Enteric glia promote visceral hypersensitivity during inflammation through intercellular signaling with gut nociceptors.

Inflammation in the intestines causes abdominal pain that is challenging to manage. The terminals of sensory neurons innervating the gut are surrounded by glia. Here, using a mouse model of acute colitis, we found that enteric glia contribute to visceral pain by secreting factors that sensitized sensory nerves innervating the gut in response to inflammation. Acute colitis induced a transient increase in the production of proinflammatory cytokines in the intestines of male and female mice. Of these, IL-1ß was produced in part by glia and augmented the opening of the intercellular communication hemichannel connexin-43 in glia, which made normally innocuous stimuli painful in female mice. Chemogenetic glial activation paired with calcium imaging in nerve terminals demonstrated that glia sensitized gut-innervating nociceptors only under inflammatory conditions. This inflammatory, glial-driven visceral hypersensitivity involved an increased abundance of the enzyme COX-2 in glia, resulting in greater production and release of prostaglandin E2 that activated EP4 receptors on sensory nerve terminals. Blocking EP4 receptors reduced nociceptor sensitivity in response to glial stimulation in tissue samples from colitis-model mice, and impairing glial connexin-43 reduced visceral hypersensitivity induced by IL-1ß in female mice. The findings suggest that therapies targeting enteric glial-neuron signaling might alleviate visceral pain caused by inflammatory disorders.

4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling.

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

Towards a central origin of nociceptive hypersensitivity in adult rats after a neonatal maternal separation.

Early life adversities influence a nervous system still in development with long-term consequences for later life. These include nociceptive circuit alterations critical to shape an adaptive pain response to protect the organism from potential damage. Adult rats with a history of neonatal maternal separation (NMS) display visceral and somatic nociceptive hypersensitivity and inefficient analgesic responses to stress. In this study, we have characterized the consequences of NMS on wide dynamic range neurons (WDR) in the spinal cord of anaesthetized adult rats during the nociceptive processing of hot and cold noxious information. We found that WDR neurons of NMS rats display an excessive coding of mechanical and thermal information applied at the rat's hindpaws. This nicely explains the hypernociceptive behaviours seen after noxious mechanical, cold and hot peripheral stimulation. A peripheral change in the expression of molecular transducers for these stimuli (i.e., TRPV1, TRPM8 and TRPA1) does not seem to account for this general hyperexcitability. Instead, a decreased chloride-mediated inhibitory tone on WDR neurons may play a role as indicated by the abnormal elevation of the type 1 Na-K-Cl cotransporter transcripts. Altogether, we propose that long-term consequences of NMS are associated with reduced spinal cord inhibition favouring the expression of pain hypersensitivity. We cannot exclude that this phenomenon is also present at supraspinal sites, as other NMS-associated symptoms include excessive anxiety and impaired sociability.

Nociceptors regulate osteoimmune transcriptomic response to infection.

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.

NaV1.7: A central role in pain.

Loss of function of sodium channel NaV1.7 produces pain insensitivity. In this issue, Deng et al.1 show that analgesia after NaV1.7 removal or pharmacological blockade is not driven by enkephalin overexpression. These results underscore the essential role, independent of endogenous opioids, of NaV1.7 for nociceptor firing and pain.

Benzydamine plays a role in limiting inflammatory pain induced by neuronal sensitization.

Benzydamine is an active pharmaceutical compound used in the oral care pharmaceutical preparation as NSAID. Beside from its anti-inflammatory action, benzydamine local application effectively reliefs pain showing analgesic and anaesthetic properties. Benzydamine mechanism of action has been characterized on inflammatory cell types and mediators highlighting its capacity to inhibit pro-inflammatory mediators' synthesis and release. On the other hand, the role of benzydamine as neuronal excitability modulator has not yet fully explored. Thus, we studied benzydamine's effect over primary cultured DRG nociceptors excitability and after acute and chronic inflammatory sensitization, as a model to evaluate relative nociceptive response. Benzydamine demonstrated to effectively inhibit neuronal basal excitability reducing its firing frequency and increasing rheobase and afterhyperpolarization amplitude. Its effect was time and dose-dependent. At higher doses, benzydamine induced changes in action potential wavelength, decreasing its height and slightly increasing its duration. Moreover, the compound reduced neuronal acute and chronic inflammatory sensitization. It inhibited neuronal excitability mediated either by an inflammatory cocktail, acidic pH or high external KCl. Notably, higher potency was evidenced under inflammatory sensitized conditions. This effect could be explained either by modulation of inflammatory and/or neuronal sensitizing signalling cascades or by direct modulation of proalgesic and action potential firing initiating ion channels. Apparently, the compound inhibited Nav1.8 channel but had no effect over Kv7.2, Kv7.3, TRPV1 and TRPA1. In conclusion, the obtained results strengthen the analgesic and anti-inflammatory effect of benzydamine, highlighting its mode of action on local pain and inflammatory signalling.

Generation of a TRPV1 knockout human pluripotent stem cell line (WAe009-A-U) using CRISPR/Cas9.

The transient receptor potential vanilloid subfamily 1 (TRPV1) is a polymodal nociceptor that is highly expressed in sensory nerves. Activation of TRPV1 receptors excites primary afferent nociceptors by opening cation channels, allowing the influx of Na+ and Ca2+ ions into the cytoplasm. Here, a TRPV1 knockout human embryonic stem cell line was generated using the CRISPR/Cas9 genome-editing technology to further study the function of TRPV1. The cell line confirmed with normal pluripotency and karyotype.

Neuroinflammation in the Dorsal Root Ganglia and Dorsal Horn Contributes to Persistence of Nociceptor Sensitization in SIV-Infected Antiretroviral Therapy-Treated Macaques.

Despite the development of antiretroviral therapy (ART), HIV-associated distal sensory polyneuropathy remains prevalent. Using SIV-infected rhesus macaques, this study examined molecular mechanisms of peripheral and central sensitization to infer chronic pain from HIV infection. Previous studies identified atrophy in nociceptive neurons during SIV infection, which was associated with monocyte infiltration into the dorsal root ganglia (DRG). However, the sensory signaling mechanism connecting this pathology to symptoms remains unclear, especially because pain persists after resolution of high viremia and inflammation with ART. We hypothesized that residual DRG and dorsal horn neuroinflammation contributes to nociceptive sensitization. Using three cohorts of macaques [uninfected (SIV-), SIV-infected (SIV+), and SIV infected with ART (SIV+/ART)], this study showed an increase in the cellular and cytokine inflammatory profiles in the DRG of SIV+/ART macaques compared with uninfected animals. It found significant increase in the expression of nociceptive ion channels, TRPV1, and TRPA1 among DRG neurons in SIV+/ART compared with uninfected animals. SIV-infected and SIV+/ART animals showed reduced innervation of the nonpeptidergic nociceptors into the dorsal horn compared with uninfected animals. Finally, there were a significantly higher number of CD68+ cells in the dorsal horn of SIV+/ART macaques compared with uninfected animals. In summary, these data demonstrate that neuroinflammation, characteristics of nociceptor sensitization, and central terminal atrophy persists in SIV+/ART animals.

Menthol excites dural afferent neurons by inhibiting leak K+ conductance in rats.

Menthol-a natural organic compound-is widely used for relieving various pain conditions including migraine. However, a high dose of menthol reportedly decreases pain thresholds and enhances pain responses. Accordingly, in the present study, we addressed the effect of menthol on the excitability of acutely isolated dural afferent neurons, which were identified with a fluorescent dye, using the whole-cell patch-clamp technique. Under a voltage-clamped condition, menthol altered the holding current levels in a concentration-dependent manner. The menthol-induced current (IMenthol) remained unaffected by the addition of selective transient receptor potential melastatin 8 antagonists. Moreover, the reversal potential of IMenthol was similar to the equilibrium potential of K+. IMenthol was accompanied by an increase in input resistance, thereby suggesting that menthol decreases the leak K+ conductance. Under a current-clamped condition, menthol caused depolarization of the membrane potential and decreased the threshold for the generation of action potential. While the IMenthol was substantially inhibited by 10 µM XE-991, a selective KV7 blocker, the M-current mediated by KV7 was not detected in the nociceptive neurons tested in the present study. Moreover, IMenthol decreased under acidic extracellular pH conditions or in the presence of 3 µM A-1899, a selective K2P3.1 and K2P9.1 blocker. The present results suggest that menthol inhibits leak K+ channels, possibly acid-sensitive two-pore domain K+ channels, thereby increasing the excitability of nociceptive sensory neurons. The resultant increase in neuron excitability may partially be responsible for the pronociceptive effect mediated by high menthol doses.

Threshold Modulative Artificial GABAergic Nociceptor.

Gamma-aminobutyric acid (GABA) is a crucial inhibitory neurotransmitter of the central nervous system. It modifies the signal threshold of the nociceptor, allowing it to react to external stimuli in various circumstances. Thus, GABAergic behaviors are critical characteristics of adaptive behavior in life. Here, a threshold-modulative artificial GABAergic nociceptor is reported for the first time at a Pt/Ti/Nb2 O5- x /Al2 O3- y /Pt/Ti (top to bottom) of the double charge trapping structure. The Al2 O3- y layer contains deep defect states that function similarly to the GABA neurotransmitter in modulating the signal threshold. Meanwhile, the Nb2 O5- x layer traps volatile charges and produces nociceptive behaviors. The combined dynamics of the two layers readily offer threshold-modulative GABAergic nociceptive behaviors. Based on these GABAergic behaviors, a method of implementing hot- and cold-sensitive thermoreceptors is demonstrated and shows its potential applications in advanced sensory devices.

Members of the CUGBP Elav-like family of RNA-binding proteins are expressed in distinct populations of primary sensory neurons.

Primary sensory dorsal root ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3' untranslated regions (3'UTRs). 3'UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) for transporting mRNAs to subcellular domains, modulating transcript stability, and regulating the rate of translation. In the current study, analysis of publicly available single-cell RNA-sequencing data generated from adult mice revealed that 17 3'UTR-binding RBPs were enriched in specific populations of DRG neurons. This included four members of the CUG triplet repeat (CUGBP) Elav-like family (CELF): CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic, and CELF3 in tyrosine hydroxylase-expressing neurons. Immunofluorescence studies confirmed that 60% of CELF4+ neurons are small-diameter C fibers and 33% medium-diameter myelinated (likely Aδ) fibers and showed that CELF4 is distributed to peripheral termini. Coexpression analyses using transcriptomic data and immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP, and the capsaicin receptor TRPV1. Reanalysis of published transcriptomic data from macaque DRG revealed a highly similar distribution of CELF members, and reanalysis of single-nucleus RNA-sequencing data derived from mouse and rat DRG after sciatic injury revealed differential expression of CELFs in specific populations of sensory neurons. We propose that CELF RBPs may regulate the fate of mRNAs in populations of nociceptors, and may play a role in pain and/or neuronal regeneration following nerve injury.

Nociceptor-Macrophage Interactions in Apical Periodontitis: How Biomolecules Link Inflammation with Pain.

Periradicular tissues have a rich supply of peripheral afferent neurons, also known as nociceptive neurons, originating from the trigeminal nerve. While their primary function is to relay pain signals to the brain, these are known to be involved in modulating innate and adaptive immunity by initiating neurogenic inflammation (NI). Studies have investigated neuroanatomy and measured the levels of biomolecules such as cytokines and neuropeptides in human saliva, gingival crevicular fluid, or blood/serum samples in apical periodontitis (AP) to validate the possible role of trigeminal nociceptors in inflammation and tissue regeneration. However, the contributions of nociceptors and the mechanisms involved in the neuro-immune interactions in AP are not fully understood. This narrative review addresses the complex biomolecular interactions of trigeminal nociceptors with macrophages, the effector cells of the innate immune system, in the clinical manifestations of AP.

Capsaicin, The Vanilloid Receptor TRPV1 Agonist in Neuroprotection: Mechanisms Involved and Significance.

Hot peppers, also called chilli, chilli pepper, or paprika of the plant genus Capsicum (family Solanaceae), are one of the most used vegetables and spices worldwide. Capsaicin (8-methyl N-vanillyl-6-noneamide) is the main pungent principle of hot green and red peppers. By acting on the capsaicin receptor or transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1), capsaicin selectively stimulates and in high doses defunctionalizes capsaicin-sensitive chemonociceptors with C and Aδ afferent fibers. This channel, which is involved in a wide range of neuronal processes, is expressed in peripheral and central branches of capsaicin-sensitive nociceptive neurons, sensory ganglia, the spinal cord, and different brain regions in neuronal cell bodies, dendrites, astrocytes, and pericytes. Several experimental and clinical studies provided evidence that capsaicin protected against ischaemic or excitotoxic cerebral neuronal injury and may lower the risk of cerebral stroke. By preventing neuronal death, memory impairment and inhibiting the amyloidogenic process, capsaicin may also be beneficial in neurodegenerative disorders such as Parkinson's or Alzheimer's diseases. Capsaicin given in systemic inflammation/sepsis exerted beneficial antioxidant and anti-inflammatory effects while defunctionalization of capsaicin-sensitive vagal afferents has been demonstrated to increase brain oxidative stress. Capsaicin may act in the periphery via the vagal sensory fibers expressing TRPV1 receptors to reduce immune oxidative and inflammatory signalling to the brain. Capsaicin given in small doses has also been reported to inhibit the experimentally-induced epileptic seizures. The aim of this review is to provide a concise account on the most recent findings related to this topic. We attempted to delineate such mechanisms by which capsaicin exerts its neuronal protective effects. We also aimed to provide the reader with the current knowledge on the mechanism of action of capsaicin on sensory receptors.

Senso-immunology: The Emerging Connection between Pain and Immunity.

The sensory and immune systems have been studied independently for a long time, whereas the interaction between the two has received little attention. We have carried out research to understand the interaction between the sensory and immune systems and have found that inflammation and bone destruction caused by fungal infection are suppressed by nociceptors. Furthermore, we have elucidated the molecular mechanism whereby fungal receptors are expressed on nociceptors and skin epithelium, how they cooperate to generate fungal pain, and how colitis and bone metabolism are regulated by mechanosensors expressed on the gut epithelium. Recently, we found that nociceptors prevent septic death by inhibiting microglia via nociceptor-derived hormones. This review summarizes our current state of knowledge on pain biology and outlines the mechanisms whereby pain and immunity interact. Our findings indicate that the sensory and immune systems share a variety of molecules and interact with each other to regulate our pathological and homeostatic conditions. This prompted us to advocate the interdisciplinary science named "senso-immunology," and this emerging field is expected to generate new ideas in both physiology and immunology, leading to the development of novel drugs to treat pain and inflammation.

"Nociplastic Pain": A Challenge to Nosology and to Nociception.

The construct of "nociplastic pain" has met with divergent receptions. On the one hand it has been enthusiastically embraced, to the extent of conflation with central sensitization of nociception and the International Classification of Diseases 11th Revision (ICD-11) entity of "primary" pain, and the promulgation of "nociplastic pain syndromes." On the other hand, it has been rejected by those whose skepticism derives from the absence, by definition, of underlying activation of nociceptors. This article seeks to dissect these divergent views and search for reconciliation between them. One line of argument is that "nociplastic" pain, "primary" pain, and "central sensitisation of nociception" reflect different domains of inquiry and should not be conflated. "Nociplastic" pain emerges as a hypothesis that confers clinical legitimacy and utility; while that hypothesis needs a minor but important modification and continues to require testing, discipline in its usage is necessary. The other line of argument discovers an unexpected impasse: the construct of "nociplastic pain" describes a phenomenon that accords with the International Association for the Study of Pain definition of pain but occurs in the absence of nociception-as-currently-defined, thus challenging the definitional link between pain and tissue damage. The article offers a resolution of this impasse by suggesting that nociception-as-currently-defined be replaced by the resurrected concept of a nociceptive apparatus, activation of which is necessary but not sufficient for the experience of pain. One consequence would be to allow the assertions underpinning "nociplastic" to be tested empirically; another would be to relate the phenomenon of pain to a more biologically plausible basis than "actual" or "resemblance to" tissue damage. PERSPECTIVE: This article explores the major challenges posed by "nociplastic pain" to nosology and to nociception. While discipline in the clinical use of the construct is required, it also emerges that the main issue is the International Association for the Study of Pain definition of nociception. A reconceptualization of nociception is proposed for logical, biological, and clinical coherence.

Understanding the initiation, delivery and processing of bone cancer pain from the peripheral to the central nervous system.

Bone cancer pain is a complex condition characterized by persistent, sudden, spontaneous pain accompanied by hyperalgesia that typically arises from bone metastases or primary bone tumors, causing severe discomfort and significantly diminishing cancer patients' quality of life and confidence in their ability to overcome the disease. It is widely known that peripheral nerves are responsible for detecting harmful stimuli, which are then transmitted to the brain via the spinal cord, resulting in the perception of pain. In the case of bone cancer, tumors and stromal cells within the bone marrow release various chemical signals, including inflammatory factors, colony-stimulating factors, chemokines, and hydrogen ions. Consequently, the nociceptors located at the nerve endings within the bone marrow sense these chemical signals, generating electrical signals that are then transmitted to the brain through the spinal cord. Subsequently, the brain processes these electrical signals in a complex manner to create the sensation of bone cancer pain. Numerous studies have investigated the transmission of bone cancer pain from the periphery to the spinal cord. However, the processing of pain information induced by bone cancer within the brain remains unclear. With the continuous advancements in brain science and technology, the brain mechanism of bone cancer pain would become more clearly understood. Herein, we focus on summarizing the peripheral nerve perception of the spinal cord transmission of bone cancer pain and provide a brief overview of the ongoing research regarding the brain mechanisms involved in bone cancer pain.

Deficient AMPK activity contributes to hyperexcitability in peripheral nociceptive sensory neurons and thermal hyperalgesia in lupus mice.

Patients with systemic lupus erythematosus (SLE) often suffer from chronic pain. Little is known about the peripheral mechanisms underlying the genesis of chronic pain induced by SLE. The aim of this study was to investigate whether and how membrane properties in nociceptive neurons in the dorsal root ganglions (DRGs) are altered by SLE. We found elevation of resting membrane potentials, smaller capacitances, lower action potential thresholds and rheobases in nociceptive neurons in the DRGs from MRL/lpr mice (an SLE mouse model) with thermal hyperalgesia. DRGs from MRL/lpr mice had increased protein expressions in TNFα, IL-1ß, and phosphorylated ERK but suppressed AMPK activity, and no changes in sodium channel 1.7 protein expression. We showed that intraplantar injection of Compound C (an AMPK inhibitor) induced thermal hyperalgesia in normal mice while intraplantar injection of AICAR (an AMPK activator) reduced thermal hyperalgesia in MRL/Lpr mice. Upon inhibition of AMPK membrane properties in nociceptive neurons from normal control mice could be rapidly switched to those found in SLE mice with thermal hyperalgesia. Our study indicates that increased excitability in peripheral nociceptive sensory neurons contributes to the genesis of thermal hyperalgesia in mice with SLE, and AMPK regulates membrane properties in nociceptive sensory neurons as well as thermal hyperalgesia in mice with SLE. Our study provides a basis for targeting signaling pathways regulating membrane properties of peripheral nociceptive neurons as a means for conquering chronic pain caused by SLE.

Calretinin-expressing islet cells are a source of pre- and post-synaptic inhibition of non-peptidergic nociceptor input to the mouse spinal cord.

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

Senso-immunology: the past, present, and future.

Pain and mechanical stimulation are thought to be alarm systems that alert the brain to physical abnormalities. When we experience unpleasant feelings in infected or traumatized tissues, our awareness is directed to the afflicted region, prompting activities such as resting or licking the tissue. Despite extensive research into the molecular biology of nociceptors, it was unclear whether their role was limited to the generation and transmission of unpleasant feelings or whether they actively modulate the pathogenesis of infected or traumatized tissues. Recently, it has become clear how the sensory and immune systems interact with one another and share similar receptors and ligands to modify the pathogenesis of various diseases. In this paper, we summarize the mechanisms of crosstalk between the sensory and immune systems and the impact of this new interdisciplinary field, which should be dubbed 'senso-immunology,' on medical science.

Meningeal P2X7 Signaling Mediates Migraine-Related Intracranial Mechanical Hypersensitivity.

Cortical spreading depolarization (CSD) is a key pathophysiological event that underlies visual and sensory auras in migraine. CSD is also thought to drive the headache phase in migraine by promoting the activation and mechanical sensitization of trigeminal primary afferent nociceptive neurons that innervate the cranial meninges. The factors underlying meningeal nociception in the wake of CSD remain poorly understood but potentially involve the parenchymal release of algesic mediators and damage-associated molecular patterns, particularly ATP. Here, we explored the role of ATP-P2X purinergic receptor signaling in mediating CSD-evoked meningeal afferent activation and mechanical sensitization. Male rats were subjected to a single CSD episode. In vivo, extracellular single-unit recording was used to measure meningeal afferent ongoing activity changes. Quantitative mechanical stimuli using a servomotor force-controlled stimulator assessed changes in the afferent's mechanosensitivity. Manipulation of meningeal P2X receptors was achieved via local administration of pharmacological agents. Broad-spectrum P2X receptor inhibition, selective blockade of the P2X7 receptor, and its related Pannexin 1 channel suppressed CSD-evoked afferent mechanical sensitization but did not affect the accompanying afferent activation response. Surprisingly, inhibition of the pronociceptive P2X2/3 receptor did not affect the activation or sensitization of meningeal afferents post-CSD. P2X7 signaling underlying afferent mechanosensitization was localized to the meninges and did not affect CSD susceptibility. We propose that meningeal P2X7 and Pannexin 1 signaling, potentially in meningeal macrophages or neutrophils, mediates the mechanical sensitization of meningeal afferents, which contributes to migraine pain by exacerbating the headache during normally innocuous physical activities.SIGNIFICANCE STATEMENT Activation and sensitization of meningeal afferents play a key role in migraine headache, but the underlying mechanisms remain unclear. Here, using a rat model of migraine with aura involving cortical spreading depolarization (CSD), we demonstrate that meningeal purinergic P2X7 signaling and its related Pannexin 1 pore, but not nociceptive P2X2/3 receptors, mediate prolonged meningeal afferent sensitization. Additionally, we show that meningeal P2X signaling does not contribute to the increased afferent ongoing activity in the wake of CSD. Our finding points to meningeal P2X7 signaling as a critical mechanism underlying meningeal nociception in migraine, the presence of distinct mechanisms underlying the activation and sensitization of meningeal afferents in migraine, and highlight the need to target both processes for effective migraine therapy.