Cold thermosensation

Below is my third and final piece of coursework, an essay entitled Multiple roles for Transient Receptor Potential Melastatin 8 (TRPM8) in cold thermosensation. This time, I discuss three recent studies which have contributed significantly to the understanding of the mechanisms by which nerve endings in the skin detect cold stimuli. I’ll post the rest of the essay over the next few days, in 3 or 4 parts. The papers I discuss will be listed as references after the discussion section.

Summary: Here I discuss three recent studies which have contributed significantly to the understanding of cold thermosensation. McKemy et al (2002) cloned and characterized TRPM8; this was the first cold receptor to be identified. Dhaka et al (2007) generated TRPM8 knockout mice, and analysed the mutant phenotype to show that TRPM8 senses and transduces innocuous cool and noxious cold stimuli, and mediates cool-induced analgesia. Takashima et al (2007) investigated the distribution in the periphery of TRPM8-expressing sensory neuron nerve terminals; their findings provide an anatomical basis for the multiple roles of TRPM8 in cold thermosensation.

Primary sensory neurons of the trigeminal and dorsal root ganglia (TG and DRG, respectively) relay tactile, noxious and hot and cold stimuli from the periphery to the central nervous. These neurons have a single process which bifurcates close to the cell body. One branch projects into the superficial layers of the spinal cord, where it forms synapses with second order sensory neurons; the other branch projects into the periphery, where it innervates the skin.

Distinct subpopulations of TG and DRG are responsive specific types of stimulus. Some respond to innocuous stimuli such as light touch and small changes in skin temperature; others respond to noxious stimuli such as intense pressure, hot and/or cold temperatures (below ~28 degrees Celsius or above ~43 degrees Celsius) and irritant chemicals released from damaged cells. Some DRG cells respond to only one type of stimulus, while others are polymodal.

TG and DRG cells are also characterised anatomically by the size of the cell body and the diameter of the fibre, and functionally by action potential conduction velocity and sensory modality. A-beta fibres are classified as proprioceptors; they have large cell bodies, are thickly myelinated, and have a high conduction velocity. C-fibres have small cell bodies, and thin, unmyelinated fibres which conduct action potentials slowly; and A-delta fibres have cell bodies of an intermediate size. Most C-fibres, and some A-delta fibres, are polymodal nociceptors, which respond to multiple stimuli.

nature05662-f1.2

Dorsal root ganglia (DRG) contain the cell bodies of primary sensory neurons. These bipolar neurons have a single process which bifurcates, with one of the branches projecting to the periphery, and the other projecting into the superficial layers of the spinal cord. In the periphery, the nerve terminals of primary sensory afferents transduce environmental stimuli of various kinds, including light touch, pain and innocuous and noxious temperatures (from Lumpkin & Catarina, 2007).

It has long been known that TG and DRG contain cells which respond to innocuous and noxious hot and cold temperatures. Until recently, however, nothing was known about the molecular mechanisms of thermoreception. But in the past decade, much progress has been made, largely because of the identification of the Transient Receptor Potential (TRP) family of ion channels. To date, six temperature-sensitive TRP channels have been identified. These channels have been named “thermoTRPs”; four of them are sensitive to heat, and two respond to cold (Dhaka et al, 2006).

McKemy et al (2002) used whole-cell patch clamping and calcium imaging to record the responses of cultured rat trigeminal ganglion neurons to cold temperatures and various cooling compounds. They found that the cells respond to menthol and cold with an increase in intracellular calcium ion concentration, and that these stimuli activate non-selective cation channels which are highly permeable to calcium. The currents measured were also found to be outwardly rectifying (i.e. much larger at positive than at negative holding potentials). Similar results were obtained from DRG neurons.

They then cloned TRPM8 from a trigeminal ganglion cDNA library and expressed it in Xenopusoocytes. Electrophysiological recordings showed that oocytes expressing TRPM8 channel were sensitive to cold, confirming that the channel is indeed a cold receptor. The cloned channel was found to have a temperature threshold of 8-28 degrees Celcius; it also conferred upon the oocytes sensitivity to menthol and eucalyptol, with the strongest response elicited by the super-cooling compound icilin.

To further investigate the role of TRPM8 in cold thermosensation, Dhaka et al (2007) generated TRPM8 knockout mice by replacing amino acid residues 2-29 of the TRPM8 gene with enhanced green fluorescent protein (EGFP). Calcium imaging showed that only 7.6% of DRG neurons from TRPM8-deficient mice, respond to a cold stimulus of 10 degrees Celcius, compared to 14.9% of cells from wild type (WT) animals.

The temperature sensitivity of the mutants was then assayed, using an apparatus consisting of multiple compartments to produce a surface temperature gradient ranging from 15-53 degrees Celcius. In this assay, both WT and TRPM8-deficient mice largely avoided severe cold (16-20 degrees Celcius) and hot (41-53 degrees Celcius) temperature compartments. However, whereas WT mice spent twice as much time in the compartment with a surface of ~35 degrees Celcius than in other the compartments, the TRPM8-/- animals spent significantly more time in the cooler zones (23-30 degrees Celcius).

A two-temperature choice assay was then performed, in which the mice were placed on a platform consisting of two identical surfaces set at different temperatures. Whereas WT mice strongly preferred warm over cold temperature surfaces, TRPM8-/- animals showed no preference to a platform set at 31 degrees Celcius to one set at 18 degrees Celcius.

The ability of TRPM8-deficient mice to detect noxious cold temperatures was then examined. When TRPM8-deficient mice were placed on a cold plate of temperature -1 degrees Celcius, their behaviour was identical to that of WTs. When placed on the cold plate following an injection of icilin into the hindpaw, the WT mice responded by rapidly by withdrawing the paw from the cold surface; this behaviour was completely abolished in the TRPM-/- mice, as was the vigorous body shaking that is normally induced by intraperitoneal injection of icilin.

Finally, it was demonstrated that TRPM8 mediates cooling-induced analgesia. A 2% formalin injection into the paw produced the same nociceptive response in both mutant and wild-type animals. However, when the animals were placed on a 17 degrees Celcius cold plate following the formalin injection, the mutant mice spent significantly more time licking and lifting the injected paw than the wild-types.

Thus, mice lacking TRPM8 have a severely impaired sensitivity to innocuous cool temperatures and have a partially impaired ability to detect noxious cold temperatures. These findings are corroborated by two independent studies in which TRPM8-/- mice were generated (Bautista et al, 2007; Colburn et al, 2007).

Takashima et al (2007) carried out one of the first investigations of the distribution of TRPM8-positive sensory nerve terminals in various peripheral structures, using transgenic mice which express enhanced green fluorescent protein under the control of the TRPM8 transcriptional promoter.

First, they confirmed that the transgene expression was neuron-specific, by showing that cultured DRG and TG neurons from the transgenic animals expressed both GFP and the pan-neuronal marker PGP-9.5. The correspondence of GFP and PGP-9.5 coexpression with TRPM8 immunoreactivity showed that the transgene expression was limited to TRPM8-positive sensory neurons. Furthermore, >80% of cultured GFP+ neurons responded to menthol by an increase in intracellular Ca2+ concentration, as determined by calcium microfluorimetry, and outwardly rectifying currents, as measured by whole-cell voltage clamp recordings.

Consistent with earlier studies, it was found that approximately 13% of DRG and TG cells expressed the transgene. The majority of these neurons had cell bodies of around 10µm diameter, and were assumed to be C-fibres, the remainder had cell bodies of up to 30µm diameter, and were assumed to be A delta fibres.

Antibody staining was then used to determine coexpression of the transgene with markers of primary sensory neurons. In DRG, approximately 25% of GFP+ cells were immunoreactive for the C-fibre marker peripherin, and ~14% stained with an antibody for NF200, an intermediate filament protein marker for A-delta fibres. In TG, >30% of GFP+ cells, expressed peripherin, and >25% were immunoreactive for NF200. Significantly, 60% of the DRG cells, and ~40% of TG cells, expressed neither peripherin nor NF200, and are, therefore characterized only by TRPM8 expression.

In the skin, GFP+ nerve terminals were found both superficially, in the stratum granulosum, and deeper, near the epidermis-dermis boundary. This is consistent with the hypothesis that innocuous cool stimuli are detected by nerve terminals located in the superficial skin layers, while noxious cold stimuli are detected terminals that are located deeper.

A complex distribution of TRPM8+ nerve terminals was also observed in other peripheral structures. In the teeth, GFP was found to be expressed in the dentin, which is known to contain A-delta fibres, and in the pulp, which contains C-fibres. In the palate, GFP+ nerve terminals were found in different layers of the epithelium.

Thus, in both DRG and TG, some TRPM8+ sensory neurons characteristics of nociceptors, but others do not. TG has a higher proportion of TRPM8+ cells than do the DRG, and, in the periphery, the terminals of TRPM8+ cells in both ganglia innervate regions of the skin and teeth that are associated with sensing different kinds of cold stimuli.

The study by McKemy et al is of great significance, as it led to the identification and characterization of the first cold receptor. This study also suggests that TRP channels have a general role in thermosensation, as all the previously identified TRP channels are sensitive to heat.

Dhaka et al (2007) show that TRPM8 is required for sensitivity to innocuous cool stimuli and is also involved in sensing noxious cold temperatures. The TRPM8 knockout mice generated in this study have only a partial deficit in sensing noxious cold stimuli, so it is most likely that at least one other cold receptor is involved. Story et al (2003) identified a second cold receptor, TRPA1/ ANKTM1, but its role under physiological conditions is still in question.

There are likely to be other, as yet unidentified, cold sensitive TRP channels. Thermosensation may involve a combinatorial code of thermoTRPs, such that a given repertoire of receptors, each activated by a specified temperature range, confers upon primary sensory afferents sensitivity to cold stimuli of different intensities.

This study also shows that TRPM8 mediates cooling-induced analgesia. TRPM8 is known to be upregulated in subsets of DRG cells following nerve injury leading to hypersensitivity and allodynia (Proudfoot et al, 2006); the channel could therefore be a useful target for novel analgesics.

Takashima et al (2007) show that TRPM8+ primary sensory neurons are neurochemically and anatomically heterogeneous. TRPM8+ sensory neurons express markers of A-delta fibres and C-fibres, and of presumptive nociceptors and non-nociceptors. A significant proportion of TRPM8+ neurons (~60%) express no other markers; thus TRPM8 may be a marker which distinguishes cold fibres from other primary sensory neurons.

This study also shows that different types of TRPM8+ sensory neurons have distinct receptive fields in the periphery, and therefore provides a neuroanatomical basis for the multiple role of TRPM8 in cold thermosensation. It also raises the possibility that TRPM8 is expressed in two labeled lines of cold fibres, one consisting of nociceptors, the other of non-nociceptors.

The three studies discussed here contribute significantly to our understanding of cold thermosensation. Together, they link events at the molecular level with behavioural responses, and provide an anatomical basis for the multiple roles of TRPM8. However, the primary sensory neurons which express TRPM8+ are not yet characterized properly, and other key questions remain unanswered. For example, how do TRP channels sense changes in skin temperature, and exactly how do these temperature changes activate a TRP channel?

TRPM8: The cold receptor

McKemy et al (2002) used whole-cell patch clamping and calcium imaging to record the responses of cultured rat trigeminal ganglion neurons to cold temperatures and various cooling compounds. They found that the cells respond to menthol and cold with an increase in intracellular calcium ion concentration, and that these stimuli activate non-selective cation channels which are highly permeable to calcium. The currents measured were also found to be outwardly rectifying (i.e. much larger at positive than at negative holding potentials). Similar results were obtained from DRG neurons.

They then cloned TRPM8 from a trigeminal ganglion cDNA library and expressed it in Xenopus oocytes. Electrophysiological recordings showed that oocytes expressing TRPM8 channel were sensitive to cold, confirming that the channel is indeed a cold receptor. The cloned channel was found to have a temperature threshold of 8-28 degrees Celcius; it also conferred upon the oocytes sensitivity to menthol and eucalyptol, with the strongest response elicited by the super-cooling compound icilin.

To further investigate the role of TRPM8 in cold thermosensation, Dhaka et al(2007) generated TRPM8 knockout mice by replacing amino acid residues 2-29 of theTRPM8 gene with enhanced green fluorescent protein (EGFP). Calcium imaging showed that only 7.6% of DRG neurons from TRPM8-deficient mice, respond to a cold stimulus of 10 degrees Celcius, compared to 14.9% of cells from wild type (WT) animals.

The temperature sensitivity of the mutants was then assayed, using an apparatus consisting of multiple compartments to produce a surface temperature gradient ranging from 15-53 degrees Celcius. In this assay, both WT and TRPM8-deficient mice largely avoided severe cold (16-20 degrees Celcius) and hot (41-53 degrees Celcius) temperature compartments. However, whereas WT mice spent twice as much time in the compartment with a surface of ~35 degrees Celcius than in other the compartments, the TRPM8-/- animals spent significantly more time in the cooler zones (23-30 degrees Celcius).

A two-temperature choice assay was then performed, in which the mice were placed on a platform consisting of two identical surfaces set at different temperatures. Whereas WT mice strongly preferred warm over cold temperature surfaces, TRPM8-/- animals showed no preference to a platform set at 31 degrees Celcius to one set at 18 degrees Celcius.

The ability of TRPM8-deficient mice to detect noxious cold temperatures was then examined. When TRPM8-deficient mice were placed on a cold plate of temperature -1 degrees Celcius, their behaviour was identical to that of WTs. When placed on the cold plate following an injection of icilin into the hindpaw, the WT mice responded by rapidly by withdrawing the paw from the cold surface; this behaviour was completely abolished in the TRPM-/- mice, as was the vigorous body shaking that is normally induced by intraperitoneal injection of icilin.

Finally, it was demonstrated that TRPM8 mediates cooling-induced analgesia. A 2% formalin injection into the paw produced the same nociceptive response in both mutant and wild-type animals. However, when the animals were placed on a 17 degrees Celcius cold plate following the formalin injection, the mutant mice spent significantly more time licking and lifting the injected paw than the wild-types.

Thus, mice lacking TRPM8 have a severely impaired sensitivity to innocuous cool temperatures and have a partially impaired ability to detect noxious cold temperatures. These findings are corroborated by two independent studies in which TRPM8-/- mice were generated (Bautista et al, 2007; Colburn et al, 2007).

Cold fibres: Neurochemistry and anatomy

Takashima et al (2007) carried out one of the first investigations of the distribution of TRPM8-positive sensory nerve terminals in various peripheral structures, using transgenic mice which express enhanced green fluorescent protein under the control of the TRPM8 transcriptional promoter.

First, they confirmed that the transgene expression was neuron-specific, by showing that cultured DRG and TG neurons from the transgenic animals expressed both GFP and the pan-neuronal marker PGP-9.5. The correspondence of GFP and PGP-9.5 coexpression with TRPM8 immunoreactivity showed that the transgene expression was limited to TRPM8-positive sensory neurons. Furthermore, >80% of cultured GFP+ neurons responded to menthol by an increase in intracellular Ca2+concentration, as determined by calcium microfluorimetry, and outwardly rectifying currents, as measured by whole-cell voltage clamp recordings.

Consistent with earlier studies, it was found that approximately 13% of DRG and TG cells expressed the transgene. The majority of these neurons had cell bodies of around 10µm diameter, and were assumed to be C-fibres, the remainder had cell bodies of up to 30µm diameter, and were assumed to be A delta fibres.

Antibody staining was then used to determine coexpression of the transgene with markers of primary sensory neurons. In DRG, approximately 25% of GFP+ cells were immunoreactive for the C-fibre marker peripherin, and ~14% stained with an antibody for NF200, an intermediate filament protein marker for A-delta fibres. In TG, >30% of GFP+ cells, expressed peripherin, and >25% were immunoreactive for NF200. Significantly, 60% of the DRG cells, and ~40% of TG cells, expressed neither peripherin nor NF200, and are, therefore characterized only by TRPM8 expression.

In the skin, GFP+ nerve terminals were found both superficially, in the stratum granulosum, and deeper, near the epidermis-dermis boundary. This is consistent with the hypothesis that innocuous cool stimuli are detected by nerve terminals located in the superficial skin layers, while noxious cold stimuli are detected terminals that are located deeper.

A complex distribution of TRPM8+ nerve terminals was also observed in other peripheral structures. In the teeth, GFP was found to be expressed in the dentin, which is known to contain A-delta fibres, and in the pulp, which contains C-fibres. In the palate, GFP+ nerve terminals were found in different layers of the epithelium.

Thus, in both DRG and TG, some TRPM8+ sensory neurons characteristics of nociceptors, but others do not. TG has a higher proportion of TRPM8+ cells than do the DRG, and, in the periphery, the terminals of TRPM8+ cells in both ganglia innervate regions of the skin and teeth that are associated with sensing different kinds of cold stimuli.

Coming in from the cold

The study by McKemy et al is of great significance, as it led to the identification and characterization of the first cold receptor. This study also suggests that TRP channels have a general role in thermosensation, as all the previously identified TRP channels are sensitive to heat.

Dhaka et al (2007) show that TRPM8 is required for sensitivity to innocuous cool stimuli and is also involved in sensing noxious cold temperatures. The TRPM8 knockout mice generated in this study have only a partial deficit in sensing noxious cold stimuli, so it is most likely that at least one other cold receptor is involved. Story et al (2003) identified a second cold receptor, TRPA1/ ANKTM1, but its role under physiological conditions is still in question.

There are likely to be other, as yet unidentified, cold sensitive TRP channels. Thermosensation may involve a combinatorial code of thermoTRPs, such that a given repertoire of receptors, each activated by a specified temperature range, confers upon primary sensory afferents sensitivity to cold stimuli of different intensities.

This study also shows that TRPM8 mediates cooling-induced analgesia. TRPM8 is known to be upregulated in subsets of DRG cells following nerve injury leading to hypersensitivity and allodynia (Proudfoot et al, 2006); the channel could therefore be a useful target for novel analgesics.

Takashima et al (2007) show that TRPM8+ primary sensory neurons are neurochemically and anatomically heterogeneous. TRPM8+ sensory neurons express markers of A-delta fibres and C-fibres, and of presumptive nociceptors and non-nociceptors. A significant proportion of TRPM8+ neurons (~60%) express no other markers; thus TRPM8 may be a marker which distinguishes cold fibres from other primary sensory neurons.

This study also shows that different types of TRPM8+ sensory neurons have distinct receptive fields in the periphery, and therefore provides a neuroanatomical basis for the multiple role of TRPM8 in cold thermosensation. It also raises the possibility that TRPM8 is expressed in two labeled lines of cold fibres, one consisting of nociceptors, the other of non-nociceptors.

The three studies discussed here contribute significantly to our understanding of cold thermosensation. Together, they link events at the molecular level with behavioural responses, and provide an anatomical basis for the multiple roles of TRPM8. However, the primary sensory neurons which express TRPM8+ are not yet characterized properly, and other key questions remain unanswered. For example, how do TRP channels sense changes in skin temperature, and exactly how do these temperature changes activate a TRP channel?

References: Bautista, D. M., et al. (2007). The menthol receptor TRMP8 is the principal detector of environmental cold. Nature 448: 204-209.

Colburn, R. W., et al. (2007). Attenuated cold sensitivity in TRPM8 null mice. Neuron 54: 379-386.

Dhaka, A., et al. (2008). Visualizing cold spots: TRPM8-expressing sensory neurons and their projections. J. Neurosci. 28: 566-575.

Dhaka, A., et al. (2007). TRPM8 is required for cold sensation in mice. Neuron 54: 371-378.

Dhaka, A., et al. (2006). TRP ion channels and temperature sensation. Annu. Rev. Neurosci. 29: 135-161.

Lumpkin, E. A. & Catarina, M. J. (2007). Mechanisms of sensory transduction in the skin. Nature 445: 858-865.

McKemy, D. D., et al. (2002). Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature416: 52-58.

Peier, A. M., et al. (2002). A TRP channel that senses cold stimuli and menthol. Cell108: 705-715.

Proudfoot, C. J., et al. (2006). Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr. Biol. 16: 1591-1605.

Story, G. M., et al. (2003). ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112: 819-829.

Takashima, Y., et al. (2007). Diversity in the neuronal circuitry of cold sensing revealed by genetic labelling of Transient Receptor Potential Melastatin 8 neurons. J. Neurosci. 27: 14147-14157.

Voets, T., et al. (2004). The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430: 748-754.

One thought on “Cold thermosensation

Comments are closed.