Cheryl L. Stucky

The menthol receptor TRPM8 is the principal detector of environmental cold

Sensory nerve fibres can detect changes in temperature over a remarkably wide range, a process that has been proposed to involve direct activation of thermosensitive excitatory transient receptor potential (TRP) ion channels. One such channel—TRP melastatin 8 (TRPM8) or cold and menthol receptor 1 (CMR1)—is activated by chemical cooling agents (such as menthol) or when ambient temperatures drop below ∼26 °C, suggesting that it mediates the detection of cold thermal stimuli by primary afferent sensory neurons. However, some studies have questioned the contribution of TRPM8 to cold detection or proposed that other excitatory or inhibitory channels are more critical to this sensory modality in vivo. Here we show that cultured sensory neurons and intact sensory nerve fibres from TRPM8-deficient mice exhibit profoundly diminished responses to cold. These animals also show clear behavioural deficits in their ability to discriminate between cold and warm surfaces, or to respond to evaporative cooling. At the same time, TRPM8 mutant mice are not completely insensitive to cold as they avoid contact with surfaces below 10 °C, albeit with reduced efficiency. Thus, our findings demonstrate an essential and predominant role for TRPM8 in thermosensation over a wide range of cold temperatures, validating the hypothesis that TRP channels are the principal sensors of thermal stimuli in the peripheral nervous system.

The mammalian sodium channel BNC1 is required for normal touch sensation

Of the vertebrate senses, touch is the least understood at the molecular level. The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.

Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1.

&NA; Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle‐induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long‐lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non‐selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.

The 5-HT3 Subtype of Serotonin Receptor Contributes to Nociceptive Processing via a Novel Subset of Myelinated and Unmyelinated Nociceptors

Serotonin is a major component of the inflammatory chemical milieu and contributes to the pain of tissue injury via an action on multiple receptor subtypes. Here we studied mice after genetic or pharmacological disruption of the 5-HT3 receptor, an excitatory serotonin-gated ion channel. We demonstrate that tissue injury-induced persistent, but not acute, nociception is significantly reduced after functional elimination of this receptor subtype. Specifically, in the setting of tissue injury, the 5-HT3receptor mediates activation of nociceptors but does not contribute to injury-associated edema. This result is explained by the localization of 5-HT3 receptor transcripts to a previously uncharacterized subset of myelinated and unmyelinated afferents, few of which express the proinflammatory neuropeptide substance P. Finally, we provide evidence that central serotonergic circuits modulate nociceptive transmission via a facilitatory action at spinal 5-HT3 receptors. We conclude that activation of both peripheral and central 5-HT3 receptors is pronociceptive and that the contribution of peripheral 5-HT3 receptors involves a novel complement of primary afferent nociceptors.

Piezo2 is required for Merkel-cell mechanotransduction

How we sense touch remains fundamentally unknown. The Merkel cell–neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aβ sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell–neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.

TRPA1 Modulates Mechanotransduction in Cutaneous Sensory Neurons

Transient receptor potential ankyrin 1 (TRPA1) is expressed by nociceptive neurons of the dorsal root ganglia (DRGs) and trigeminal ganglia, but its roles in cold and mechanotransduction are controversial. To determine the contribution of TRPA1 to cold and mechanotransduction in cutaneous primary afferent terminals, we used the ex vivo skin–nerve preparation from Trpa1+/+, Trpa1+/−, and Trpa1−/− adult mouse littermates. Cutaneous fibers from TRPA1-deficient mice showed no deficits in acute cold sensitivity, but they displayed striking deficits in mechanical response properties. C-fiber nociceptors from Trpa1−/− mice exhibited action potential firing rates 50% lower than those in wild-type C-fibers across a wide range of force intensities. Aδ-fiber mechanonociceptors also had reduced firing, but only at high intensity forces (>100 mN). Surprisingly, the firing rates of low-threshold Aβ and D-hair mechanoreceptive fibers were also altered. TRPA1 protein and mRNA expression was assessed in DRG neurons and cutaneous innervation by using Trpa1 in situ hybridization, an antibody for TRPA1, and an antibody for placental alkaline phosphatase (PLAP) in mice in which PLAP was substituted for Trpa1. DRG neurons of all sizes expressed Trpa1 mRNA or PLAP immunoreactivity. TRPA1 or PLAP immunolabeling was detected not only on many thin-caliber axons and intraepidermal endings but also on many large-caliber axons as well as lanceolate and Meissner endings. Epidermal and hair follicle keratinocytes also express TRPA1 message and protein. We propose that TRPA1 modulates mechanotransduction via a cell-autonomous mechanism in nociceptor terminals and possibly through a modulatory role in keratinocytes, which may interact with sensory terminals to modify their mechanical firing properties.

POINT MUTATION IN TRKB CAUSES LOSS OF NT4-DEPENDENT NEURONS WITHOUT MAJOR EFFECTS ON DIVERSE BDNF RESPONSES

Neurotrophins are a family of soluble ligands that promote the survival and differentiation of peripheral and central neurons and regulate synaptic function. The two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), bind and activate a single high-affinity receptor, TrkB. Experiments in cell culture have revealed that an intact Shc adaptor binding site on TrkB and subsequent activation of the Ras/MAPK pathway are important for neuronal survival and neurite outgrowth. To elucidate the intracellular signaling pathways that mediate the diverse effects of BDNF and NT4 in vivo, we have mutated in the mouse germline the Shc binding site in the trkB gene. This trkB(shc) mutation revealed distinctive responses to BDNF and NT4. While nearly all NT4-dependent sensory neurons were lost in trkB(shc/shc) mutant mice, BDNF-dependent neurons were only modestly affected. Activation of MAP kinases and in vitro survival of cultured trkB(shc/shc) neurons were reduced in response to both neurotrophins, with NT4 being less potent than BDNF, suggesting differential activation of TrkB by the two ligands. Moreover, while the Ras/MAPK pathway is required for in vitro differentiation of neuronal cells, trkB(shc/shc) mutant mice do not show any defects in BDNF-dependent differentiation of CNS neurons or in the function of sensory neurons that mediate innocuous touch.

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.

Species and strain differences in rodent sciatic nerve anatomy: Implications for studies of neuropathic pain

&NA; Hindlimb pain models developed in rats have been transposed to mice, but assumed sciatic nerve neuroanatomic similarities have not been examined. We compared sciatic nerve structural organization in mouse strains (C57BL/6J, DBA/2J, and B6129PF2/J) and rat strains (Wistar, Brown Norway, and Sprague–Dawley). Dissection and retrograde labeling showed mouse sciatic nerve origins predominantly from the third lumbar (L3) and L4 spinal nerves, unlike the L4 and L5 in rats. Proportionate contributions by each level differed significantly between strains in both mice and rats. Whereas all rats had six lumbar vertebrae, variable patterns in mice included mostly five vertebrae in DBA/2J, mostly six vertebrae in C57BL/6J, and a mix in B6129PF2/J. Mice with a short lumbar vertebral column showed a rostral shift in relative contributions to the sciatic nerve by L3 and L4. Ligation of the mouse L4 nerve created hyperalgesia similar to that in rats after L5 ligation, and motor changes were similar after mouse L4 and rat L5 ligation (foot cupping) and after mouse L3 and rat L4 ligation (flexion weakness). Thus, mouse L3 and L4 neural segments are anatomically and functionally homologous with rat L4 and L5 segments. Neuronal changes after distal injury or inflammation should be sought in the mouse L3 and L4 ganglia, and the spinal nerve ligation model in mice should involve ligation of the L4 nerve while L3 remains intact. Strain‐dependent variability in segmental contributions to the sciatic nerve may account in part for genetic differences in pain behavior after spinal nerve ligation.

Peripheral inflammation selectively increases TRPV1 function in IB4-positive sensory neurons from adult mouse

C‐fiber nociceptors can be divided into two groups based on growth factor dependency and isolectin B4 (IB4) binding. IB4‐negative nociceptors have been proposed to contribute to inflammatory pain. Since the TRPV1 receptor is critical for inflammatory heat hyperalgesia, we hypothesized that inflammation would sensitize IB4 negative but not IB4‐positive small‐diameter neurons to TRPV1 stimuli. Two days after complete Freunds adjuvant (CFA)‐induced inflammation in the hind paw of mice, lumbar 4/5 ganglia were dissociated and small‐diameter (≤26 μm) neurons were quantified for responsiveness to the TRPV1 agonists, capsaicin and protons using patch clamp recordings. Surprisingly, inflammation did not alter the responsiveness of IB4‐negative neurons to capsaicin or protons. Conversely, inflammation increased the percentage of IB4‐positive neurons that responded to 1 μM capsaicin from 24 to 80% and increased the percentage that responded to pH 5.0 from 54 to 85%. In parallel, inflammation increased the percentage of IB4‐positive neurons that was TRPV1‐immunoreactive. The inflammation‐induced increase in capsaicin‐ and proton‐responsiveness was entirely mediated by TRPV1 because IB4‐positive neurons from inflamed TRPV1−/− mice were capsaicin‐insensitive and unaltered in proton‐responsiveness. Interestingly, comparison of neurons from TRPV1+/+ and TRPV1−/− mice revealed that the sustained proton‐evoked currents in IB4‐positive neurons were independent of TRPV1 whereas the sustained‐only proton currents in IB4‐negative neurons were TRPV1‐dependent. Together, these data indicate that TRPV1 function and expression are selectively increased in IB4‐positive neurons during inflammation in mouse and suggest a novel role for IB4‐positive C‐fibers during inflammation.