, the lowest temperature tested in the 2-place assay (Figure 4b). Thus we hypothesized that TRPA1-input (and TRPV1-cells) might be involved in triggering aversion to painful cold in TRPM8-DTR mice.
To test this hypothesis, we first confirmed that TRPA1-expression is eliminated in TRPV1-DTR mice (Figure 5a and and2a).2a). Next we used a cold plantar assay (Brenner et al., 2012) to investigate whether TRPV1-cell input affects an animal’s tolerance of noxious cold. In this assay, paw withdrawal latency is measured following application of a piece of dry ice to the glass plate directly under the mouse’s hind paw (rapid cooling of the plantar surface). As expected knockout of TRPM8 or ablation of TRPM8 cells significantly increased the time it took for mice to respond (Figure 5b), and in agreement with the two-plate preference assay, ablation of the TRPM8 cells resulted in a more profound deficit in cold sensing than was observed in TRPM8−/− mice. In contrast, TRPV1-DTR mice displayed reaction times that were no different from control animals and more importantly, TRPV1-DTR/TRPM8-DTR animals were indistinguishable in their response from TRPM8-DTR animals (Figure 5b). Therefore TRPA1 has no major role in this type of cold reflex responses.
Which cells mediate the residual responses to cold in TRPM8-DTR animals? Previously we have shown that TRPV1-DTA mice, which lack several classes of sensory neuron because the TRPV1-lineage is ablated during development in this strain, were completely insensitive to cold in the two plate assay (Mishra et al., 2011). Therefore we suspected that other nociceptors that are lost in TRPV1-DTA mice might be involved and set out to examine if Mrgprd-expressing neurons augment cold sensation through TRPM8 neurons at very low temperatures. We obtained the previously reported Mrgprd-DTR knockin-line (Cavanaugh et al., 2009) (a generous gift from David Anderson) and confirmed that these mice have no significant cold sensing phenotype (see Figure 6b, light gray bar). Next we crossed the Mrgprd-DTR allele into a TRPM8-DTR background to ablate both sets of neurons (Figure 6a) and found that these mice exhibited significantly less cold sensitivity than TRPM8-DTR mice in the cold plantar assay of noxious cold (Figure 6b, dark gray bar). Thus Mrgprd-cells not only detect mechanical pain (Cavanaugh et al., 2009) but also respond to extreme cold. Nonetheless, the TRPV1-DTA mice that we described previously (Mishra et al., 2011) have an even greater deficit in cold sensation than the Mrgprd-DTR/TRPM8-DTR double mutants implying that additional TRPV1-lineage cells may also respond to painfully cold temperatures. Could functional responses of TRPV1-cells to cold, unmasked by ablating both TRPM8 and Mrgprd-neurons, account for this difference? To answer this question we generated and tested triple mutants lacking TRPV1-, TRPM8- and Mrgprd-neurons. These mice (Figure 6b, hatched bar) were indistinguishable in their response to cold to the double mutants that still had normal TRPV1- input (Figure 6b, dark grey bar). Thus our data demonstrate that TRPV1-cells play a very minor role (if any) in cold detection and since TRPA1 is exclusively expressed in TRPV1-cells, further limit the role this protein can have as a cold detector.
We also examined responses of mutant animals to painful heat. Because, exposure to temperatures above 50 °C needs to be strictly limited to prevent injury, we could not use the two-place preference test and instead used paw withdrawal from a 55 °C heated block with a 30 s exposure maximum to assay noxious heat. As shown previously (Mishra et al., 2011) TRPV1-DTA mice exhibit no escape reaction within the cutoff (Figure 7b, black bar). However, although TRPV1-DTR mice are far less sensitive to heat than controls, they still exhibit paw-withdrawal within the 30 s cutoff (Figure 7b, red bar). Do Mrgprd cells contribute to responses to painful heat as well as noxious cold? Again we generated double mutant animals, this time TRPV1-DTR/Mrgprd-DTR, demonstrated efficient ablation of both populations of sensory neurons (Figure 7a) and tested behavioral responses. Importantly, just as observed for cold sensation, ablation of Mrgprd cells alone had no effect on paw withdrawal latency, but when combined with ablation of TRPV1-cells, it significantly increased reaction time to high temperature (Figure 7b). Indeed TRPV1-DTR/Mrgprd-DTR mice were almost completely insensitive to 55 °C exposure just like the TRPV1-DTA animals. Taken together these results show that Mrgprd neurons respond to extremes of heat and cold and may enhance escape reactions (as well as modify the sensory experience) to these noxious and painful stimuli.
Interestingly, TRPM8-DTR mice not only showed reduced responses to cold temperatures in the two-plate preference assay, but also a marked shift in their preferred temperature from 35 °C in control animals to 25 °C in the mutants (Figure 4). Thus we set out to investigate how TRPV1- and TRPM8-neurons affect the perception of warmth (i.e. the temperature range that a normal mouse actively seeks out). To do this, we again used the two plate assay but initially decreased the temperature of the fixed-plate to 25 °C, the preference maximum for TRPM8-DTR mice (Figure 4). Under these conditions control mice preferred to remain on the warmer block at temperatures up to and including 40 °C but still strongly disliked higher temperatures (Figure 8a). TRPV1-DTR animals behaved indistinguishably from controls in the warm (30 – 40 °C) regime but unlike controls, continued to show increased preference for the test-plate even temperatures that are normally strongly aversive (up to 50 °C, Figure 8a, red squares). As predicted TRPM8-DTR mice exhibited a completely different phenotype. These animals never were attracted to the warmer environment and importantly showed progressively increasing aversion at 35 °C and above (Figure 8a).
We also subjected animals to the opposite scenario where the fixed-block was set at a modestly aversive elevated temperature (45 °C). In this assay, normal mice chose to stay at cooler test temperatures far longer than when the fixed-block is set at 30 °C (compare Figure 8b and Figure 4). As expected, TRPV1-DTRs showed strong preference for 45 °C at all temperatures tested (Figure 8b) while TRPM8-DTR mice always preferred the colder temperature even when the test-block was at 0 °C (Figure 8b, blue squares). Taken together these data indicate that the threshold for TRPM8-mediated aversion is around 35 °C (body temperature), and suggest that TRPV1 and TRPM8 neurons provide all thermosensitive input from the periphery. If this is true, then the simplest explanation for the sensation of warmth would be that it reflects the absence of aversive signaling from either cold or hot sensing neurons rather than a positive signal from another class of thermoreceptor. This model predicts that ablation of both hot and cold sensing neurons should yield mice that cannot detect warmth. Figure 8 (panels c & d) demonstrates that TRPV1-DTR/TRPM8-DTR animals fail to exhibit attraction to temperatures that normal animals actively seek out and indeed are essentially indifferent to temperatures between 0 and 50 °C. Thus input through the TRPM8 and TRPV1 cells not only alert mice to unpleasant or noxious temperatures but also specify their preferred temperature range and control responses to warmth.
Here, we used a selective ablation strategy to explore the contribution of individual, molecularly defined classes of sensory neuron to the mammalian sense of temperature. Importantly, the DTR-transgenes that we developed are very selectively expressed in sensory neurons (see Methods for details) and ablation of each class of cell was specific and had no detectable effects on other sensory neurons (Figure 1 & 2) or the wider somatosensory circuitry (Figure 3). Moreover, diphtheria-toxin treatment of the different transgenic mice resulted in reproducible, highly selective and non-overlapping effects on animal behavior ruling out non-specific effects of this experimental technique. Indeed, similar approaches have been extensively validated in previous studies of the peripheral nervous system (Gogos et al., 2000, Cavanaugh et al., 2009), CNS (Luquet et al., 2005), and for other sensory systems (Huang et al., 2006). Thus our results demonstrate that thermosensation almost exclusively uses two differentially tuned populations of sensory neurons: TRPV1 cells that respond to high temperature and TRPM8 cells that detect cold to encode a full range of sensory percepts (cold, cool, warm and hot). Interestingly, thermoreception in fruit flies closely parallels its mammalian counterpart and also uses separate populations of hot and cold responsive neurons to encode the full temperature range (Gallio et al., 2011), perhaps reflecting a common evolutionary need of animals to avoid temperature extremes.
In our experiments, ablating TRPV1-cells but not knockout of the TRPV1-channel itself had major effects on an animal’s ability to detect elevated temperatures as high as 50 °C implying that these cells must express novel heat sensors in addition to TRPV1. Similarly, although TRPM8 serves as an important receptor for cooling (Bautista et al., 2007, Colburn et al., 2007, Dhaka et al., 2007) the greater thermal deficit of TRPM8-DTR mice compared to knockouts (Figures 4 & 5) indicates that additional cold receptors are activated in these neurons when temperatures fall below 15 °C. Our data also demonstrate that extremes of heat and cold recruit at least one more class of nociceptors, the cells expressing Mrgprd, which are not normally thought to respond to thermal cues (Cavanaugh et al., 2009). While it is possible that these neurons in fact are a class of thermosensor that only respond to temperature extremes we favor a model where the known sensitivity of Mrgprd-nociceptors to inflammatory and algesic compounds (Dussor et al., 2008, Rau et al., 2009) accounts for their recruitment by both noxious heat and noxious cold. In contrast, no role for TRPV1-cells (and by implication the TRPA1 channel) was detected in responses to low temperatures (Figures 4 – 6).
Are TRPV1 and TRPA1 required for any types of thermal response? Our study was primarily designed to uncover the role of specific cells types in somatosensation rather than the role of specific receptors. Nonetheless, we found little evidence that either channel is directly involved in detecting thermal cues over a wide, physiologically relevant temperature range. Notably our data are largely consistent with previous reports (Caterina et al., 2000, Bautista et al., 2006) in the temperature range (0 – 50 °C) that we studied. Indeed, the biggest effects of TRPV1 on heat detection appear at more extreme temperatures and after injury or inflammation (Caterina et al., 2000), conditions not investigated in our study. Similarly, TRPA1 may have a more significant role as a cold transducer after injury (del Camino et al., 2010) and it is possible that cellular coding is also modified in these circumstances. However, in normal mice TRPA1 neither affects temperature preference nor withdrawal responses from cold thus challenging the notion (Kwan et al., 2006, Karashima et al., 2009) that TRPA1 is a cold-temperature sensor in the absence of injury.
Interestingly ablation of TRPM8-neurons caused major temperature detection deficits in the warm temperature range (up to 35 °C, Figures 4 & 8) in line with previous in vivo studies (de la Pena et al., 2005, Madrid et al., 2009, Sarria et al., 2012) that found similar tuning using functional modulation of these cells. Together with the flat temperature response profile of TRPV1-DTR/TRPM8-DTR double mutant animals, the most parsimonious explanation for the feeling of warmth is that this percept actually reflects minimal signaling through the TRPM8-cells (and little or no input from TRPV1-cells). However, because TRPV1-expressing neurons (and TRPM8-counterparts) may not be homogeneous populations of thermosensors, we cannot completely exclude more complex scenarios e.g., where a subset of TRPV1-neurons is tuned to respond to warm temperatures and provides attractive rather than aversive input.
How would the simple model (lack of aversive input through TRPM8 and TRPV1 neurons) fit with our own experience? While it is practically impossible to extrapolate human perception to behavioral studies of mice, it remains likely that a very similar peripheral mechanism accounts for temperature detection in both species. The separate inputs of cooling and heating nicely explain our ability to distinguish cold from hot and why inflammation and injury, which increase responses through TRPV1-cells (Caterina and Julius, 2001), make normally pleasant heat unbearable. Other familiar experiences like the soothing effects of a cool breeze or menthol as well as the thermal grill illusion (Craig, 2002) or the painful effects of going from a freezing environment to a warm one point to significant and complex interactions between these two distinct sensory lines. Finally, our evaluation of temperature (and the valence assigned to stimuli) can vary greatly according to our core-temperature. Thus we anticipate that future studies mapping the connections and circuitry of the afferent TRPM8 and TRPV1 sensory lines will be useful in explaining how sensation drives temperature perception and how this information interacts with internal and emotional state to control behavior.
Author Contributions: We are grateful to Nick Ryba for encouragement and helpful advice and Drs. Minh Nguyen and Lars von Buchholtz as well as members of MGU for valuable suggestions. Transgenic mice were generated by Andrew Cho in the NIDCR-core. We are very grateful to Dr. David Anderson for providing Mrgprd-DTR mice. This research was supported by the intramural research program of the NIH, NIDCR (MAH).