Inflammation, the body's primary defensive response system to injury and infection, is triggered by molecular signatures of microbes and tissue injury. These molecules also stimulate specialized sensory neurons, termed nociceptors. Activation of nociceptors mediates inflammation through antidromic release of neuropeptides into infected or injured tissue, producing neurogenic inflammation. Because HMGB1 is an important inflammatory mediator that is synthesized by neurons, we reasoned nociceptor release of HMGB1 might be a component of the neuroinflammatory response. In support of this possibility, we show here that transgenic nociceptors expressing channelrhodopsin-2 (ChR2) directly release HMGB1 in response to light stimulation. Additionally, HMGB1 expression in neurons was silenced by crossing synapsin-Cre (Syn-Cre) mice with floxed HMGB1 mice (HMGB1f/f). When these mice undergo sciatic nerve injury to activate neurogenic inflammation, they are protected from the development of cutaneous inflammation and allodynia as compared to wild-type controls. Syn-Cre/HMGB1fl/fl mice subjected to experimental collagen antibody-induced arthritis, a disease model in which nociceptor-dependent inflammation plays a significant pathological role, are protected from the development of allodynia and joint inflammation. Thus, nociceptor HMGB1 is required to mediate pain and inflammation during sciatic nerve injury and collagen antibody-induced arthritis.

OnabotulinumtoxinA (onabotA) is approved globally for prevention of chronic migraine; however, the classical mechanism of action of onabotA in motor and autonomic neurons cannot fully explain the effectiveness of onabotulinumtoxinA in this sensory neurological disease. We sought to explore the direct effects of onabotulinumtoxinA on mouse trigeminal ganglion sensory neurons using an inflammatory soup-based model of sensitization.

Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.

Recently, we reported the isolation of the Kv3.4 current in dorsal root ganglion (DRG) neurons and described dysregulation of this current in a spinal cord injury (SCI) model of chronic pain. These studies strongly suggest that rat Kv3.4 channels are major regulators of excitability in DRG neurons from pups and adult females, where they help determine action potential (AP) repolarization and spiking properties. Here, we characterized the Kv3.4 current in rat DRG neurons from adult males and show that it transfers 40-70% of the total repolarizing charge during the AP across all ages and sexes. Following SCI, we also found remodeling of the repolarizing currents during the AP. In the light of these studies, homomeric Kv3.4 channels expressed in DRG nociceptors are emerging novel targets that may help develop new approaches to treat neuropathic pain.

The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.

Osteoimmune diseases, such as apical periodontitis, are prevalent, often painful, inflammatory conditions resulting in bone loss and reduced quality of life. There is growing evidence that the nociceptive fibers densely innervating affected tissues regulate disease progression; therefore, we hypothesized that nociceptors regulate the transcriptomic profile of the periapical osteolytic lesion in a mouse model of apical periodontitis. Male control and nociceptor-ablated mice underwent pulp exposures, and after 0, 7, or 14 days, total RNA from periapical tissues was submitted for sequencing and bioinformatic analysis. Pulp exposure triggers the differential expression of hundreds of genes over the course of infection. At 14 days post pulp exposure, 422 genes, including Tnf, Il1a, and Il1b, were differentially expressed between nociceptor-ablated and control mice with greater enrichment of biological processes related to inflammation in nociceptor-ablated mice. Nociceptor ablation regulates the transcriptomic profile of periapical lesions in a mouse model of apical periodontitis, shifting the gene expression profile to a greater enrichment of inflammatory genes, suggesting nociceptors play a role in the kinetics of the immune response. This newly uncovered neuro-immune axis and its mechanisms in apical periodontitis can be an important therapeutic target for the treatment of this prevalent disease.

Mechanical nociception is an evolutionarily conserved sensory process required for the survival of living organisms. Previous studies have revealed much about the neural circuits and sensory molecules in mechanical nociception, but the cellular mechanisms adopted by nociceptors in force detection remain elusive. To address this issue, we study the mechanosensation of a fly larval nociceptor (class IV da neurons, c4da) using a customized mechanical device. We find that c4da are sensitive to mN-scale forces and make uniform responses to the forces applied at different dendritic regions. Moreover, c4da showed a greater sensitivity to localized forces, consistent with them being able to detect the poking of sharp objects, such as wasp ovipositor. Further analysis reveals that high morphological complexity, mechanosensitivity to lateral tension and possibly also active signal propagation in dendrites contribute to the sensory features of c4da. In particular, we discover that Piezo and Ppk1/Ppk26, two key mechanosensory molecules, make differential but additive contributions to the mechanosensitivity of c4da. In all, our results provide updates into understanding how c4da process mechanical signals at the cellular level and reveal the contributions of key molecules.

Transient receptor potential vanilloid I (TRPV1) sensitization in peripheral nociceptors is a prominent phenomenon that occurs in inflammatory pain conditions. Pro-algesic agents can potentiate TRPV1 activity in nociceptors through both stimulation of its channel gating and mobilization of channels to the neuronal surface in a context dependent manner. A recent study reported that ATP-induced TRPV1 sensitization in peptidergic nociceptors involves the exocytotic release of channels trafficked by large dense core vesicles (LDCVs) that cargo alpha-calcitonin gene related peptide alpha (αCGRP). We hypothesized that, similar to ATP, bradykinin may also use different mechanisms to sensitize TRPV1 channels in peptidergic and non-peptidergic nociceptors. We found that bradykinin notably enhances the excitability of peptidergic nociceptors, and sensitizes TRPV1, primarily through the bradykinin receptor 2 pathway. Notably, bradykinin sensitization of TRPV1 in peptidergic nociceptors was significantly blocked by inhibiting Ca(2+)-dependent neuronal exocytosis. In addition, silencing αCGRP gene expression, but not substance P, drastically reduced bradykinin-induced TRPV1 sensitization in peptidergic nociceptors. Taken together, these findings indicate that bradykinin-induced sensitization of TRPV1 in peptidergic nociceptors is partially mediated by the exocytotic mobilization of new channels trafficked by αCGRP-loaded LDCVs to the neuronal membrane. Our findings further imply a central role of αCGRP peptidergic nociceptors in peripheral algesic sensitization, and substantiate that inhibition of LDCVs exocytosis is a valuable therapeutic strategy to treat pain, as it concurrently reduces the release of pro-inflammatory peptides and the membrane recruitment of thermoTRP channels.

Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates inflammatory heat hyperalgesia, but the underlying mechanisms are unknown. We induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, and augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. In particular, inflammation provoked a pronounced increase in nociceptors with functional co-expression of TRPM3, TRPV1 and TRPA1. Finally, pharmacological inhibition of TRPM3 dampened TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw, but not in those innervating healthy tissue. These insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.

Itch-specific neurons have been sought for decades. The existence of such neurons has been doubted recently as a result of the observation that itch-mediating neurons also respond to painful stimuli. We genetically labeled and manipulated MrgprA3(+) neurons in the dorsal root ganglion (DRG) and found that they exclusively innervated the epidermis of the skin and responded to multiple pruritogens. Ablation of MrgprA3(+) neurons led to substantial reductions in scratching evoked by multiple pruritogens and occurring spontaneously under chronic itch conditions, whereas pain sensitivity remained intact. Notably, mice in which TRPV1 was exclusively expressed in MrgprA3(+) neurons exhibited itch, but not pain, behavior in response to capsaicin. Although MrgprA3(+) neurons were sensitive to noxious heat, activation of TRPV1 in these neurons by noxious heat did not alter pain behavior. These data suggest that MrgprA3 defines a specific subpopulation of DRG neurons mediating itch. Our study opens new avenues for studying itch and developing anti-pruritic therapies.

Non-opioid targets are needed for addressing osteoarthritis pain, which is mechanical in nature and associated with daily activities such as walking and climbing stairs. Piezo2 has been implicated in the development of mechanical pain, but the mechanisms by which this occurs remain poorly understood, including the role of nociceptors. Here we show that nociceptor-specific Piezo2 conditional knock-out mice were protected from mechanical sensitization associated with inflammatory joint pain in female mice, joint pain associated with osteoarthritis in male mice, as well as both knee swelling and joint pain associated with repeated intra-articular injection of nerve growth factor in male mice. Single cell RNA sequencing of mouse lumbar dorsal root ganglia and in situ hybridization of mouse and human lumbar dorsal root ganglia revealed that a subset of nociceptors co-express Piezo2 and Ntrk1 (the gene that encodes the nerve growth factor receptor TrkA). These results suggest that nerve growth factor-mediated sensitization of joint nociceptors, which is critical for osteoarthritic pain, is also dependent on Piezo2, and targeting Piezo2 may represent a therapeutic option for osteoarthritis pain control.

Mechanical nociception is essential for animal survival. However, the forces involved in nociceptor activation and the underlying mechanotransduction mechanisms remain elusive. Here, we address these problems by investigating nocifensive behavior in Drosophila larvae. We show that strong poking stimulates nociceptors with a mixture of forces including shear stress and stretch. Unexpectedly, nociceptors are selectively activated by shear stress, but not stretch. Both the shear stress responses of nociceptors and nocifensive behavior require transient receptor potential A1 (TrpA1), which is specifically expressed in nociceptors. We further demonstrate that expression of mammalian or Drosophila TrpA1 in heterologous cells confers responses to shear stress but not stretch. Finally, shear stress activates TrpA1 in a membrane-delimited manner, through modulation of membrane fluidity. Together, our study reveals TrpA1 as an evolutionarily conserved mechanosensitive channel specifically activated by shear stress and suggests a critical role of shear stress in activating nociceptors to drive mechanical nociception.

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.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.

Clinical and preclinical studies have revealed that local administration of opioid agonists into peripheral tissue attenuates inflammatory pain. However, few studies have examined whether peripherally restricted opioids are effective in reducing mechanical allodynia and hyperalgesia that usually follows nerve injury. The aim of the present study was to determine whether the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic pain conditions is depressed by direct activation of delta opioid receptors (DORs) on their peripheral terminals. A murine model of peripheral neuropathic pain was induced with a spared nerve (tibial) injury, in which mice survived 7 or 28 days after surgery before electrophysiological testing began. Control groups comprised naïve and sham-operated animals. An ex vivo preparation of mouse plantar skin with attached tibial nerve was used to examine electrophysiologically the effects of the selective DOR agonist, deltorphin II, on the response properties of individual cutaneous C-fiber nociceptors. In contrast to naïve and sham-operated animals, deltorphin II induced an inhibition of the mechanical responsiveness of C-fiber mechanical nociceptors innervating skin under neuropathic conditions. The effects of deltorphin II were concentration-dependent and prevented by pretreatment with naltrindole indicating DOR-mediated inhibitory effects of deltorphin II. Our results provide the first direct evidence for expression of functional DORs on mechanical nociceptors innervating skin in an animal model of neuropathic pain.

Inflammation is a part of the body's natural response to tissue injury which initiates the healing process. Unfortunately, inflammation is frequently painful and leads to hypersensitivity to mechanical stimuli, which is difficult to treat clinically. While it is well established that altered sensory processing in the spinal cord contributes to mechanical hypersensitivity (central sensitization), it is still debated whether primary afferent neurons become sensitized to mechanical stimuli after tissue inflammation. We induced inflammation in C57BL/6 mice via intraplantar injection of Complete Freund's Adjuvant. Cutaneous C fibers exhibited increased action potential firing to suprathreshold mechanical stimuli. We found that abnormal responses to intense mechanical stimuli were completely suppressed by acute incubation of the receptive terminals with the TRPA1 inhibitor, HC-030031. Further, elevated responses were predominantly exhibited by a specific subgroup of C fibers, which we determined to be C-Mechano Cold sensitive fibers. Thus, in the presence of HC-030031, C fiber mechanical responses in inflamed mice were not different than responses in saline-injected controls. We also demonstrate that injection of the HC-030031 compound into the hind paw of inflamed mice alleviates behavioral mechanical hyperalgesia without affecting heat hyperalgesia. Further, we pharmacologically anesthetized the TRPA1-expressing fibers in vivo by co-injecting the membrane-impermeable sodium channel inhibitor QX-314 and the TRPA1 agonist cinnamaldehyde into the hind paw. This approach also alleviated behavioral mechanical hyperalgesia in inflamed mice but left heat hypersensitivity intact. Our findings indicate that C-Mechano Cold sensitive fibers exhibit enhanced firing to suprathreshold mechanical stimuli in a TRPA1-dependent manner during inflammation, and that input from these fibers drives mechanical hyperalgesia in inflamed mice.

1. Experiments were performed on anaesthetized cats to investigate the receptive properties of regenerated cutaneous tibial nerve nociceptors, and to obtain evidence for coupling between them and other afferent fibres as being possible peripheral mechanisms involved in neuropathic pain. These properties were studied 6-7 months after nerve section and repair. 2. Recordings were made from 25 regenerated nociceptors; 14 were A fibres and the remainder were C fibres. Their receptive field sizes and conduction velocities were similar to controls. There was no significant difference between their mechanical thresholds and those of a control population of nociceptors. 3. Regenerated nociceptors were significantly more responsive to suprathreshold mechanical stimuli than were uninjured control fibres. This increase in mechanical sensitivity occurred in both A and C fibres, although A fibres showed a greater increase in mechano-sensitivity than C fibres. Over half of the regenerated nociceptors (13/25) showed after-discharge to mechanical stimuli which was never seen in controls; the mean firing rate during this period of after-discharge was significantly related to both stimulus intensity and stimulus area. 4. There was no significant difference between the heat encoding properties of regenerated nociceptors and control nociceptors. Cold sensitivity was similarly unchanged. Thus, abnormal peripheral sprouting was unlikely to account for the increased mechanical sensitivity of the regenerated fibres. None of the regenerated nociceptors were found to be coupled to other fibres. 5. These results suggest that the clinical observation of mechanical hyperalgesia in patients after nerve injury may have a peripheral basis. Based on this model, other signs of neuropathic pain (i.e. tactile or thermal allodynia) are more likely to be due to altered central processing.

Nerve growth factor (NGF) induces local hyperalgesia for a few days after intramuscular injection, but longer-lasting muscle pain upon systemic administration. As the muscle fascia is densely innervated by free nerve endings, we hypothesized a lasting sensitization of fascia nociceptors by NGF. We administered 1 μg NGF (dissolved in 100 μL saline) ultrasound-guided to the fascia of the Musculus erector spinae muscle at the lumbar level of 14 male volunteers and assessed hypersensitivity after 6 hours, and 1, 3, 7, 14, and 21 days. Pain upon mechanical stimuli (constant pressure and dynamic impact), upon exercise and electrically induced M. erector spinae contraction, and upon injection of 100 μL phosphate buffer pH4 (at day 7 and 14 only) to the fascia of both NGF- and saline-treated muscles, was investigated. Injections into the muscle fascia did not cause acute pain. Local heat pain thresholds were unchanged following NGF and saline (control) administration. NGF evoked a lasting (days 1-7) and significant reduction of pressure pain, pressure thresholds, exercise-evoked muscle pain, and hyperalgesia to impact stimuli (12 m/s). Pain upon injected protons was significantly elevated (P<0.04) for 2 weeks. NGF induced a sensitization of the muscle fascia to mechanical and chemical stimuli lasting for up to 2 weeks. As nociceptors in the fascia appear to be particularly prone to sensitization, they may contribute to acute or chronic muscle pain.

A comprehensive understanding of the molecular machinery important for nociception is essential to improving the treatment of pain. Here, we show that the BMP signaling pathway regulates nociception downstream of the E3 ubiquitin ligase highwire (hiw). hiw loss of function in nociceptors caused antagonistic and pleiotropic phenotypes with simultaneous insensitivity to noxious heat but sensitized responses to optogenetic activation of nociceptors. Thus, hiw functions to both positively and negatively regulate nociceptors. We find that a sensory reception-independent sensitization pathway was associated with BMP signaling. BMP signaling in nociceptors was up-regulated in hiw mutants, and nociceptor-specific expression of hiw rescued all nociception phenotypes including the increased BMP signaling. Blocking the transcriptional output of the BMP pathway with dominant negative Mad suppressed nociceptive hypersensitivity that was induced by interfering with hiw. The up-regulated BMP signaling phenotype in hiw genetic mutants could not be suppressed by mutation in wallenda suggesting that hiw regulates BMP in nociceptors via a wallenda independent pathway. In a newly established Ca2+ imaging preparation, we observed that up-regulated BMP signaling caused a significantly enhanced Ca2+ signal in the axon terminals of nociceptors that were stimulated by noxious heat. This response likely accounts for the nociceptive hypersensitivity induced by elevated BMP signaling in nociceptors. Finally, we showed that 24-hour activation of BMP signaling in nociceptors was sufficient to sensitize nociceptive responses to optogenetically-triggered nociceptor activation without altering nociceptor morphology. Overall, this study demonstrates the previously unrevealed roles of the Hiw-BMP pathway in the regulation of nociception and provides the first direct evidence that up-regulated BMP signaling physiologically sensitizes responses of nociceptors and nociception behaviors.

We have previously described a rat model for the contribution of neuroplastic changes in nociceptors to the transition from acute to chronic pain. In this model a prior injury activates protein kinase C epsilon (PKCepsilon), inducing a chronic state characterized by marked prolongation of the hyperalgesia induced by inflammatory cytokines, prototypically prostaglandin E(2) (PGE(2)), referred to as hyperalgesic priming. In this study we evaluated the population of nociceptors involved in priming, by lesioning isolectin B4-positive (IB4(+)) nociceptors with intrathecal administration of a selective neurotoxin, IB4-saporin. To confirm that the remaining, TrkA(+)/IB4(-), nociceptors are still functional, we evaluated if nerve growth factor (NGF) induced hyperalgesia. While pretreatment with IB4-saporin eliminated the acute mechanical hyperalgesia induced by glia-derived neurotrophic factor (GDNF), NGF and PsiepsilonRACK, a highly selective activator of PKCepsilon, induced robust hyperalgesia. After injection of NGF, GDNF or PsiepsilonRACK, at a time at which hyperalgesia induced by PGE(2) is markedly prolonged (hyperalgesic priming) in control rats, in IB4-saporin-pretreated rats PGE(2) failed to produce this prolonged hyperalgesia. Thus, while PKCepsilon is present in most dorsal root ganglion neurons, where it can contribute to acute mechanical hyperalgesia, priming is restricted to IB4(+)-nociceptors, including those that are TrkA(+). While PKCepsilon activation can induce acute hyperalgesia in the IB4(+) population, it fails to induce priming. We suggest that hyperalgesic priming occurs only in IB4(+) nociceptors, and that in the peripheral terminals of nociceptors separate intracellular pools of PKCepsilon mediate nociceptor sensitization and the induction of hyperalgesic priming.