Innocuous Mechanical Stimuli Research Articles

Topical capsaicin modulates the two-point discrimination threshold-Modulation depends on stimulation modality and intensity.

Spatial acuity concerns the ability to localize and discriminate sensory input and is often tested using the two-point discrimination threshold (2PDT). Sensitization of the pain system can affect the spatial acuity, but it is unclear how 2PDTs of different testing modalities are affected. The aim was to investigate if the 2PDTs for mechanical and heat stimulation at different intensities were modulated by topical capsaicin sensitization. 30 healthy subjects were divided into either a capsaicin or a placebo group. The 2PDT was tested using two different modalities, mechanical and thermal (laser) delivered at innocuous and noxious intensities. The 2PDT were determined at baseline and re-assessed 48 h later. In the follow-up session, the subjects either had a capsaicin patch (8%) or placebo patch placed in the testing area for 30 min before re-testing the 2PDT. The 2PDT was highly dependent on stimulation modality and intensity. The lowest 2PDT was found for innocuous mechanical stimuli (40.0 mm, 95% CI 38.1-41.9 mm), and the highest 2PDT was found for innocuous thermal stimuli (81.7 mm, 95% CI 73.9-89.5 mm). Topical capsaicin generally increased the 2PDT, but this was only significant for innocuous mechanical stimuli. The perceived intensity of the stimuli was increased following capsaicin and was generally higher for noxious stimuli than for innocuous stimuli (ANOVA, p < 0.001). This study showed that capsaicin provoked pain sensitization increased the 2PDT. The 2PDT tested using innocuous mechanical stimuli showed less variable results indicating that this test is most suitable to detect this aspect of spatial acuity. This study investigated how the two-point discrimination threshold (2PDT) can be modulated by topical capsaicin. The 2PDT was assessed for two different modalities (thermal and mechanical) and for two different intensities (innocuous and noxious) before and after capsaicin. The results showed that the 2PDT was generally impaired following capsaicin, but this was only significant for mechanical innocuous stimuli. Furthermore, it was shown that mechanical innocuous stimuli assessed the 2PDT with lower variability than other combinations.

High-Fat Diet-Induced Blood-Brain Barrier Dysfunction: Impact on Allodynia and Motor Coordination in Rats.

The associations among increased pain sensitivity, obesity, and systemic inflammation have not been described as related to BBB dysfunctions. To analyze the metabolic, behavioral, and inflammatory effects of a high-fat diet (HFD) and ultrastructural modifications in brain regions, we used an in vivo experimental model. Adult male Wistar rats were randomly assigned to one of two conditions, an ad libitum control group or an HFD (60%)-fed group, for eight weeks. At the end of the protocol, glucose and insulin tolerance tests were performed. Additionally, we analyzed the response to a normally innocuous mechanical stimulus and changes in motor coordination. At the end of the protocol, HFD-fed rats presented increased HOMA-IR and metabolic syndrome (MetS) prevalence. HFD-fed rats also developed an increased nociceptive response to mechanical stimuli and neurological injury, resulting in impaired motor function. Hypothalamus and cerebellum neurons from HFD-fed rats presented with nuclear swelling, an absence of nucleoli, and karyolysis. These results reveal that HFD consumption affects vital brain structures such as the cerebellum, hippocampus, and hypothalamus. This, in turn, could be producing neuronal damage, impairing cellular communication, and consequently altering motricity and pain sensitivity. Although direct evidence of a causal link between BBB dysfunction and sensory-motor changes was not observed, understanding the association uncovered in this study could lead to targeted therapeutic strategies.

The role of spinal neurons targeted by corticospinal neurons in central poststroke neuropathic pain.

Central poststroke pain (CPSP) is one of the primary sequelae following stroke, yet its underlying mechanisms are poorly understood. By lesioning the lateral thalamic nuclei, we first established a CPSP model that exhibits mechanical and thermal hypersensitivity. Innocuous mechanical stimuli following the thalamic lesion evoked robust neural activation in somatosensory corticospinal neurons (CSNs), as well as in the deep dorsal horn, where low threshold mechanosensory afferents terminate. In this study, we used viral-based mapping and intersectional functional manipulations to decipher the role of somatosensory CSNs and their spinal targets in the CPSP pathophysiology. We first mapped the post-synaptic spinal targets of lumbar innervating CSNs using an anterograde trans-synaptic AAV1-based strategy and showed these spinal interneurons were activated by innocuous tactile stimuli post-thalamic lesion. Functionally, tetanus toxin-based chronic inactivation of spinal neurons targeted by CSNs prevented the development of CPSP. Consistently, transient chemogenetic silencing of these neurons alleviated established mechanical pain hypersensitivity and innocuous tactile stimuli evoked aversion linked to the CPSP. In contrast, chemogenetic activation of these neurons was insufficient to induce robust mechanical allodynia typically observed in the CPSP. The CSNs and their spinal targets are required but insufficient for the establishment of CPSP hypersensitivity. Our study provided novel insights into the neural mechanisms underlying CPSP and potential therapeutic interventions to treat refractory central neuropathic pain conditions.

Spinal Nmur2-positive Neurons Play a Crucial Role in Mechanical Itch

The dorsal spinal cord is crucial for the transmission and modulation of multiple somatosensory modalities, such as itch, pain, and touch. Despite being essential for the well-being and survival of an individual, itch and pain, in their chronic forms, have increasingly been recognized as clinical problems. Although considerable progress has been made in our understanding of the neurochemical processing of nociceptive and chemical itch sensations, the neural substrate that is crucial for mechanical itch processing is still unclear. Here, using genetic and functional manipulation, we identified a population of spinal neurons expressing neuromedin U receptor 2 (Nmur2+) as critical elements for mechanical itch. We found that spinal Nmur2+ neurons are predominantly excitatory neurons, and are enriched in the superficial laminae of the dorsal horn. Pharmacogenetic activation of cervical spinal Nmur2+ neurons evoked scratching behavior. Conversely, the ablation of these neurons using a caspase-3-based method decreased von Frey filament-induced scratching behavior without affecting responses to other somatosensory modalities. Similarly, suppressing the excitability of cervical spinal Nmur2+ neurons via the overexpression of functional Kir2.1 potassium channels reduced scratching in response to innocuous mechanical stimuli, but not to pruritogen application. At the lumbar level, pharmacogenetic activation of these neurons evoked licking and lifting behaviors. However, ablating these neurons did not affect the behavior associated with acute pain. Thus, these results revealed the crucial role of spinal Nmur2+ neurons in mechanical itch. Our study provides important insights into the neural basis of mechanical itch, paving the way for developing novel therapies for chronic itch. PerspectiveExcitatory Nmur2+ neurons in the superficial dorsal spinal cord are essential for mechanical but not chemical itch information processing. These spinal Nmur2+ neurons represent a potential cellular target for future therapeutic interventions against chronic itch. Spinal and supraspinal Nmur2+ neurons may play different roles in pain signal processing.

Transcriptional profiling of dental sensory and proprioceptive trigeminal neurons using single-cell RNA sequencing

Dental primary afferent (DPA) neurons and proprioceptive mesencephalic trigeminal nucleus (MTN) neurons, located in the trigeminal ganglion and the brainstem, respectively, are essential for controlling masticatory functions. Despite extensive transcriptomic studies on various somatosensory neurons, there is still a lack of knowledge about the molecular identities of these populations due to technical challenges in their circuit-validated isolation. Here, we employed high-depth single-cell RNA sequencing (scRNA-seq) in combination with retrograde tracing in mice to identify intrinsic transcriptional features of DPA and MTN neurons. Our transcriptome analysis revealed five major types of DPA neurons with cell type-specific gene enrichment, some of which exhibit unique mechano-nociceptive properties capable of transmitting nociception in response to innocuous mechanical stimuli in the teeth. Furthermore, we discovered cellular heterogeneity within MTN neurons that potentially contribute to their responsiveness to mechanical stretch in the masseter muscle spindles. Additionally, DPA and MTN neurons represented sensory compartments with distinct molecular profiles characterized by various ion channels, receptors, neuropeptides, and mechanoreceptors. Together, our study provides new biological insights regarding the highly specialized mechanosensory functions of DPA and MTN neurons in pain and proprioception.

A glutamatergic DRN–VTA pathway modulates neuropathic pain and comorbid anhedonia-like behavior in mice

Chronic pain causes both physical suffering and comorbid mental symptoms such as anhedonia. However, the neural circuits and molecular mechanisms underlying these maladaptive behaviors remain elusive. Here using a mouse model, we report a pathway from vesicular glutamate transporter 3 neurons in the dorsal raphe nucleus to dopamine neurons in the ventral tegmental area (VGluT3DRN→DAVTA) wherein population-level activity in response to innocuous mechanical stimuli and sucrose consumption is inhibited by chronic neuropathic pain. Mechanistically, neuropathic pain dampens VGluT3DRN → DAVTA glutamatergic transmission and DAVTA neural excitability. VGluT3DRN → DAVTA activation alleviates neuropathic pain and comorbid anhedonia-like behavior (CAB) by releasing glutamate, which subsequently promotes DA release in the nucleus accumbens medial shell (NAcMed) and produces analgesic and anti-anhedonia effects via D2 and D1 receptors, respectively. In addition, VGluT3DRN → DAVTA inhibition produces pain-like reflexive hypersensitivity and anhedonia-like behavior in intact mice. These findings reveal a crucial role for VGluT3DRN → DAVTA → D2/D1NAcMed pathway in establishing and modulating chronic pain and CAB.

Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception.

Calcium (Ca2+) plays a pivotal role in modulating neuronal-mediated responses to modality-specific sensory stimuli. Recent studies in Drosophila reveal class III (CIII) multidendritic (md) sensory neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Functional analyses revealed CIII-mediated multimodal behavioral output is dependent upon activation levels with stimulus-evoked Ca2+ displaying relatively low vs. high intracellular levels in response to gentle touch vs. noxious cold, respectively. However, the mechanistic bases underlying modality-specific differential Ca2+ responses in CIII neurons remain incompletely understood. We hypothesized that noxious cold-evoked high intracellular Ca2+ responses in CIII neurons may rely upon Ca2+ induced Ca2+ release (CICR) mechanisms involving transient receptor potential (TRP) channels and/or metabotropic G protein coupled receptor (GPCR) activation to promote cold nociceptive behaviors. Mutant and/or CIII-specific knockdown of GPCR and CICR signaling molecules [GABAB-R2, Gαq, phospholipase C, ryanodine receptor (RyR) and Inositol trisphosphate receptor (IP3R)] led to impaired cold-evoked nociceptive behavior. GPCR mediated signaling, through GABAB-R2 and IP3R, is not required in CIII neurons for innocuous touch evoked behaviors. However, CICR via RyR is required for innocuous touch-evoked behaviors. Disruptions in GABAB-R2, IP3R, and RyR in CIII neurons leads to significantly lower levels of cold-evoked Ca2+ responses indicating GPCR and CICR signaling mechanisms function in regulating Ca2+ release. CIII neurons exhibit bipartite cold-evoked firing patterns, where CIII neurons burst during rapid temperature change and tonically fire during steady state cold temperatures. GABAB-R2 knockdown in CIII neurons resulted in disorganized firing patterns during cold exposure. We further demonstrate that application of GABA or the GABAB specific agonist baclofen potentiates cold-evoked CIII neuron activity. Upon ryanodine application, CIII neurons exhibit increased bursting activity and with CIII-specific RyR knockdown, there is an increase in cold-evoked tonic firing and decrease in bursting. Lastly, our previous studies implicated the TRPP channel Pkd2 in cold nociception, and here, we show that Pkd2 and IP3R genetically interact to specifically regulate cold-evoked behavior, but not innocuous mechanosensation. Collectively, these analyses support novel, modality-specific roles for metabotropic GABAergic signaling and CICR mechanisms in regulating intracellular Ca2+ levels and cold-evoked behavioral output from multimodal CIII neurons.

Peripheral endomorphins drive mechanical alloknesis under the enzymatic control of CD26/DPPIV

Mechanical alloknesis (or innocuous mechanical stimuli-evoked itch) often occurs in dry skin-based disorders such as atopic dermatitis and psoriasis. However, the molecular and cellular mechanisms underlying mechanical alloknesis remain unclear. We recently reported the involvement of CD26 in the regulation of psoriatic itch. This molecule exhibits dipeptidyl peptidase IV (DPPIV) enzyme activity and exerts its biologic effects by processing various substances, including neuropeptides. The aim of the present study was to investigate the peripheral mechanisms of mechanical alloknesis by using CD26/DPPIV knockout (CD26KO) mice. We applied innocuous mechanical stimuli to CD26KO or wild-type mice. The total number of scratching responses was counted as the alloknesis score. Immunohistochemical and behavioral pharmacologic analyses were then performed to examine the physiologic activities of CD26/DPPIV or endomorphins (EMs), endogenous agonists of μ-opioid receptors. Mechanical alloknesis was more frequent in CD26KO mice than in wild-type mice. The alloknesis score in CD26KO mice was significantly reduced by the intradermal administration of recombinant DPPIV or naloxone methiodide, a peripheral μ-opioid receptor antagonist, but not by that of mutant DPPIV without enzyme activity. EMs (EM-1 and EM-2), selective ligands for μ-opioid receptors, are substrates for DPPIV. Immunohistochemically, EMs were located in keratinocytes, fibroblasts, and peripheral sensory nerves. Behavioral analyses revealed that EMs preferentially provoked mechanical alloknesis over chemical itch. DPPIV-digested forms of EMs did not induce mechanical alloknesis. The present results suggest that EMs induce mechanical alloknesis at the periphery under the enzymatic control of CD26/DPPIV.

Optogenetic Manipulations of Amygdala Neurons Modulate Spinal Nociceptive Processing and Behavior Under Normal Conditions and in an Arthritis Pain Model.

The amygdala is an important neural substrate for the emotional–affective dimension of pain and modulation of pain. The central nucleus (CeA) serves major amygdala output functions and receives nociceptive and affected–related information from the spino-parabrachial and lateral–basolateral amygdala (LA–BLA) networks. The CeA is a major site of extra–hypothalamic expression of corticotropin releasing factor (CRF, also known as corticotropin releasing hormone, CRH), and amygdala CRF neurons form widespread projections to target regions involved in behavioral and descending pain modulation. Here we explored the effects of modulating amygdala neurons on nociceptive processing in the spinal cord and on pain-like behaviors, using optogenetic activation or silencing of BLA to CeA projections and CeA–CRF neurons under normal conditions and in an acute pain model. Extracellular single unit recordings were made from spinal dorsal horn wide dynamic range (WDR) neurons, which respond more strongly to noxious than innocuous mechanical stimuli, in normal and arthritic adult rats (5–6 h postinduction of a kaolin/carrageenan–monoarthritis in the left knee). For optogenetic activation or silencing of CRF neurons, a Cre–inducible viral vector (DIO–AAV) encoding channelrhodopsin 2 (ChR2) or enhanced Natronomonas pharaonis halorhodopsin (eNpHR3.0) was injected stereotaxically into the right CeA of transgenic Crh–Cre rats. For optogenetic activation or silencing of BLA axon terminals in the CeA, a viral vector (AAV) encoding ChR2 or eNpHR3.0 under the control of the CaMKII promoter was injected stereotaxically into the right BLA of Sprague–Dawley rats. For wireless optical stimulation of ChR2 or eNpHR3.0 expressing CeA–CRF neurons or BLA–CeA axon terminals, an LED optic fiber was stereotaxically implanted into the right CeA. Optical activation of CeA–CRF neurons or of BLA axon terminals in the CeA increased the evoked responses of spinal WDR neurons and induced pain-like behaviors (hypersensitivity and vocalizations) under normal condition. Conversely, optical silencing of CeA–CRF neurons or of BLA axon terminals in the CeA decreased the evoked responses of spinal WDR neurons and vocalizations, but not hypersensitivity, in the arthritis pain model. These findings suggest that the amygdala can drive the activity of spinal cord neurons and pain-like behaviors under normal conditions and in a pain model.

Inhibition of angiotensin converting enzyme induces mechanical allodynia through increasing substance P expression in mice

Although emerging evidence shows that angiotensin converting enzyme (ACE) is associated with pain, it is not clear whether inhibition of ACE could affect to nociceptive transmission and which mediators are involved in this process. Here we investigated whether administration of the ACE inhibitors, captopril and enalapril increases the expression of substance P (SP) and whether this increase contributes to the induction of mechanical allodynia in mice. ACE was expressed in the lumbar dorsal root ganglion (DRG) and the superficial dorsal horn (SDH) region of the spinal cord in mice. Either intraperitoneal or intrathecal administration of the ACE inhibitors, captopril and enalapril for 10 days significantly increased the paw withdrawal frequency to innocuous mechanical stimuli and the levels of SP in both the lumbar DRG and the SDH region of the spinal cord dorsal horn. In addition, intraperitoneal administration of the SP receptor (neurokinin-1 receptor) antagonist, L-733,060 suppressed mechanical allodynia that was induced by pretreatment of captopril and enalapril. Intraplantar administration of SP for 3 days induces mechanical allodynia, and this effect was reduced by exogenous ACE administration. These findings demonstrate that inhibition of ACE increases the levels of SP in both the lumbar DRG and spinal cord dorsal horn, ultimately contributing to the induction of mechanical allodynia in mice.

The two-point discrimination threshold depends both on the stimulation noxiousness and modality.

The two-point discrimination threshold (2PDT) has been used to investigate the integration of sensory information, especially in relation to spatial acuity. The 2PDT has been investigated for both innocuous mechanical stimuli and noxious thermal stimuli; however, previous studies used different stimulation modalities to compare innocuous and noxious stimuli. This study investigated the 2PDT in 19 healthy participants, using both thermal (laser) and mechanical stimulation modalities. Within each modality, both innocuous and noxious intensities were applied. Concurrent point stimuli were applied to the right volar forearm, with separation distances of 0-120mm, in steps of 10mm. 0mm corresponds to a single point. Following each stimulus, the participants indicated the number of perceived points (1 or 2) and the perceived intensity (NRS: 0: no perception, 3: pain threshold, 10: maximum pain). The order of stimulation modality, intensity and distance was randomized. The 2PDT for innocuous and noxious mechanical stimuli was 34.7mm and 47.1mm, respectively. For thermal stimuli, the 2PDT was 80.5mm for innocuous stimuli and 66.9mm for noxious stimuli. The average NRS for thermal stimuli was 1.6 for innocuous intensities and 4.0 for noxious intensities, while for mechanical stimuli, the average NRS was 0.9 for innocuous intensities and 3.6 for noxious intensities. This study showed that the 2PDT highly depends on both stimulation modality and intensity. Within each modality, noxious intensities modulates the 2PDT differently, i.e., noxious intensities lowers the 2PDT for thermal stimuli, but increases the 2PDT for mechanical stimuli.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch.

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFP mice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.SIGNIFICANCE STATEMENT Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

Combination of a Gellan Gum-Based Hydrogel With Cell Therapy for the Treatment of Cervical Spinal Cord Injury.

Cervical spinal cord trauma represents more than half of the spinal cord injury (SCI) cases worldwide. Respiratory compromise, as well as severe limb motor deficits, are among the main consequences of cervical lesions. In the present work, a Gellan Gum (GG)-based hydrogel modified with GRGDS peptide, together with adipose tissue-derived stem/stromal cells (ASCs) and olfactory ensheathing cells (OECs), was used as a therapeutic strategy after a C2 hemisection SCI in rats. Hydrogel or cells alone, and a group without treatment, were also tested. Four weeks after injury, compound muscle action potentials (CMAPs) were performed to assess functional phrenic motor neuron (PhMN) innervation of the diaphragm; no differences were observed amongst groups, confirming that the PhMN pool located between C3 and C5 was not affected by the C2 injury or by the treatments. In the same line, the vast majority of diaphragmatic neuromuscular junctions remained intact. Five weeks post-injury, inspiratory bursting of the affected ipsilateral hemidiaphragm was evaluated through EMG recordings of dorsal, medial and ventral subregions of the muscle. All treatments significantly increased EMG amplitude at the ventral portion in comparison to untreated animals, but only the combinatorial group presented increased EMG amplitude at the medial portion of the hemidiaphragm. No differences were observed in forelimb motor function, neither in markers for axonal regrowth (neuronal tracers), astrogliosis (GFAP) and inflammatory cells (CD68). Moreover, using Von Frey testing of mechanical allodynia, it was possible to find a significant effect of the group combining hydrogel and cells on hypersensitivity; rats with a SCI displayed an increased response of the contralateral forelimb to a normally innocuous mechanical stimulus, but after treatment with the combinatorial therapy this behavior was reverted almost to the levels of uninjured controls. These results suggest that our therapeutic approach may have beneficial effects on both diaphragmatic recovery and sensory function.

New approach for investigating neuropathic allodynia by optogenetics.

Mechanical allodynia is pain caused by normally innocuous mechanical stimuli and is a cardinal and intractable symptom of neuropathic pain. Roles of low-threshold mechanoreceptors (LTMRs), including Aβ fibers, in mechanical allodynia have previously been proposed, but the necessity and sufficiency of LTMRs in allodynia have not been fully determined. Recent technological advances have made it possible to achieve subpopulation-specific ablation, silencing or stimulation, and to dissect and elucidate complex neuronal circuitry. Recent studies using an optogenetic approach have shown that activation of LTMRs, including Aβ fibers that genetically express channelrhodopsin-2, by illuminating blue light to the skin elicit morphine-resistant withdrawal behaviors after nerve damage. Whole-cell recording has revealed that optical Aβ stimulation after nerve injury causes excitation of lamina I dorsal horn neurons, which are normally silent by this stimulation. Moreover, Aβ stimulation after nerve injury results in activation of central amygdaloid neurons and produces aversive behaviors. In summary, these findings indicate that optogenetics is a powerful approach for investigating LTMR-derived pain (resembling mechanical allodynia) with sensory and emotional features after nerve injury and for discovering novel and effective drugs to treat neuropathic pain.

Tiling and somatotopic alignment of mammalian low-threshold mechanoreceptors

Innocuous mechanical stimuli acting on the skin are detected by sensory neurons, known as low-threshold mechanoreceptors (LTMRs). LTMRs are classified based on their response properties, action potential conduction velocity, rate of adaptation to static indentation of the skin, and terminal anatomy. Here, we report organizational properties of the cutaneous and central axonal projections of the five principal hairy skin LTMR subtypes. We find that axons of neurons within a particular LTMR class are largely nonoverlapping with respect to their cutaneous end organs (e.g., hair follicles), with Aβ rapidly adapting-LTMRs being the sole exception. Individual neurons of each LTMR class are mostly nonoverlapping with respect to their associated hair follicles, with the notable exception of C-LTMRs, which exhibit multiple branches that redundantly innervate individual hair follicles. In the spinal cord, LTMR central projections exhibit rostrocaudal elongation and mediolateral compression, compared with their cutaneous innervation patterns, and these central projections also exhibit a fine degree of homotypic topographic adjacency. These findings thus reveal homotypic tiling of LTMR subtype axonal projections in hairy skin and a remarkable degree of spatial precision of spinal cord axonal termination patterns, suggesting a somatotopically precise tactile encoding capability of the mechanosensory dorsal horn.

Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia

Chronic pain presents a major unmet clinical problem. One of the most common symptoms of chronic pain is tactile allodynia, where previously innocuous mechanical stimuli are perceived as painful. To help develop more effective analgesics to treat this condition, a better understanding of the neuronal circuits underlying sensory perception in necessary. We have shown that inhibitory interneurons in the spinal dorsal horn that express parvalbumin (PV) form axoaxonic synapses on to the central terminals of low threshold mechanoreceptive (LTMR) afferents. Here, we define a spinal microcircuit comprising of LTMR afferents, inhibitory PV interneurons, and glutamatergic vertical cells whose axons project to lamina I, that explains how LTMR input can activate pain circuits under pathological conditions. We show that these PV cells receive monosynaptic input from myelinated LTMR afferents from both hairy and glabrous skin. We also determine that these interneurons are the source of axoaxonic synapses on to the central terminals of LTMR afferents that appose vertical cells, forming triadic synaptic arrangements. Furthermore, we provide evidence that these interneurons generate both GABAergic presynaptic inhibition of myelinated LTMR afferents, and postsynaptic inhibition of vertical cells through the release of GABA and glycine. In neuropathic mice, PV cell excitability is reduced, but we find no evidence of structural plasticity. Taken together, these findings show that PV cell-mediated presynaptic inhibition of LTMR afferents and postsynaptic inhibition of vertical cells both play critical roles in the normal perception of mechanical stimuli, and identify this microcircuit as a target for therapeutic intervention to alleviate allodynia.

5-HT2A Receptor-Induced Morphological Reorganization of PKCγ-Expressing Interneurons Gates Inflammatory Mechanical Allodynia in Rat.

Mechanical allodynia, a widespread pain symptom that still lacks effective therapy, is associated with the activation of a dorsally directed polysynaptic circuit within the spinal dorsal horn (SDH) or medullary dorsal horn (MDH), whereby tactile inputs into deep SDH/MDH can gain access to superficial SDH/MDH, eliciting pain. Inner lamina II (IIi) interneurons expressing the γ isoform of protein kinase C (PKCγ+) are key elements for allodynia circuits, but how they operate is still unclear. Combining behavioral, ex vivo electrophysiological, and morphological approaches in an adult rat model of facial inflammatory pain (complete Freund's adjuvant, CFA), we show that the mechanical allodynia observed 1 h after CFA injection is associated with the following (1) sensitization (using ERK1/2 phosphorylation as a marker) and (2) reduced dendritic arborizations and enhanced spine density in exclusively PKCγ+ interneurons, but (3) depolarized resting membrane potential (RMP) in all lamina IIi PKCγ+/PKCγ- interneurons. Blocking MDH 5HT2A receptors (5-HT2AR) prevents facial mechanical allodynia and associated changes in the morphology of PKCγ+ interneurons, but not depolarized RMP in lamina IIi interneurons. Finally, activation of MDH 5-HT2AR in naive animals is enough to reproduce the behavioral allodynia and morphological changes in PKCγ+ interneurons, but not the electrophysiological changes in lamina IIi interneurons, induced by facial inflammation. This suggests that inflammation-induced mechanical allodynia involves strong morphological reorganization of PKCγ+ interneurons via 5-HT2AR activation that contributes to open the gate for transmission of innocuous mechanical inputs to superficial SDH/MDH pain circuitry. Preventing 5-HT2AR-induced structural plasticity in PKCγ+ interneurons might represent new avenues for the specific treatment of inflammation-induced mechanical hypersensitivity.SIGNIFICANCE STATEMENT Inflammatory or neuropathic pain syndromes are characterized by pain hypersensitivity such as mechanical allodynia (pain induced by innocuous mechanical stimuli). It is generally assumed that mechanisms underlying mechanical allodynia, because they are rapid, must operate at only the level of functional reorganization of spinal or medullary dorsal horn (MDH) circuits. We discovered that facial inflammation-induced mechanical allodynia is associated with rapid and strong structural remodeling of specifically interneurons expressing the γ isoform of protein kinase C (PKCγ) within MDH inner lamina II. Moreover, we elucidated a 5-HT2A receptor to PKCγ/ERK1/2 pathway leading to the behavioral allodynia and correlated morphological changes in PKCγ interneurons. Therefore, descending 5-HT sensitize PKCγ interneurons, a putative "gate" in allodynia circuits, via 5-HT2A receptor-induced structural reorganization.

Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch.

The somatosensory system relays many signals ranging from light touch to pain and itch. Touch is critical to spatial awareness and communication. However, in disease states, innocuous mechanical stimuli can provoke pathologic sensations such as mechanical itch (alloknesis). The molecular and cellular mechanisms that govern this conversion remain unknown. We found that in mice, alloknesis in aging and dry skin is associated with a loss of Merkel cells, the touch receptors in the skin. Targeted genetic deletion of Merkel cells and associated mechanosensitive Piezo2 channels in the skin was sufficient to produce alloknesis. Chemogenetic activation of Merkel cells protected against alloknesis in dry skin. This study reveals a previously unknown function of the cutaneous touch receptors and may provide insight into the development of alloknesis.

Transient receptor potential vanilloid 4 (TRPV4) expression on the nerve fibers of human dental pulp is upregulated under inflammatory condition

ObjectiveTransient receptor potential vanilloid 4 (TRPV4) has been considered as a mechano-, thermo- and osmo-receptor. Under inflammatory conditions in dental pulp, teeth can become sensitive upon exposure to a variety of innocuous stimuli. The objective of the present study was to investigate the expression of the TRPV4 channel on nerve fibers in human dental pulp of non-symptomatic and symptomatic teeth associated with inflammatory conditions. DesignDental pulp from extracted human permanent teeth was processed for fluorescence immunohistochemistry. Ten asymptomatic (normal) and 10 symptomatic (symptoms associated with pulpitis) teeth were used in this study. Nerve fibers were identified by immunostaining for a marker, protein gene product 9.5, and the cells were counterstained with 4′,6-diamidino-2-phenylindole. An anti-TRPV4 antibody was used to trace TRPV4 expression. ResultsTRPV4 expression was co-localized with the nerve fiber marker. Immunoreactivity for TRPV4 was more intense (p < 0.05) in the nerves of symptomatic teeth than those of normal teeth. The number of co-localization spots was increased significantly (p < 0.05) in the dental pulp of symptomatic teeth compared with that of asymptomatic (normal) teeth. ConclusionsThere is expression of TRPV4 channels on the nerve fibers of human dental pulp. Our findings suggest upregulation of TRPV4 expression under inflammatory conditions in the pulp. The upregulation of TRPV4 channels may be associated with the exaggerated response of dental pulp to innocuous mechanical, thermal and osmotic stimuli under inflammatory conditions.