Enhanced behavioral responses to cold stimuli following CGRPα sensory neuron ablation are dependent on TRPM8
© McCoy and Zylka; licensee BioMed Central Ltd. 2014
Received: 26 August 2014
Accepted: 4 November 2014
Published: 19 November 2014
Calcitonin gene-related peptide-α (CGRPα) is a classic marker of peptidergic nociceptive neurons and is expressed in myelinated and unmyelinated dorsal root ganglia (DRG) neurons. Recently, we found that ablation of Cgrpα-expressing sensory neurons reduced noxious heat sensitivity and enhanced sensitivity to cold stimuli in mice. These studies suggested that the enhanced cold responses were due to disinhibition of spinal neurons that receive inputs from cold-sensing/TRPM8 primary afferents; although a direct role for TRPM8 was not examined at the time.
Here, we ablated Cgrpα-expressing sensory neurons in mice lacking functional TRPM8 and evaluated sensory responses to noxious heat, cold temperatures, and cold mimetics (acetone evaporative cooling and icilin). We also evaluated thermoregulation in these mice following an evaporative cold challenge. We found that ablation of Cgrpα-expressing sensory neurons in a Trpm8 -/- background reduced sensitivity to noxious heat but did not enhance sensitivity to cold stimuli. Thermoregulation following the evaporative cold challenge was not affected by deletion of Trpm8 in control or Cgrpα-expressing sensory neuron-ablated mice.
Our data indicate that the enhanced behavioral responses to cold stimuli in CGRPα sensory neuron-ablated mice are dependent on functional TRPM8, whereas the other sensory and thermoregulatory phenotypes caused by CGRPα sensory neuron ablation are independent of TRPM8.
Somatosensory stimuli are detected by neurons located in the dorsal root ganglia (DRG), which then transmit this information to the spinal cord for processing . In the DRG, nociceptive neurons sense noxious thermal and mechanical stimuli and are broadly characterized as peptidergic or non-peptidergic, with peptidergic neurons commonly marked by calcitonin gene-related peptide immunoreactivity (CGRP-IR). CGRP-IR reflects expression of two closely linked genes, Cgrpα (Calca) and Cgrpβ (Calcb). Both genes are expressed in DRG neurons; although Cgrpα is expressed at higher levels .
We recently generated a mouse line in which Cgrpα–expressing sensory neurons could be ablated in an inducible fashion, thus allowing us to examine how somatosensation is impaired in the absence of these neurons . To accomplish this, we crossed mice with a LoxP-stopped human diphtheria receptor (hDTR) knocked-in to the Cgrpα locus with Advillin-Cre mice, generating “Cgrpα-DTR +/- ” mice . Advillin-Cre was used to restrict hDTR expression to Cgrpα-expressing sensory neurons [3–5]. CGRPα sensory neurons were then ablated in adult mice following two sequential intraperitoneal (i.p.) injections of diphtheria toxin (DTX). Following ablation, we found that sensitivity to noxious heat, capsaicin, and two pruritogens (histamine and chloroquine) were greatly reduced and thermoregulation following an evaporative cold challenge was impaired. Mechanical sensitivity was unaffected. In contrast, CGRPα sensory neuron-ablated mice showed enhanced behavioral responses to cold stimuli and cold mimetics, including enhanced responses to icilin at a dose that selectively activates TRPM8 in vivo . This enhanced responsiveness was not due to a change in the number of TRPM8 immunoreactive (TRPM8+) DRG neurons or to increased peripheral nerve responses to cold stimuli. Instead, our findings suggested that CGRPα primary afferents cross-inhibited postsynaptic spinal neurons that were cold/icilin sensitive. Thus, ablation of CGRPα sensory neurons led to disinhibition of spinal neurons that were postsynaptic to TRPM8 afferents, suggesting a spinal mechanism for enhanced cold responsiveness. Moreover, our data suggested that TRPM8 activity might be responsible for driving enhanced cold sensitivity when CGRPα sensory neurons were ablated, although we did not test this possibility directly.
Here, we sought to directly evaluate whether enhanced cold sensory responses in CGRPα sensory neuron-ablated mice were dependent on TRPM8. To accomplish this, we crossed Cgrpα-DTR +/- mice with Trpm8 -/- mice to produce “DTR-Trpm8 -/- ” mice. With these mice, we were able to inducibly ablate CGRPα sensory neurons in a genetic background that lacks functional TRPM8. We then compared somatosensory responses in these mice, pre- and post-ablation, to Cgrpα-DTR +/- mice—mice with functional TRPM8.
Results and discussion
The total number of NF200+ DRG neurons is reduced following CGRPα sensory neuron ablation
The enhanced behavioral responses to cold stimuli in CGRPα sensory neuron-ablated mice are dependent on TRPM8
TRPM8 neurons respond to cold stimuli, including cold mimetics like icilin, and are expressed in myelinated or unmyelinated neurons [8, 9]. We previously found that ablation of Cgrpα-expressing DRG neurons resulted in enhanced sensitivity to cold stimuli, including cold mimetics like icilin. Moreover, following ablation, there was increased activity in spinal neurons that are postsynaptic to icilin-responsive, TRPM8+ primary afferents . These data suggested that the enhanced sensitivity to cold stimuli might be driven by enhanced tonic and evoked activity in TRPM8+ neurons. To test this possibility, we took advantage of the fact that tonic and evoked activity in TRPM8 neurons can be greatly reduced by deleting Trpm8 . Thus, to directly assess whether the enhanced cold sensory responses were TRPM8-dependent, we introduced the Trpm8 -/- allele into our Cgrpα-DTR +/- line (see Methods for details), generating DTR-Trpm8 -/- mice. We then behaviorally tested these mice, along with Cgrpα-DTR +/- mice (positive controls), before and after ablating CGRPα sensory neurons (by injecting 100 μg/kg DTX; two i.p. injections separated by 72 h). Additional groups of mice were injected with saline and served as controls.
Weight loss and thermoregulatory deficits following CGRPα sensory neuron ablation are TRPM8 independent
Next, we placed mice from each genotype in a 37°C water bath for 2 min and measured rectal body temperature at 5 min increments for 60 min (Figure 6E). Immersing the mouse in the water bath increased core body temperature, which is followed by a decrease in body temperature because of evaporative cooling. Both the DTX-treated Cgrpα-DTR +/- mice and DTX-treated DTR-Trpm8 -/- mice had difficulty with thermoregulation, as evidenced by a greater drop in core body temperature following evaporative cooling and a slower return to baseline relative to saline-injected mice (Figure 6E). Since there were no significant differences in rectal temperature between Cgrpα-DTR +/- mice and DTR-Trpm8 -/- mice post DTX-treatment, ablation of CGRPα neurons appears to impair thermoregulation independent of TRPM8. Note there was no significant difference between saline-treated genotypes (effectively wild-type versus Trpm8 -/- ), indicating that loss of TRPM8 does not impair thermoregulation in this evaporative cooling assay.
We previously found that ablation of CGRPα-containing DRG neurons in mice resulted in decreased sensitivity to noxious heat , presumably because TRPV1 is expressed in ~50% of CGRP+ DRG neurons  and at least half of all TRPV1 neurons were ablated following DTX injection. This decreased sensitivity to noxious heat was coupled with enhanced sensitivity to noxious and innocuous cold. Here, we found that this enhanced sensitivity to cold stimuli was dependent on TRPM8, as enhanced cold sensitivity was lost when CGRPα sensory neurons were ablated in a Trpm8 -/- background. In contrast, both Cgrpα-DTR +/- and DTR-Trpm8 -/- mice showed reduced responses to noxious and innocuous heat stimuli and equivalent responses following an evaporative cold challenge, showing that the heat phenotype and thermoregulatory phenotype following CGRPα sensory neuron ablation is independent of TRPM8. Our data thus strongly support the importance of TRPM8 in mediating enhanced cold sensory responses when myelinated and unmyelinated CGRPα sensory neurons are ablated.
Intriguingly, when humans underwent nerve compression to inhibit nerve impulse conduction in myelinated fibers, stimuli that were perceived as cool or cold prior to nerve compression felt burning hot and painful [15–17]. These findings suggested that loss of myelinated fiber inputs can centrally enhance cold sensation in humans, transforming cold into a stimulus that is perceived as painful. Our findings in mice may thus have parallels to human sensory biology.
Animal care and use
All vertebrate animals and procedures used in this study were approved by the Institutional Animal Care and Use Committee at The University of North Carolina at Chapel Hill. Mice were maintained on a 12 h:12 h light:dark cycle, were fed DietGel 76A (72-03-502; clearH2O.com) and water ad libitum, and were tested during the light phase. Mice were acclimated to the testing room, equipment and experimenter 1-3 days prior to testing. Cgrpα-DTR +/- mice were generated by crossing Cgrpα-GFP -/- female mice  with Advillin-Cre -/- male mice ; and hence are heterozygous for the Advillin-Cre and Cgrpα-DTR alleles. DTR-TRPM8 -/- mice were generated as follows: Trpm8 -/- mice (The Jackson Laboratory, B6.129P2-Trpm8 tm1Jul /J; stock #008198) were crossed with Cgrpα-GFP -/- mice  and with Advillin-Cre -/- mice  to generate heterozygous mice. The heterozygous offspring were crossed to generate Cgrpα-GFP -/- :Trpm8 -/- or Advillin-Cre -/- :Trpm8 -/- mice. Cgrpα-GFP -/- :Trpm8 -/- female mice were bred with Advillin-Cre -/- :Trpm8 -/- male mice, to generate mice with the following alleles Cgrpα-GFP +/- :Advillin-Cre +/- :Trpm8 -/- . Diphtheria toxin (DTX, List Biologicals) was injected as described previously , and does not affect sensory responses or body temperature when administered to wild-type mice, as previously described .
Male mice (10 weeks old) were perfused with cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. L4 dorsal root ganglia were removed from each animal and post-fixed in the same fixative for 3.5 h. One DRG from each pair was sectioned at 20 μm and collected onto Superfrost Plus slides. Every fourth section from each DRG was immunostained for NeuN (mouse IgG1; Millipore, MAB377, 1:250), CGRP (sheep IgG; Enzo Life Sciences, BML-CA1137, 1:300), and NF200 (rabbit IgG; Sigma, N4142, 1:800). Donkey anti-sheep IgG – Alexa-568 and a donkey anti-rabbit IgG – Alexa-647 (Invitrogen) were used at 1:200; a rat anti-mouse IgG1 – FITC (Invitrogen) was used at 1:10. A high-salt (2.7% NaCl) 0.05 M Tris-buffer containing 0.3% Triton X-100, pH 7.6 was used for all steps, except for final rinses in phosphate buffered saline before adding coverslips. All DRG sections were imaged on a Zeiss LSM 510 confocal microscope.
For all behavior experiments, 8-12 week old male mice were used. Heat sensitivity was measured by heating each hindpaw once per day using the Plantar Test apparatus (IITC) with a cut-off time of 20 s. For the tail immersion assay, each mouse was gently restrained in a towel, and the distal one-third of the tail was immersed into a water bath heated to 46.5°C or 49°C or into 75% ethanol cooled to -10°C . The latency to flick or withdraw the tail was measured once per mouse. The cut-off was set at 40 s, 30 s, and 60 s, respectively. For the hot plate test, the latency to jump, shake, or lick a hindpaw was measured within a 30 s cut-off. To measure mechanical sensitivity, we used an electronic von Frey apparatus (IITC) with semi-flexible tips. Two measurements for each hindpaw were taken and averaged to determine the paw withdrawal threshold in grams. The cotton swab assay (innocuous mechanical) was performed as described . For the acetone test , each mouse was placed into a Plexiglas chamber with a wire mesh floor, 50 μL of acetone was placed onto the glabrous surface of the left hindpaw, and the time spent licking was measured for 1 min. Icilin (60 μg in 25 μL injection volume) was injected into one hindpaw, and the number of flinches in 10 min was counted. The cold plantar assay was performed with mice resting on the glass surface of the Plantar Test apparatus (IITC) . For the two-temperature discrimination assay, each mouse was placed into a Plexiglas chamber covering two metal surfaces that were set at the same or different temperatures [10, 11]. The amount of time mice spent on each side over a 10 min period was recorded. Hot and cold sensitivities were assessed on a metal plate heated/cooled to a range of temperatures (5-55°C), with a cut-off time of 30 s, as described . For the water repulsion assay , the mouse was immersed in a 37°C water bath for 2 min. The mouse was removed from the water and placed onto a paper towel for 5 s, then its rectal temperature (deep body temperature, Tb, measured using a digital thermometer, Acorn Temp TC Thermocouple) was measured every 5 min for 60 min. at approximately the same time each day.
We would like to thank Gabriela Salazar for technical assistance and Bonnie Taylor-Blake for DRG dissections, immunostaining, imaging, and cell counts. The UNC Confocal Imaging Core is funded by grants from NINDS (P30NS045892) and NICHD (P30HD03110). This work was supported by grants to M.J.Z. from NINDS (R01NS081127, R01NS067688).
- Basbaum AI, Bautista DM, Scherrer G, Julius D: Cellular and molecular mechanisms of pain. Cell 2009, 139: 267–284. 10.1016/j.cell.2009.09.028PubMed CentralPubMedView ArticleGoogle Scholar
- Schutz B, Mauer D, Salmon AM, Changeux JP, Zimmer A: Analysis of the cellular expression pattern of beta-CGRP in alpha-CGRP-deficient mice. J Comp Neurol 2004, 476: 32–43. 10.1002/cne.20211PubMedView ArticleGoogle Scholar
- McCoy ES, Taylor-Blake B, Street SE, Pribisko AL, Zheng J, Zylka MJ: Peptidergic CGRPalpha primary sensory neurons encode heat and itch and tonically suppress sensitivity to cold. Neuron 2013, 78: 138–151. 10.1016/j.neuron.2013.01.030PubMed CentralPubMedView ArticleGoogle Scholar
- Hasegawa H, Abbott S, Han BX, Qi Y, Wang F: Analyzing somatosensory axon projections with the sensory neuron-specific Advillin gene. J Neurosci 2007, 27: 14404–14414. 10.1523/JNEUROSCI.4908-07.2007PubMedView ArticleGoogle Scholar
- Minett MS, Nassar MA, Clark AK, Passmore G, Dickenson AH, Wang F, Malcangio M, Wood JN: Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons. Nat Commun 2012, 3: 791.PubMed CentralPubMedView ArticleGoogle Scholar
- Knowlton WM, Bifolck-Fisher A, Bautista DM, McKemy DD: TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. Pain 2010, 150: 340–350. 10.1016/j.pain.2010.05.021PubMed CentralPubMedView ArticleGoogle Scholar
- McCoy ES, Taylor-Blake B, Zylka MJ: CGRPalpha-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch. PLoS One 2012, 7: e36355. 10.1371/journal.pone.0036355PubMed CentralPubMedView ArticleGoogle Scholar
- Cain DM, Khasabov SG, Simone DA: Response properties of mechanoreceptors and nociceptors in mouse glabrous skin: an in vivo study. J Neurophysiol 2001, 85: 1561–1574.PubMedGoogle Scholar
- Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K: Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors. J Comp Neurol 2005, 493: 596–606. 10.1002/cne.20794PubMedView ArticleGoogle Scholar
- Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL, Jordt SE, Julius D: The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 2007, 448: 204–208. 10.1038/nature05910PubMedView ArticleGoogle Scholar
- Dhaka A, Murray AN, Mathur J, Earley TJ, Petrus MJ, Patapoutian A: TRPM8 is required for cold sensation in mice. Neuron 2007, 54: 371–378. 10.1016/j.neuron.2007.02.024PubMedView ArticleGoogle Scholar
- Colburn RW, Lubin ML, Stone DJ Jr, Wang Y, Lawrence D, D'Andrea MR, Brandt MR, Liu Y, Flores CM, Qin N: Attenuated cold sensitivity in TRPM8 null mice. Neuron 2007, 54: 379–386. 10.1016/j.neuron.2007.04.017PubMedView ArticleGoogle Scholar
- Knowlton WM, Daniels RL, Palkar R, McCoy DD, McKemy DD: Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice. PLoS One 2011, 6: e25894. 10.1371/journal.pone.0025894PubMed CentralPubMedView ArticleGoogle Scholar
- Feketa VV, Balasubramanian A, Flores CM, Player MR, Marrelli SP: Shivering and tachycardic responses to external cooling in mice are substantially suppressed by TRPV1 activation but not by TRPM8 inhibition. Am J Physiol Regul Integr Comp Physiol 2013, 305: R1040–1050. 10.1152/ajpregu.00296.2013PubMed CentralPubMedView ArticleGoogle Scholar
- Wahren LK, Torebjork E, Jorum E: Central suppression of cold-induced C fibre pain by myelinated fibre input. Pain 1989, 38: 313–319. 10.1016/0304-3959(89)90218-2PubMedView ArticleGoogle Scholar
- Yarnitsky D, Ochoa JL: Release of cold-induced burning pain by block of cold-specific afferent input. Brain 1990,113(Pt 4):893–902.PubMedView ArticleGoogle Scholar
- Chakour MC, Gibson SJ, Bradbeer M, Helme RD: The effect of age on A delta- and C-fibre thermal pain perception. Pain 1996, 64: 143–152. 10.1016/0304-3959(95)00102-6PubMedView ArticleGoogle Scholar
- Wang JJ, Ho ST, Hu OY, Chu KM: An innovative cold tail-flick test: the cold ethanol tail-flick test. Anesth Analg 1995, 80: 102–107.PubMedGoogle Scholar
- Garrison SR, Dietrich A, Stucky CL: TRPC1 contributes to light-touch sensation and mechanical responses in low-threshold cutaneous sensory neurons. J Neurophysiol 2012, 107: 913–922. 10.1152/jn.00658.2011PubMed CentralPubMedView ArticleGoogle Scholar
- Brenner DS, Golden JP, Gereau RW: A novel behavioral assay for measuring cold sensation in mice. PLoS One 2012, 7: e39765. 10.1371/journal.pone.0039765PubMed CentralPubMedView ArticleGoogle Scholar
- Gentry C, Stoakley N, Andersson DA, Bevan S: The roles of iPLA2, TRPM8 and TRPA1 in chemically induced cold hypersensitivity. Mol Pain 2010, 6: 4. 10.1186/1744-8069-6-4PubMed CentralPubMedView ArticleGoogle Scholar
- Westerberg R, Tvrdik P, Unden AB, Mansson JE, Norlen L, Jakobsson A, Holleran WH, Elias PM, Asadi A, Flodby P, Toftgård R, Capecchi MR, Jacobsson A: Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem 2004, 279: 5621–5629. 10.1074/jbc.M310529200PubMedView ArticleGoogle Scholar
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