Neuron-specific RNA-sequencing reveals different responses in peripheral neurons after nerve injury.

Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.

Sulfide and polysulfide as pronociceptive mediators: Focus on Cav3.2 function enhancement and TRPA1 activation.

Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Cav3.2 T-type Ca2+ channels by eliminating Zn2+ bound to the channels, thereby promoting somatic and visceral pain. Cav3.2 is under inhibition by Zn2+ in physiological conditions, so that sulfides function to reboot Cav3.2 from Zn2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Cav3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Cav3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain.

STINGing away the pain: the role of interferon-stimulated genes.

Pain and inflammation are biologically intertwined responses that warn the body of potential danger. In this issue of the JCI, Defaye, Bradaia, and colleagues identified a functional link between inflammation and pain, demonstrating that inflammation-induced activation of stimulator of IFN genes (STING) in dorsal root ganglia nociceptors reduced pain-like behaviors in a rodent model of inflammatory pain. Utilizing mice with a gain-of-function STING mutation, Defaye, Bradaia, and colleagues identified type I IFN regulation of voltage-gated potassium channels as the mechanism of this pain relief. Further investigation into mechanisms by which proinflammatory pathways can reduce pain may reveal druggable targets and insights into new approaches for treating persistent pain.

Induction of antiviral interferon-stimulated genes by neuronal STING promotes the resolution of pain in mice.

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-ß response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.

An Artificial LiSiOx Nociceptor with Neural Blockade and Self-Protection Abilities.

An artificial nociceptor, as a critical and special bionic receptor, plays a key role in a bioelectronic device that detects stimuli and provides warnings. However, fully exploiting bioelectronic applications remains a major challenge due to the lack of the methods of implementing basic nociceptor functions and nociceptive blockade in a single device. In this work, we developed a Pt/LiSiOx/TiN artificial nociceptor. It had excellent stability under the 104 endurance test with pulse stimuli and exhibited a significant threshold current of 1 mA with 1 V pulse stimuli. Other functions such as relaxation, inadaptation, and sensitization were all realized in a single device. Also, the pain blockade function was first achieved in this nociceptor with over a 25% blocking degree, suggesting a self-protection function. More importantly, an obvious depression was activated by a stimulus over 1.6 V due to the cooperative effects of both lithium ions and oxygen ions in LiSiOx and the dramatic accumulation of Joule heat. The conducting channel ruptured partially under sequential potentiation, thus achieving nociceptive blockade, besides basic functions in one single nociceptor, which was rarely reported. These results provided important guidelines for constructing high-performance memristor-based artificial nociceptors and opened up an alternative approach to the realization of bioelectronic systems for artificial intelligence.

CXCL5 activates CXCR2 in nociceptive sensory neurons to drive joint pain and inflammation in experimental gouty arthritis.

Gouty arthritis evokes joint pain and inflammation. Mechanisms driving gout pain and inflammation remain incompletely understood. Here we show that CXCL5 activates CXCR2 expressed on nociceptive sensory neurons to drive gout pain and inflammation. CXCL5 expression was increased in ankle joints of gout arthritis model mice, whereas CXCR2 showed expression in joint-innervating sensory neurons. CXCL5 activates CXCR2 expressed on nociceptive sensory neurons to trigger TRPA1 activation, resulting in hyperexcitability and pain. Neuronal CXCR2 coordinates with neutrophilic CXCR2 to contribute to CXCL5-induced neutrophil chemotaxis via triggering CGRP- and substance P-mediated vasodilation and plasma extravasation. Neuronal Cxcr2 deletion ameliorates joint pain, neutrophil infiltration and gait impairment in model mice. We confirmed CXCR2 expression in human dorsal root ganglion neurons and CXCL5 level upregulation in serum from male patients with gouty arthritis. Our study demonstrates CXCL5-neuronal CXCR2-TRPA1 axis contributes to gouty arthritis pain, neutrophil influx and inflammation that expands our knowledge of immunomodulation capability of nociceptive sensory neurons.

Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs.

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

Nociceptor spontaneous activity is responsible for fragmenting non-rapid eye movement sleep in mouse models of neuropathic pain.

Spontaneous pain, a major complaint of patients with neuropathic pain, has eluded study because there is no reliable marker in either preclinical models or clinical studies. Here, we performed a comprehensive electroencephalogram/electromyogram analysis of sleep in several mouse models of chronic pain: neuropathic (spared nerve injury and chronic constriction injury), inflammatory (Freund's complete adjuvant and carrageenan, plantar incision) and chemical pain (capsaicin). We find that peripheral axonal injury drives fragmentation of sleep by increasing brief arousals from non-rapid eye movement sleep (NREMS) without changing total sleep amount. In contrast to neuropathic pain, inflammatory or chemical pain did not increase brief arousals. NREMS fragmentation was reduced by the analgesics gabapentin and carbamazepine, and it resolved when pain sensitivity returned to normal in a transient neuropathic pain model (sciatic nerve crush). Genetic silencing of peripheral sensory neurons or ablation of CGRP+ neurons in the parabrachial nucleus prevented sleep fragmentation, whereas pharmacological blockade of skin sensory fibers was ineffective, indicating that the neural activity driving the arousals originates ectopically in primary nociceptor neurons and is relayed through the lateral parabrachial nucleus. These findings identify NREMS fragmentation by brief arousals as an effective proxy to measure spontaneous neuropathic pain in mice.

Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae.

An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues, however the mechanisms by which epidermal interactions shape processing of noxious inputs is still poorly understood. Here, we identify a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites, but not at other sites in the epidermis. From a genetic screen we identified miR-14 as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. We found that miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, we found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.

Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

From pain to tumor immunity: influence of peripheral sensory neurons in cancer.

The nervous and immune systems are the primary sensory interfaces of the body, allowing it to recognize, process, and respond to various stimuli from both the external and internal environment. These systems work in concert through various mechanisms of neuro-immune crosstalk to detect threats, provide defense against pathogens, and maintain or restore homeostasis, but can also contribute to the development of diseases. Among peripheral sensory neurons (PSNs), nociceptive PSNs are of particular interest. They possess a remarkable capability to detect noxious stimuli in the periphery and transmit this information to the brain, resulting in the perception of pain and the activation of adaptive responses. Pain is an early symptom of cancer, often leading to its diagnosis, but it is also a major source of distress for patients as the disease progresses. In this review, we aim to provide an overview of the mechanisms within tumors that are likely to induce cancer pain, exploring a range of factors from etiological elements to cellular and molecular mediators. In addition to transmitting sensory information to the central nervous system, PSNs are also capable, when activated, to produce and release neuropeptides (e.g., CGRP and SP) from their peripheral terminals. These neuropeptides have been shown to modulate immunity in cases of inflammation, infection, and cancer. PSNs, often found within solid tumors, are likely to play a significant role in the tumor microenvironment, potentially influencing both tumor growth and anti-tumor immune responses. In this review, we discuss the current state of knowledge about the degree of sensory innervation in tumors. We also seek to understand whether and how PSNs may influence the tumor growth and associated anti-tumor immunity in different mouse models of cancer. Finally, we discuss the extent to which the tumor is able to influence the development and functions of the PSNs that innervate it.

Hypothalamic Paraventricular Stimulation Inhibits Nociceptive Wide Dynamic Range Trigeminocervical Complex Cells via Oxytocinergic Transmission.

Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.

CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Effects of systemic oxytocin administration on ultraviolet B-induced nociceptive hypersensitivity and tactile hyposensitivity in mice.

Ultraviolet B (UVB) radiation induces cutaneous inflammation, leading to thermal and mechanical hypersensitivity. Here, we examine the mechanical properties and profile of tactile and nociceptive peripheral afferents functionally disrupted by this injury and the role of oxytocin (OXT) as a modulator of this disruption. We recorded intracellularly from L4 afferents innervating the irradiated area (5.1 J/cm2) in 4-6 old week male mice (C57BL/6J) after administering OXT intraperitoneally, 6 mg/Kg. The distribution of recorded neurons was shifted by UVB radiation to a pattern observed after acute and chronic injuries and reduced mechanical thresholds of A and C- high threshold mechanoreceptors while reducing tactile sensitivity. UVB radiation did not change somatic membrane electrical properties or fiber conduction velocity. OXT systemic administration rapidly reversed these peripheral changes toward normal in both low and high-threshold mechanoreceptors and shifted recorded neuron distribution toward normal. OXT and V1aR receptors were present on the terminals of myelinated and unmyelinated afferents innervating the skin. We conclude that UVB radiation, similar to local tissue surgical injury, cancer metastasis, and peripheral nerve injury, alters the distribution of low and high threshold mechanoreceptors afferents and sensitizes nociceptors while desensitizing tactile units. Acute systemic OXT administration partially returns all of those effects to normal.

Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors.

In vivo analysis of protein function in nociceptor subpopulations using antisense oligonucleotides and short interfering RNAs is limited by their non-selective cellular uptake. To address the need for selective transfection methods, we covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors. Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA were found to have: (a) less PKCε in dorsal root ganglia (DRG), (b) reduced PKCε expression in IB4(+) but not IB4(-) DRG neurons, and (c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)-nociceptors. These results establish that IB4-streptavidin can be used to study protein function in a defined subpopulation of nociceptive C-fiber afferents.

Human Foot Outperforms the Hand in Mechanical Pain Discrimination.

Tactile discrimination has been extensively studied, but mechanical pain discrimination remains poorly characterized. Here, we measured the capacity for mechanical pain discrimination using a two-alternative forced choice paradigm, with force-calibrated indentation stimuli (Semmes-Weinstein monofilaments) applied to the hand and foot dorsa of healthy human volunteers. In order to characterize the relationship between peripheral nociceptor activity and pain perception, we recorded single-unit activity from myelinated (A) and unmyelinated (C) mechanosensitive nociceptors in the skin using microneurography. At the perceptual level, we found that the foot was better at discriminating noxious forces than the hand, which stands in contrast to that for innocuous force discrimination, where the hand performed better than the foot. This observation of superior mechanical pain discrimination on the foot compared to the hand could not be explained by the responsiveness of individual nociceptors. We found no significant difference in the discrimination performance of either the myelinated or unmyelinated class of nociceptors between skin regions. This suggests the possibility that other factors such as skin biophysics, receptor density or central mechanisms may underlie these regional differences.

Few-layered graphene increases the response of nociceptive neurons to irritant stimuli.

The unique properties of few-layered graphene (FLG) make it interesting for a variety of applications, including biomedical applications, such as tissue engineering and drug delivery. Although different studies focus on applications in the central nervous system, its interaction with the peripheral nervous system has been so far overlooked. Here, we investigated the effects of exposure to colloidal dispersions of FLG on the sensory neurons of the rat dorsal root ganglia (DRG). We found that the FLG flakes were actively internalized by sensory neurons, accumulated in large intracellular vesicles, and possibly degraded over time, without major toxicological concerns, as neuronal viability, morphology, protein content, and basic electrical properties of DRG neurons were preserved. Interestingly, in our electrophysiological investigation under noxious stimuli, we observed an increased functional response upon FLG treatment of the nociceptive subpopulation of DRG neurons in response to irritants specific for chemoreceptors TRPV1 and TRPA1. The observed effects of FLG on DRG neurons may open-up novel opportunities for applications of these materials in specific disease models.

SARS-CoV-2 sneezing explained.

Viral protein prods nerve cells, may be treatment target.

Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-α.

Tumor necrosis factor α (TNF-α) is a major pro-inflammatory cytokine, important in many diseases, that sensitizes nociceptors through its action on a variety of ion channels, including voltage-gated sodium (NaV) channels. We show here that TNF-α acutely upregulates sensory neuron excitability and current density of threshold channel NaV1.7. Using electrophysiological recordings and live imaging, we demonstrate that this effect on NaV1.7 is mediated by p38 MAPK and identify serine 110 in the channel's N terminus as the phospho-acceptor site, which triggers NaV1.7 channel insertion into the somatic membrane. We also show that the N terminus of NaV1.7 is sufficient to mediate this effect. Although acute TNF-α treatment increases NaV1.7-carrying vesicle accumulation at axonal endings, we did not observe increased channel insertion into the axonal membrane. These results identify molecular determinants of TNF-α-mediated regulation of NaV1.7 in sensory neurons and demonstrate compartment-specific effects of TNF-α on channel insertion in the neuronal plasma membrane.