Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

Immunoglobulin somatic hypermutation in a defined biochemical system recapitulates affinity maturation and permits antibody optimization.

We describe a purified biochemical system to produce monoclonal antibodies (Abs) in vitro using activation-induced deoxycytidine deaminase (AID) and DNA polymerase η (Polη) to diversify immunoglobulin variable gene (IgV) libraries within a phage display format. AID and Polη function during B-cell affinity maturation by catalyzing somatic hypermutation (SHM) of immunoglobulin variable genes (IgV) to generate high-affinity Abs. The IgV mutational motif specificities observed in vivo are conserved in vitro. IgV mutations occurred in antibody complementary determining regions (CDRs) and less frequently in framework (FW) regions. A unique feature of our system is the use of AID and Polη to perform repetitive affinity maturation on libraries reconstructed from a preceding selection step. We have obtained scFv Abs against human glucagon-like peptide-1 receptor (GLP-1R), a target in the treatment of type 2 diabetes, and VHH nanobodies targeting Fatty Acid Amide Hydrolase (FAAH), involved in chronic pain, and artemin, a neurotropic factor that regulates cold pain. A round of in vitro affinity maturation typically resulted in a 2- to 4-fold enhancement in Ab-Ag binding, demonstrating the utility of the system. We tested one of the affinity matured nanobodies and found that it reduced injury-induced cold pain in a mouse model.

Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFRα3.

Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin-GFRα3 signaling can be targeted to selectively treat cold pain.

TRPM8 activation attenuates inflammatory responses in mouse models of colitis.

Transient Receptor Potential Melastatin-8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8(GFP) mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid-induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene-related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene-related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

Artemin, a glial cell line-derived neurotrophic factor family member, induces TRPM8-dependent cold pain.

Chronic pain associated with injury or disease can result from dysfunction of sensory afferents whereby the threshold for activation of pain-sensing neurons (nociceptors) is lowered. Neurotrophic factors control nociceptor development and survival, but also induce sensitization through activation of their cognate receptors, attributable, in part, to the modulation of ion channel function. Thermal pain is mediated by channels of the transient receptor potential (TRP) family, including the cold and menthol receptor TRPM8. Although it has been shown that TRPM8 is involved in cold hypersensitivity, the molecular mechanisms underlying this pain modality are unknown. Using microarray analyses to identify mouse genes enriched in TRPM8 neurons, we found that the glial cell line-derived neurotrophic factor (GDNF) family receptor GFRα3 is expressed in a subpopulation of TRPM8 sensory neurons that have the neurochemical profile of cold nociceptors. Moreover, we found that artemin, the specific GFRα3 ligand that evokes heat hyperalgesia, robustly sensitized cold responses in a TRPM8-dependent manner in mice. In contrast, GFRα1 and GFRα2 are not coexpressed with TRPM8 and their respective ligands GDNF and neurturin did not induce cold pain, whereas they did evoke heat hyperalgesia. Nerve growth factor induced mild cold sensitization, consistent with TrkA expression in TRPM8 neurons. However, bradykinin failed to alter cold sensitivity even though its receptor expresses in a subset of TRPM8 neurons. These results show for the first time that only select neurotrophic factors induce cold sensitization through TRPM8 in vivo, unlike the broad range of proalgesic agents capable of promoting heat hyperalgesia.

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

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.

Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models.

A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

The neurotrophic factor artemin and its receptor GFRα3 mediate migraine-like pain via the ion channel TRPM8.

Background: Migraine has a strong genetic foundation, including both monogenic and polygenic types. The former are rare, with most migraine considered polygenic, supported by genome-wide association studies (GWAS) identifying numerous genetic variants associated with migraine risk. Surprisingly, some of the most common mutations are associated with TRPM8, a non-selective cation channel that is the primary sensor of cold temperatures in primary afferent neurons of the somatosensory system. However, it is unlikely that the temperature sensitivity of TRPM8 underlies its role in migraine pathogenesis. To define the basis of the channel's involvement, we reasoned that cellular processes that increase cold sensitivity in the skin, such as the neurotrophic factor artemin, via its receptor GFRα3, also mediate TRPM8-associated migraine-like pain in the meninges. Methods: To investigate the role of artemin and GFRα3 in preclinical rodent migraine models, we infused nitroglycerin acutely and chronically, and measured changes in periorbital and hind paw mechanical sensitivity in male and female mice lacking GFRα3, after neutralization of free artemin with specific monoclonal antibodies, or by systemic treatment with a TRPM8-specific antagonist. Further, in wildtypes and mice lacking either GFRα3 or TRPM8, we tested the effects of supradural infusions of a mix of inflammatory mediators, artemin, and a TRPM8-specific agonist on migraine-related pain in mice. Results: We find that mechanical allodynia induced by systemic nitroglycerin, or supradural infusion of inflammatory mediators, involves GFRα3. In addition, neutralization of circulating artemin reduces the nitroglycerin phenotype, demonstrating the importance of this neurotrophic pathway. Further, we show TRPM8 expression in the meninges and that direct supradural infusion of either a TRPM8-specific agonist or artemin itself produces mechanical allodynia, the latter dependent on TRPM8 and ameliorated by concurrent treatment with sumatriptan. Conclusions: These results indicate that neuroinflammatory events in the meninges can produce migraine-like pain in mice via artemin and GFRα3, likely acting upstream of TRPM8, providing a novel pathway that may contribute to migraine pathogenesis.

Enhanced insulin clearance in mice lacking TRPM8 channels.

Blood glucose concentration is tightly regulated by the rate of insulin secretion and clearance, a process partially controlled by sensory neurons serving as metabolic sensors in relevant tissues. The activity of these neurons is regulated by the products of metabolism which regulate transmitter release, and recent evidence suggests that neuronally expressed ion channels of the transient receptor potential (TRP) family function in this critical process. Here, we report the novel finding that the cold and menthol-gated channel TRPM8 is necessary for proper insulin homeostasis. Mice lacking TRPM8 respond normally to a glucose challenge while exhibiting prolonged hypoglycemia in response to insulin. Additionally, Trpm8-/- mice have increased rates of insulin clearance compared with wild-type animals and increased expression of insulin-degrading enzyme in the liver. TRPM8 channels are not expressed in the liver, but TRPM8-expressing sensory afferents innervate the hepatic portal vein, suggesting a TRPM8-mediated neuronal control of liver insulin clearance. These results demonstrate that TRPM8 is a novel regulator of serum insulin and support the role of sensory innervation in metabolic homeostasis.

Endogenous inflammatory mediators produced by injury activate TRPV1 and TRPA1 nociceptors to induce sexually dimorphic cold pain that is dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as CGRP and substance P, inducing neurogenic inflammation which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked if inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 and TRPA1 lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid (LPA) or 4-hydroxy-2-nonenal (4HNE), finding each induces cold pain that is dependent on the cold-gated channel TRPM8. Inhibition of either CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Lastly, we find that cold pain induced by inflammatory mediators and neuropeptides requires the neurotrophin artemin and its receptor GFRα3. These results demonstrate that tissue damage alters cold sensitivity via neurogenic inflammation, likely leading to localized artemin release that induces cold pain via GFRα3 and TRPM8. Significance Statement: The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 and the neurotrophin receptor GFRα3 that leads to cold pain, providing select targets for potential therapies for this pain modality.

Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice.

ABSTRACT: Migraine is a complex neurovascular disorder that is one of the leading causes of disability and a reduced quality of life. Even with such a high societal impact, our understanding of the cellular and molecular mechanisms that contribute to migraine headaches is limited. To address this complex disorder, several groups have performed genome-wide association studies to elucidate migraine susceptibility genes, with many identifying transient receptor potential melastatin 8 (TRPM8), a cold-sensitive cation channel expressed in peripheral afferents innervating the trigeminovascular system, and the principal mediator of cold and cold pain associated with injury and disease. Interestingly, these migraine-associated single-nucleotide polymorphisms reside in noncoding regions of TRPM8, with those correlated with reduced migraine risk exhibiting lower TRPM8 expression and decreased cold sensitivity. Nonetheless, as a role for TRPM8 in migraine has yet to be defined, we sought to address this gap in our knowledge using mouse genetics and TRPM8 antagonism to determine whether TRPM8 channels or neurons are required for migraine-like pain (mechanical allodynia and facial grimace) in inducible migraine models. Our results show that both evoked and spontaneous pain behaviors are dependent on both TRPM8 channels and neurons, as well as required in both acute and chronic migraine models. Moreover, inhibition of TRPM8 channels prevented acute but not established chronic migraine-like pain. These results are consistent with its association with migraine in genetic analyses and establish that TRPM8 channels are a component of the underlying mechanisms of migraine.

Scraping through the ice: uncovering the role of TRPM8 in cold transduction.

The proper detection of environmental temperatures is essential for the optimal growth and survival of organisms of all shapes and phyla, yet only recently have the molecular mechanisms for temperature sensing been elucidated. The discovery of temperature-sensitive ion channels of the transient receptor potential (TRP) superfamily has been pivotal in explaining how temperatures are sensed in vivo, and here we will focus on the lone member of this cohort, TRPM8, which has been unequivocally shown to be cold sensitive. TRPM8 is expressed in somatosensory neurons that innervate peripheral tissues such as the skin and oral cavity, and recent genetic evidence has shown it to be the principal transducer of cool and cold stimuli. It is remarkable that this one channel, unlike other thermosensitive TRP channels, is associated with both innocuous and noxious temperature transduction, as well as cold hypersensitivity during injury and, paradoxically, cold-mediated analgesia. With ongoing research, the field is getting closer to answering a number of fundamental questions regarding this channel, including the cellular mechanisms of TRPM8 modulation, the molecular context of TRPM8 expression, as well as the full extent of the role of TRPM8 in cold signaling in vivo. These findings will further our understanding of basic thermotransduction and sensory coding, and may have important implications for treatments for acute and chronic pain.

Nociceptive afferent phenotyping reveals that transient receptor potential ankyrin 1 promotes cold pain through neurogenic inflammation upstream of the neurotrophic factor receptor GFRα3 and the menthol receptor transient receptor potential melastatin 8.

ABSTRACT: The proper detection and behavioral response to painfully cold temperatures is critical for avoiding potentially harmful tissue damage. Cold allodynia and hyperalgesia, pain associated with innocuous cooling and exaggerated pain with noxious cold, respectively, are common in patients with chronic pain. In peripheral somatosensory afferents, the ion channels transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) are candidate receptors for innocuous and noxious cold temperatures, respectively. However, the role of TRPA1 as a cold sensor has remained controversial, and recent evidence suggests that TRPM8 channels and afferents mediate the detection of both pleasant and painful cold. To determine the role of TRPA1 afferents in cold-induced mouse behaviors in vivo, we used functional phenotyping by targeted nerve conduction block with the cell-impermeant lidocaine derivative QX-314. Surprisingly, we find that injection of QX-314 with TRPA1 agonists reduces cold-induced behaviors in mice, but does so in a TRPM8-dependent manner. Moreover, this effect is sexually dimorphic and requires the glial cell line-derived neurotrophic factor receptor GFRα3, as does cold hypersensitivity produced by the activation of TRPA1 channels. Taken together, these results suggest that under conditions of neurogenic inflammation, TRPA1 works upstream of GFRα3 and TRPM8 to produce cold hypersensitivity, providing novel insights into the role of TRPA1 channels in cold pain.

Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1.

Wasabi, horseradish and mustard owe their pungency to isothiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust hypersensitivity to thermal and mechanical stimuli. Despite their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Here we show that mustard oil depolarizes a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the TRP ion channel family recently implicated in the detection of noxious cold. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors.

Roles of transient receptor potential channels in pain.

Pain perception begins with the activation of primary sensory nociceptors. Over the past decade, flourishing research has revealed that members of the Transient Receptor Potential (TRP) ion channel family are fundamental molecules that detect noxious stimuli and transduce a diverse range of physical and chemical energy into action potentials in somatosensory nociceptors. Here we highlight the roles of TRP vanilloid 1 (TRPV1), TRP melastatin 8 (TRPM8) and TRP ankyrin 1 (TRPA1) in the activation of nociceptors by heat and cold environmental stimuli, mechanical force, and by chemicals including exogenous plant and environmental compounds as well as endogenous inflammatory molecules. The contribution of these channels to pain and somatosensation is discussed at levels ranging from whole animal behavior to molecular modulation by intracellular signaling proteins. An emerging theme is that TRP channels are not simple ion channel transducers of one or two stimuli, but instead serve multidimensional roles in signaling sensory stimuli that are exceptionally diverse in modality and in their environmental milieu.

Design and construction of a two-temperature preference behavioral assay for undergraduate neuroscience laboratories.

Behavioral assays in the undergraduate neuroscience laboratory are useful for illustrating a variety of physiological concepts. An example is homeostatic temperature regulation (thermoregulation). Many model organisms, from flies to mice, regulate internal temperatures in part by moving to suitable climates (thermotaxis). A particularly reliable method of quantifying temperature-dependent thermotactic behaviors is the two-temperature preference behavioral assay. In this preparation, an organism is free to move between two temperature-controlled surfaces, thus revealing its preferred thermal environment. Here we present the design and construction of a two-temperature preference assay chamber. The device uses Peltier-based thermoelectric modules (TECs) for heating and cooling, and is capable of precision control of temperatures from -5ºC to 60ºC. Our approach can be easily adapted for use in a variety of physiological and behavioral assays that require precise temperature control over a wide range of temperatures.

Action potential initiation in the peripheral terminals of cold-sensitive neurones innervating the guinea-pig cornea.

The site at which action potentials initiate within the terminal region of unmyelinated sensory axons has not been resolved. Combining recordings of nerve terminal impulses (NTIs) and collision analysis, the site of action potential initiation in guinea-pig corneal cold receptors was determined. For most receptors (77%), initiation mapped to a point in the time domain that was closer to the nerve terminal than to the site of electrical stimulation at the back of the eye. Guinea-pig corneal cold receptors are Adelta-neurones that lose their myelin sheath at the point where they enter the cornea, and therefore their axons conduct more slowly within the cornea. Allowing for this inhomogeneity in conduction speed, the resulting spatial estimates of action potential initiation sites correlated with changes in NTI shape predicted by simulation of action potentials initiating within a nerve terminal. In some receptors, more than one NTI shape was observed. Simulations of NTI shape suggest that the origin of differing NTI shapes result from action potentials initiating at different, spatially discrete, locations within the nerve terminal. Importantly, the relative incidence of NTI shapes resulting from action potential initiation close to the nerve termination increased during warming when nerve activity decreased, indicating that the favoured site of action potential initiation shifts toward the nerve terminal when it hyperpolarizes. This finding can be explained by a hyperpolarization-induced relief of Na(+) channel inactivation in the nerve terminal. The results provide direct evidence that the molecular entities responsible for stimulus transduction and action potential initiation reside in parallel with one another in the unmyelinated nerve terminals of cold receptors.

Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons.

Sensory nerves detect an extensive array of somatosensory stimuli, including environmental temperatures. Despite activating only a small cohort of sensory neurons, cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Psychophysical and functional data show that cold responses are mediated by both C- and A delta-fibers with separate peripheral receptive zones, each of which likely provides one or more of these distinct cold sensations. With this diversity in the neural basis for cold, it is remarkable that the majority of cold responses in vivo are dependent on the cold and menthol receptor transient receptor potential melastatin 8 (TRPM8). TRPM8-null mice are deficient in temperature discrimination, detection of noxious cold temperatures, injury-evoked hypersensitivity to cold, and nocifensive responses to cooling compounds. To determine how TRPM8 plays such a critical yet diverse role in cold signaling, we generated mice expressing a genetically encoded axonal tracer in TRPM8 neurons. Based on tracer expression, we show that TRPM8 neurons bear the neurochemical hallmarks of both C- and A delta-fibers, and presumptive nociceptors and non-nociceptors. More strikingly, TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and first- and second-cold pain. These results further demonstrate that the peripheral neural circuitry of cold sensing is cellularly and anatomically complex, yet suggests that cold fibers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations.

Molecular basis of peripheral innocuous cold sensitivity.

Of somatosensory modalities cold is one of the more ambiguous percepts, evoking the pleasant sensation of cooling, the stinging bite of cold pain, and welcome relief from chronic pain. Moreover, unlike the precipitous thermal thresholds for heat activation of thermosensitive afferent neurons, thresholds for cold fibers are across a range of cool to cold temperatures that spans over 30°C. Until recently, how cold produces this myriad of biologic effects was unknown. However, recent advances in our understanding of cold mechanisms at the behavioral, physiologic, and cellular level have begun to provide insights into this sensory modality. The identification of a number of ion channels that either serve as the principal detectors of a cold stimulus in the peripheral nervous system, or are part of a differential expression pattern of channels that maintain cell excitability in the cold, endows select neurons with properties that are amenable to electric signaling in the cold. This chapter highlights the current understanding of the molecules involved in cold transduction in the mammalian peripheral nervous system, as well as presenting a hypothetic model to account for the broad range of cold thermal thresholds and distinct functions of cold fibers in perception, pain, and analgesia.