Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.

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.

Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1-6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2-4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.

Recent work demonstrates that epidermal keratinocytes are critical for normal touch sensation. However, it is unknown if keratinocytes contribute to touch evoked pain and hypersensitivity following tissue injury. Here, we used inhibitory optogenetic and chemogenetic techniques to determine the extent to which keratinocyte activity contributes to the severe neuropathic pain that accompanies chemotherapeutic treatment. We found that keratinocyte inhibition largely alleviates paclitaxel-induced mechanical hypersensitivity. Furthermore, we found that paclitaxel exposure sensitizes mouse and human keratinocytes to mechanical stimulation through the keratinocyte mechanotransducer Piezo1. These findings demonstrate the contribution of non-neuronal cutaneous cells to neuropathic pain and pave the way for the development of new pain-relief strategies that target epidermal keratinocytes and Piezo1.

The GFR alpha2 receptor is the cognate co-receptor for the neurotrophic factor neurturin and GFR alpha2 is selectively expressed by isolectin B(4) (IB(4))-binding nociceptive sensory neurons. Here, we used two physiological approaches in combination with mice that have a targeted deletion of the GFR alpha2 gene (GFR alpha2 -/- mice) in order to determine whether GFR alpha2/neurturin signalling regulates the functional properties or the survival of IB(4)-binding nociceptors. Because 50 % of IB(4)-binding neurons respond to noxious heat and because patch clamp recordings of isolated dorsal root ganglion sensory neurons allow one to neurochemically identify subpopulations of neurons, we analysed the noxious heat responsiveness of IB(4)-positive and -negative small-diameter neurons isolated from adult GFR alpha2 -/- and littermate control mice. The percentage of IB(4)-positive neurons that had large (> 100 pA) heat-evoked inward currents was severely reduced in GFR alpha2 -/- mice (12 %) compared to wild-type littermates (47 %), and this loss in large-magnitude heat currents was accounted for by an increase in neurons with very small (< 100 pA) heat-evoked currents as well as an increase in neurons with no detectable heat current. Counts of IB(4)-positive and -negative neurons, as well as counts of unmyelinated axons in the saphenous nerve, confirmed that the loss in neurons with large-amplitude heat currents was due to a deficit in heat transduction and not a decrease in cell survival. The effect was modality specific for heat because mechanical transduction of all fibre types, including IB(4)-positive C fibres, was normal. Our data are the first to indicate a transduction-function role for GFR alpha2/neurturin signalling in a specific class of sensory neurons.

TRPM8 has been implicated in pain and migraine based on dorsal root- and trigeminal ganglion-enriched expression, upregulation in preclinical models of pain, knockout mouse studies, and human genetics. Here, we evaluated the therapeutic potential in pain of AMG2850 ((R)-8-(4-(trifluoromethyl)phenyl)-N-((S)-1,1,1-trifluoropropan-2-yl)-5,6-dihydro-1,7-naphthyridine-7(8H)-carboxamide), a small molecule antagonist of TRPM8 by in vitro and in vivo characterization. AMG2850 is potent in vitro at rat TRPM8 (IC90 against icilin activation of 204 ± 28 nM), highly selective (>100-fold IC90 over TRPV1 and TRPA1 channels), and orally bioavailable (F po > 40 %). When tested in a skin-nerve preparation, AMG2850 blocked menthol-induced action potentials but not mechanical activation in C fibers. AMG2850 exhibited significant target coverage in vivo in a TRPM8-mediated icilin-induced wet-dog shake (WDS) model in rats (at 10 mg/kg p.o.). However, AMG2850 did not produce a significant therapeutic effect in rat models of inflammatory mechanical hypersensitivity or neuropathic tactile allodynia at doses up to 100 mg/kg. The lack of efficacy suggests that either TRPM8 does not play a role in mediating pain in these models or that a higher level of target coverage is required. The potential of TRPM8 antagonists as migraine therapeutics is yet to be determined.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

TRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal.

Therapeutic use of general sodium channel blockers, such as lidocaine, can substantially reduce the enhanced activity in sensory neurons that accompanies chronic pain after nerve or tissue injury. However, because these general blockers have significant side effects, there is great interest in developing inhibitors that specifically target subtypes of sodium channels. Moreover, some idiopathic small-fiber neuropathies are driven by gain-of-function mutations in specific sodium channel subtypes. In the current study, we focus on one subtype, the voltage-gated sodium channel 1.8 (Nav1.8). Nav1.8 is preferentially expressed in nociceptors, and gain-of-function mutations in Nav1.8 result in painful mechanical hypersensitivity in humans. Here, we used the recently developed gain-of-function Nav1.8 transgenic mouse strain, Possum, to investigate Nav1.8-mediated peripheral afferent hyperexcitability. This gain-of-function mutation resulted in markedly increased mechanically evoked action potential firing in subclasses of Aβ, Aδ, and C fibers. Moreover, mechanical stimuli initiated bursts of action potential firing in specific subpopulations that continued for minutes after removal of the force and were not susceptible to conduction failure. Surprisingly, despite the intense afferent firing, the behavioral effects of the Nav1.8 mutation were quite modest, as only frankly noxious stimuli elicited enhanced pain behavior. These data demonstrate that a Nav1.8 gain-of-function point mutation contributes to intense hyperexcitability along the afferent axon within distinct sensory neuron subtypes.

Many patients with sickle cell disease (SCD) suffer from chronic pain, which is often described as neuropathic in nature. Although vascular and inflammatory pathology undoubtedly contribute to the SCD pain experience, the nociceptive signals that ultimately drive symptoms are detected and transmitted by peripheral sensory neurons. To date, no systematic histological examination of peripheral nerves has been completed in patients or mouse models of SCD to diagnose disease-related neuropathy.

Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund's adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.

Fabry disease (FD) causes life-long pain, the mechanisms of which are unclear. Patients with FD have chronic pain that mirrors symptoms of other painful peripheral neuropathies. However, it is unclear what underlying damage occurs in FD peripheral nerves that may contribute to chronic pain. Here, we characterized myelinated and unmyelinated fiber pathology in peripheral nerves of a rat model of FD. Decreased nerve fiber density and increased nerve fiber pathology were noted in unmyelinated and myelinated fibers from FD rats; both observations were dependent on sampled nerve fiber modality and anatomical location. FD myelinated axons exhibited lipid accumulations that were determined to be the FD-associated lipid globotriaosylceramide (Gb3), and to a lesser extent lysosomes. These findings suggest that axonal Gb3 accumulation may drive peripheral neuron dysfunction and subsequent pain in FD.

The first point of our body's contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions.

Chronic pain is a disabling disease with limited treatment options. While animal models have revealed important aspects of pain neurobiology, therapeutic translation of this knowledge requires our understanding of these cells and networks of pain in humans. We propose a multi-institutional collaboration to rigorously and ethically address this challenge.

The spared nerve injury (SNI) model of neuropathic pain produces robust and reproducible behavioral mechanical hypersensitivity. Although this rodent model of neuropathic pain has been well established and widely used, peripheral mechanisms underlying this phenotype remain incompletely understood. Here we investigated the role of cutaneous sensory fibers in the maintenance of mechanical hyperalgesia in mice post-SNI.

Epidermal keratinocytes mediate touch sensation by detecting and encoding tactile information to sensory neurons. However, the specific mechanotransducers that enable keratinocytes to respond to mechanical stimulation are unknown. Here, we found that the mechanically-gated ion channel PIEZO1 is a key keratinocyte mechanotransducer. Keratinocyte expression of PIEZO1 is critical for normal sensory afferent firing and behavioral responses to mechanical stimuli in mice.

Keratinocytes are the most abundant cell type in the epidermis, the most superficial layer of skin. Historically, epidermal-innervating sensory neurons were thought to be the exclusive detectors and transmitters of environmental stimuli. However, recent work from our lab (Moehring et al., 2018) and others (Baumbauer et al., 2015) has demonstrated that keratinocytes are also critical for normal mechanotransduction and mechanically-evoked behavioral responses in mice. Here, we asked whether keratinocyte activity is also required for normal cold and heat sensation. Using calcium imaging, we determined that keratinocyte cold activity is conserved across mammalian species and requires the release of intracellular calcium through one or more unknown cold-sensitive proteins. Both epidermal cell optogenetic inhibition and interruption of ATP-P2X4 signaling reduced reflexive behavioral responses to cold and heat stimuli. Based on these data and our previous findings, keratinocyte purinergic signaling is a modality-conserved amplification system that is required for normal somatosensation in vivo.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

Severe neuropathic pain is a hallmark of Fabry disease, a genetic disorder caused by a deficiency in lysosomal α-galactosidase A. Pain experienced by these patients significantly impacts their quality of life and ability to perform everyday tasks. Patients with Fabry disease suffer from peripheral neuropathy, sensory abnormalities, acute pain crises, and lifelong ongoing pain. Although treatment of pain through medication and enzyme replacement therapy exists, pain persists in many of these patients. Some has been learned in the past decades regarding clinical manifestations of pain in Fabry disease and the pathological effects of α-galactosidase A insufficiency in neurons. Still, it is unclear how pain and sensory abnormalities arise in patients with Fabry disease and how these can be targeted with therapeutics. Our knowledge is limited in part due to the lack of adequate preclinical models to study the disease. This review will detail the types of pain, sensory abnormalities, influence of demographics on pain, and current strategies to treat pain experienced by patients with Fabry disease. In addition, we discuss the current knowledge of Fabry pain pathogenesis and which aspects of the disease preclinical models accurately recapitulate. Understanding the commonalities and divergences between humans and preclinical models can be used to further interrogate mechanisms causing the pain and sensory abnormalities as well as advance development of the next generation of therapeutics to treat pain in patients with Fabry disease.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund's adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.