Activity-dependent extracellular potassium changes in unmyelinated versus myelinated areas in olfactory regions of the isolated female guinea-pig brain.

The potassium released in the extracellular space during neuronal activity is rapidly removed by glia and neurons to maintain tissue homeostasis. Oligodendrocyte-derived myelin axonal coating contributes to potassium buffering and is therefore crucial to control brain excitability. We studied activity-dependent extracellular potassium ([K+]o) changes in the piriform cortex (PC), a region that features highly segregated bundles of myelinated and unmyelinated fibers. Four-aminopyridine (4AP; 50 µM) treatment or patterned high-frequency stimulations (hfST) were utilized to generate [K+]o changes measured with potassium-sensitive electrodes in the myelinated lateral olfactory tract (LOT), in the unmyelinated PC layer I and in the myelinated deep PC layers in the ex vivo isolated guinea-pig brain. Seizure-like events induced by 4AP are initiated by the abrupt [K+]o rise in the layer I formed by unmyelinated fibers (Uva et al., 2017). Larger [K+]o shifts occurred in unmyelinated layers compared to the myelinated LOT. LOT hfST that mimicks pre-seizure discharges also generated higher [K+]o changes in unmyelinated PC layer I than in LOT and deep PC layers. The treatment with the Kir4.1 potassium channel blocker BaCl2 (100 µM) enhanced the [K+]o changes generated by hfST in myelinated structures. Our data show that activity-dependent [K+]o changes are intrinsically different in myelinated vs unmyelinated cortical regions. The larger [K+]o shifts generated in unmyelinated structures may represent a vehicle for seizure generation.

Identifying vagal bronchopulmonary afferents mediating cough response to inhaled sulfur dioxide in mice.

Sulfur dioxide (SO2), a common environmental and industrial air pollutant, possesses a potent effect in eliciting cough reflex, but the primary type of airway sensory receptors involved in its tussive action has not been clearly identified. This study was carried out to determine the relative roles of three major types of vagal bronchopulmonary afferents [slowly adapting receptors (SARs), rapidly adapting receptors (RARs), and C-fibers] in regulating the cough response to inhaled SO2. Our results showed that inhalation of SO2 (300 or 600 ppm for 8 min) evoked an abrupt and intense stimulatory effect on bronchopulmonary C-fibers, which continued for the entire duration of inhalation challenge and returned toward the baseline in 1-2 min after resuming room air-breathing in anesthetized and mechanically ventilated mice. In stark contrast, the same SO2 inhalation challenge generated a distinct and consistent inhibitory effect on both SARs and phasic RARs; their phasic discharges synchronized with respiratory cycles during the baseline (breathing room air) began to decline progressively within 1-3 min after the onset of SO2 inhalation, ceased completely before termination of the 8-min inhalation challenge, and then slowly returned toward the baseline after >40 min. In a parallel study in awake mice, inhalation of SO2 at the same concentration and duration as that in the nerve recording experiments evoked cough responses in a pattern and time course similar to that observed in the C-fiber responses. Based on these results, we concluded that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough response to inhaled SO2.NEW & NOTEWORTHY This study demonstrated that inhalation of a high concentration of sulfur dioxide, an irritant gas and common air pollutant, completely and reversibly inhibited the neural activities of both slowly adapting receptor and rapidly adapting receptor, two major types of mechanoreceptors in the lungs with their activities conducted by myelinated fibers. Furthermore, the results of this study suggested that stimulation of vagal bronchopulmonary C-fibers is primarily responsible for triggering the cough reflex responses to inhaled sulfur dioxide.

Hush, little baby: The role of C-tactile afferents in babywearing infants with neonatal opioid withdrawal.

Social touch through infant holding, skin-to-skin contact, and infant carrying (babywearing) decreases infant distress and promotes secure attachment. Unknown is the extent to which these effects are the result of the activation of C-Tactile afferents (CTs), the constellation of nerve fibers associated with affective touch, primarily located in the head and trunk of the body. The purpose of the present study was to compare dynamic touch (CTs activated) to static touch (CTs less activated) during a babywearing procedure among infants experiencing Neonatal Opioid Withdrawal Syndrome (NOWS). NOWS is a spectrum of clinical symptoms, including elevated heart rate (HR), associated with withdrawal from intrauterine opioid exposure. We hypothesized that stroking an infant's head during babywearing would amplify the pleasurable effect of babywearing as measured by changes in infant HR. Twenty-nine infants in a Neonatal Intensive Care Unit (NICU) in the Southwestern USA were worn in an infant carrier starting at five days old (M = 5.4, SD = 2.6; 46.2 % White, 26.9 % Latinx, 11.5 % Native American) and physiological readings were conducted daily; heart rates of infants and caregivers were taken every 15-seconds for 5-minutes, before, during, and after babywearing (30 min per phase). Each day infants alternated (randomly) in a static touch (hands-free babywearing) or dynamic touch condition (stroking the top of the infants' head at a velocity of 3 cm/s while babywearing). On average, infants completed 3 dynamic and 3 static babywearing sessions. Hospital and research staff participated in babywearing when a parent was not available (31.0 % of infants were exclusively worn by volunteers, 27.6 % were exclusively worn by parents). We analyzed the data using Hierarchical Linear Models due to the 3-level nested design (N = 29 infants, N = 191 readings, N = 11,974 heart rates). Compared to baseline (infant calm/asleep and without contact), infant's HRs significantly declined during and after babywearing, controlling for pharmacological treatment. These effects were significantly stronger during the dynamic touch condition (reduction in HR of 11.17 bpm) compared to the static touch condition (reduction in HR of 3.74 bpm). These effects did not significantly vary by wearer (mother, father, volunteer). However, differences between the dynamic and static conditions were significantly stronger in earlier babywearing sessions, potentially indicating a learning effect. There was evidence for a calming effect among caregivers as well, particularly in the dynamic touch condition, when caregivers were engaged in active touch. Activation of CTs appears to be an important mechanism in the physiological benefits of babywearing and in the symbiotic role of caregiver-infant attachment.

Eliciting the rubber hand illusion by the activation of nociceptive C and Aδ fibers.

ABSTRACT: The coherent perceptual experience of one's own body depends on the processing and integration of signals from multiple sensory modalities, including vision, touch, and proprioception. Although nociception provides critical information about damage to the tissues of one's body, little is known about how nociception contributes to own-body perception. A classic experimental approach to investigate the perceptual and neural mechanisms involved in the multisensory experience of one's own body is the rubber hand illusion (RHI). During the RHI, people experience a rubber hand as part of their own body (sense of body ownership) caused by synchronized stroking of the rubber hand in the participant's view and the hidden participant's real hand. We examined whether the RHI can be elicited by visual and "pure" nociceptive stimulation, ie, without tactile costimulation, and if so, whether it follows the basic perceptual rules of the illusion. In 6 separate experiments involving a total of 180 healthy participants, we used a Nd:YAP laser stimulator to specifically target C and Aδ fibers in the skin and compared the illusion condition (congruent visuonociceptive stimulation) to control conditions of incongruent visuonociceptive, incongruent visuoproprioceptive, and no nociceptive stimulation. The illusion was quantified through direct (questionnaire) and indirect (proprioceptive drift) behavioral measures. We found that a nociceptive rubber hand illusion (N-RHI) could be elicited and that depended on the spatiotemporal congruence of visuonociceptive signals, consistent with basic principles of multisensory integration. Our results suggest that nociceptive information shapes multisensory bodily awareness and contributes to the sense of body ownership.

Selective intrafascicular stimulation of myelinated and unmyelinated nerve fibers through a longitudinal electrode: A computational study.

Carbon nanotube (CNT) fiber electrodes have demonstrated exceptional spatial selectivity and sustained reliability in the context of intrafascicular electrical stimulation, as evidenced through rigorous animal experimentation. A significant presence of unmyelinated C fibers, known to induce uncomfortable somatosensory experiences, exists within peripheral nerves. This presence poses a considerable challenge to the excitation of myelinated Aß fibers, which are crucial for tactile sensation. To achieve nuanced tactile sensory feedback utilizing CNT fiber electrodes, the selective stimulation of Aß sensory afferents emerges as a critical factor. In confronting this challenge, the present investigation sought to refine and apply a rat sciatic-nerve model leveraging the capabilities of the COMSOL-NEURON framework. This approach enables a systematic evaluation of the influence exerted by stimulation parameters and electrode geometry on the activation dynamics of both myelinated Aß and unmyelinated C fibers. The findings advocate for the utilization of current pulses featuring a pulse width of 600 µs, alongside the deployment of CNT fibers characterized by a diminutive diameter of 10 µm, with an exclusively exposed cross-sectional area, to facilitate reduced activation current thresholds. Comparative analysis under monopolar and bipolar electrical stimulation conditions revealed proximate activation thresholds, albeit with bipolar stimulation exhibiting superior fiber selectivity relative to its monopolar counterpart. Concerning pulse waveform characteristics, the adoption of an anodic-first biphasic stimulation modality is favored, taking into account the dual criteria of activation threshold and fiber selectivity optimization. Consequently, this investigation furnishes an efficacious stimulation paradigm for the selective activation of touch-related nerve fibers, alongside provisioning a comprehensive theoretical foundation for the realization of natural tactile feedback within the domain of prosthetic hand applications.

Slow touch and ultrafast pain fibres: Revisiting peripheral nerve classification.

One hundred years ago, Erlanger and Gasser demonstrated that conduction velocity is correlated with the diameter of a peripheral nerve axon. Later, they also demonstrated that the functional role of the axon is related to its diameter: touch is signalled by large-diameter axons, whereas pain and temperature are signalled by small-diameter axons. Certain discoveries in recent decades prompt a modification of this canonical classification. Here, we review the evidence for unmyelinated (C) fibres signalling touch at a slow conduction velocity and likely contributing to affective aspects of tactile information. We also review the evidence for large-diameter Aß afferents signalling pain at ultrafast conduction velocity and likely contributing to the rapid nociceptive withdrawal reflex. These discoveries imply that conduction velocity is not as clear-cut an indication of the functional role of the axon as previously thought. We finally suggest that a future taxonomy of the peripheral afferent nervous system might be based on the combination of the axons molecular expression and electrophysiological response properties.

Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aß-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aß-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aß-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

Multiple averaged records to identify Aδ-fibers in sensory nerves.

BACKGROUND: Existing methods identify only ≈10 Aδ-fibers in human sensory nerves per recording. This study examines methods to increase the detection of Aδ-fibers. NEW METHOD: Two to 20 averages of 500 replicate responses to epidermal nerve stimulation are obtained. Pairs of different averages are constructed. Each pair is analyzed with algorithms applied to amplitude and frequency to detect replication of responses to stimulation as "simultaneous similarities in two averages" (SS2AVs) at ≥99.5th percentile of control. In a pair of averages the latencies of amplitude and frequency SS2AVs for the same response to stimulation may differ by ≤0.25 ms. Therefore, Aδ-fibers are identified by the 0.25 ms moving sum of SS2AV latencies of the pairs of averages. RESULTS: Increasing averages increases pairs of different averages and detection of Aδ-fibers: from 2 to 10 Aδ-fibers with two averages (one pair) to >50 Aδ-fibers with 12-20 averages (66-190 pairs). COMPARISON WITH EXISTING METHOD(S): Existing methods identify ≤10 Aδ-fibers in 10 averages/45 pairs with the medians of amplitude and frequency algorithms applied to all 45 pairs. This study identifies Aδ-fibers (i) by applying these algorithms at the 99.5th percentile of control, (ii) to each pair of averages and (iii) by the 0.25 ms sum of algorithm identified events (SS2AVs) in all pairs. These three changes significantly increase the detection of Aδ-fibers, e.g., in 10 averages/45pairs from 10 to 45. CONCLUSIONS: Three modifications of existing methods can increase the detection of Aδ-fibers to an amount suitable (>50 with ≥12 averages) for statistical comparison of different nerves.

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.

Identification of corneal and intra-epidermal axonal swellings in amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: In patients with amyotrophic lateral sclerosis (ALS), axonal spheroids in motor axons have been identified in post-mortem studies. In this study, axonal spheroids and swellings on C-fibers of ALS patients were investigated using corneal confocal microscopy (CCM) and skin biopsy, respectively. METHODS: Thirty-one ALS patients and 20 healthy subjects were evaluated with CCM to assess corneal nerve-fiber length (CNFL), -fiber density (CNFD), -branch density (CNBD), dendritic cell (DC) density, and axonal spheroids originating from C-fibers (>100 µm2 ). In addition, intraepidermal nerve fiber density (IENFD) and axonal swellings (>1.5 µm) were assessed in skin biopsies obtained from the arms and legs of 22 patients and 17 controls. RESULTS: In ALS patients, IENFD, CNFD, CNFL, and CNBD were not different from controls. The density of DCs and the number of patients with increased DC density were higher in ALS patients than controls (p = .0005 and p = .008). The number of patients with axonal spheroids was higher than controls (p = .03). DISCUSSION: Evaluation of DCs and axonal bulbs in C-fibers of ALS patients could provide insights into pathophysiology or potentially serve as biomarkers in ALS.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

Hypersensitivity of myelinated A-fibers via toll-like receptor 5 promotes mechanical allodynia in tenascin-X-deficient mice associated with Ehlers-Danlos syndrome.

Deficiency of an extracellular matrix glycoprotein tenascin-X (TNX) leads to a human heritable disorder Ehlers-Danlos syndrome, and TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. We previously reported that TNX-deficient (Tnxb-/-) mice exhibit mechanical allodynia and hypersensitivity to myelinated A-fibers. Here, we investigated the pain response of Tnxb-/- mice using pharmacological silencing of A-fibers with co-injection of N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314), a membrane-impermeable lidocaine analog, plus flagellin, a toll-like receptor 5 (TLR5) ligand. Intraplantar co-injection of QX-314 and flagellin significantly increased the paw withdrawal threshold to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aß fiber responses), but not 5 Hz (C fiber responses) in wild-type mice. The QX-314 plus flagellin-induced silencing of Aδ- and Aß-fibers was also observed in Tnxb-/- mice. Co-injection of QX-314 and flagellin significantly inhibited the mechanical allodynia and neuronal activation of the spinal dorsal horn in Tnxb-/- mice. Interestingly, QX-314 alone inhibited the mechanical allodynia in Tnxb-/- mice, and it increased the paw withdrawal threshold to stimuli at frequencies of 250 Hz and 2000 Hz in Tnxb-/- mice, but not in wild-type mice. The inhibition of mechanical allodynia induced by QX-314 alone was blocked by intraplantar injection of a TLR5 antagonist TH1020 in Tnxb-/- mice. These results suggest that mechanical allodynia due to TNX deficiency is caused by the hypersensitivity of Aδ- and Aß-fibers, and it is induced by constitutive activation of TLR5.

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (https://github.com/Microneurography/APTrack). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during in vivo human microneurography, for the first time, and during ex vivo mouse electrophysiological recordings of C-fibers and Aδ-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

What are C-tactile afferents and how do they relate to "affective touch"?

Since their initial discovery in cats, low-threshold C-fiber mechanoreceptors have become a central interest of scientists studying the affective aspects of touch. Their pursuit in humans, here termed C-tactile (CT) afferents, has led to the establishment of a research field referred to as "affective touch", which is differentiated from "discriminative touch". Presently, we review these developments based on an automated semantic analysis of more than 1000 published abstracts as well as empirical evidence and the solicited opinions of leading experts in the field. Our review provides a historical perspective and update of CT research, it reflects on the meaning of "affective touch", and discusses how current insights challenge established views on the relation between CTs and affective touch. We conclude that CTs support gentle, affective touch, but that not every affective touch experience relies on CTs or must necessarily be pleasant. Moreover, we speculate that currently underappreciated aspects of CT signaling will prove relevant for the manner in which these unique fibers support how humans connect both physically and emotionally.

Peripheral neuroplasticity of respiratory chemoreflexes, induced by prenatal nicotinic exposure: Implication for SIDS.

Sudden Infant Death Syndrome (SIDS) occurs during sleep in seemingly healthy infants. Maternal cigarette smoking and hypoxemia during sleep are assumed to be the major causal factors. Depressed hypoxic ventilatory response (dHVR) is observed in infants with high risk of SIDS, and apneas (lethal ventilatory arrest) appear during the fatal episode of SIDS. Disturbance of the respiratory center has been proposed to be involved, but the pathogenesis of SIDS is still not fully understood. Peripherally, the carotid body is critical to generate HVR, and bronchopulmonary and superior laryngeal C-fibers (PCFs and SLCFs) are important for triggering central apneas; however, their roles in the pathogenesis of SIDS have not been explored until recently. There are three lines of recently accumulated evidence to show the disorders of peripheral sensory afferent-mediated respiratory chemoreflexes in rat pups with prenatal nicotinic exposure (a SIDS model) in which acute severe hypoxia leads to dHVR followed by lethal apneas. (1) The carotid body-mediated HVR is suppressed with a reduction of the number and sensitivity of glomus cells. (2) PCF-mediated apneic response is largely prolonged via increased PCF density, pulmonary IL-1ß and serotonin (5-hydroxytryptamine, 5-HT) release, along with the enhanced expression of TRPV1, NK1R, IL1RI and 5-HT3R in pulmonary C-neurons to strengthen these neural responses to capsaicin, a selective stimulant to C-fibers. (3) SLCF-mediated apnea and capsaicin-induced currents in superior laryngeal C-neurons are augmented by upregulation of TRPV1 expression in these neurons. These results, along with hypoxic sensitization/stimulation of PCFs, gain insight into the mechanisms of prenatal nicotinic exposure-induced peripheral neuroplasticity responsible for dHVR and long-lasting apnea during hypoxia in rat pups. Therefore, in addition to the disturbance in the respiratory center, the disorders of peripheral sensory afferent-mediated chemoreflexes may also be involved in respiratory failure and death denoted in SIDS victims.

The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats.

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

The evaluation of small fibers in multiple sclerosis.

BACKGROUND: Dysesthetic or ongoing extremity pain is a common symptom in all multiple sclerosis (MS) types. Although the pathology of the disease is the demyelination of central neurons, the patients may also complain of neuropathic pain in distal extremities that is generally related to A-delta and C fiber dysfunction. It is not known whether thinly myelinated and unmyelinated fibers are affected in MS patients. We aim to investigate the small fiber loss and its length dependency. METHODS: We evaluated the skin biopsy taken from proximal and distal leg of MS patients with neuropathic pain. Six patients with primary progressive MS (PPMS), seven with relapsing-remitting MS (RRMS), seven with secondary progressive MS (SPMS) and as a control group ten age and sex-matched healthy controls were included. Neurological examination, electrophysiological evaluation and DN4 questionnaire were performed. Subsequently, skin punch biopsy from 10 cm above the lateral malleolus and proximal thigh were done. The biopsy samples were stained with PGP9.5 antibody and intraepidermal nerve fiber density (IENFD) was determined. RESULTS: The mean proximal IENFD was 8.58±3.58 fibers/mm among MS patients and 14.72±2.89 fiber/mm among healthy controls (p=0.001). However, the mean distal IENFD did not differ between MS patients and healthy controls (9.26±3.24 and 9.75±1.6 fiber/mm respectively. Although proximal and distal IENFD tends to be lower in MS patients with neuropathic pain, there was no statistically significant difference between MS patients with and without neuropathic pain CONCLUSION: Although MS is a demyelinating disease, unmyelinated fibers can also be affected. Our findings suggest non-length dependent small fiber neuropathy in MS patients.

Human pain ratings to electrical sinusoids increase with cooling through a cold-induced increase in C-fibre excitability.

ABSTRACT: Low-frequency sinusoidal current applied to human skin evokes local axon reflex flare and burning pain, indicative of C-fibre activation. Because topical cooling works well as a local analgesic, we examined the effect of cooling on human pain ratings to sinusoidal and rectangular profiles of constant current stimulation. Unexpectedly, pain ratings increased upon cooling the skin from 32 to 18°C. To explore this paradoxical observation, the effects of cooling on C-fibre responses to stimulation with sinusoidal and rectangular current profiles were determined in ex vivo segments of mouse sural and pig saphenous nerve. As expected by thermodynamics, the absolute value of electrical charge required to activate C-fibre axons increased with cooling from 32°C to 20°C, irrespective of the stimulus profile used. However, for sinusoidal stimulus profiles, cooling enabled a more effective integration of low-intensity currents over tens of milliseconds resulting in a delayed initiation of action potentials. Our findings indicate that the paradoxical cooling-induced enhancement of electrically evoked pain in people can be explained by an enhancement of C-fibre responsiveness to slow depolarization at lower temperatures. This property may contribute to symptoms of enhanced cold sensitivity, especially cold allodynia, associated with many forms of neuropathic pain.

Contralateral Afferent Input to Lumbar Lamina I Neurons as a Neural Substrate for Mirror-Image Pain.

Mirror-image pain arises from pathologic alterations in the nociceptive processing network that controls functional lateralization of the primary afferent input. Although a number of clinical syndromes related to dysfunction of the lumbar afferent system are associated with the mirror-image pain, its morphophysiological substrate and mechanism of induction remain poorly understood. Therefore, we used ex vivo spinal cord preparation of young rats of both sexes to study organization and processing of the contralateral afferent input to the neurons in the major spinal nociceptive projection area Lamina I. We show that decussating primary afferent branches reach contralateral Lamina I, where 27% of neurons, including projection neurons, receive monosynaptic and/or polysynaptic excitatory drive from the contralateral Aδ-fibers and C-fibers. All these neurons also received ipsilateral input, implying their involvement in the bilateral information processing. Our data further show that the contralateral Aδ-fiber and C-fiber input is under diverse forms of inhibitory control. Attenuation of the afferent-driven presynaptic inhibition and/or disinhibition of the dorsal horn network increased the contralateral excitatory drive to Lamina I neurons and its ability to evoke action potentials. Furthermore, the contralateral Aßδ-fibers presynaptically control ipsilateral C-fiber input to Lamina I neurons. Thus, these results show that some lumbar Lamina I neurons are wired to the contralateral afferent system whose input, under normal conditions, is subject to inhibitory control. A pathologic disinhibition of the decussating pathways can open a gate controlling contralateral information flow to the nociceptive projection neurons and, thus, contribute to induction of hypersensitivity and mirror-image pain.SIGNIFICANCE STATEMENT We show that contralateral Aδ-afferents and C-afferents supply lumbar Lamina I neurons. The contralateral input is under diverse forms of inhibitory control and itself controls the ipsilateral input. Disinhibition of decussating pathways increases nociceptive drive to Lamina I neurons and may cause induction of contralateral hypersensitivity and mirror-image pain.

Expanding the genetic causes of small-fiber neuropathy: SCN genes and beyond.

Small-fiber neuropathy (SFN) is a disorder that exclusively affects the small nerve fibers, sparing the large nerve fibers. Thinly myelinated Aδ-fibers and unmyelinated C-fibers are damaged, leading to development of neuropathic pain, thermal dysfunction, sensory symptoms, and autonomic disturbances. Although many SFNs are secondary and due to immunological causes or metabolic disturbances, the etiology is unknown in up to half of the patients. Over the years, this proportion of "idiopathic SFN" has decreased, as familial and genetic causes have been discovered, thus shifting a proportion of once "idiopathic" cases to the genetic category. After the discovery of SCN9A-gene variants in 2012, SCN10A and SCN11A variants have been found to be pathogenic in SFN. With improved accessibility of SFN diagnostic tools and genetic tests, many non-SCN variants and genetically inherited systemic diseases involving the small nerve fibers have also been described, but only scattered throughout the literature. There are 80 SCN variants described as causing SFN, 8 genes causing hereditary sensory autonomic neuropathies (HSAN) described with pure SFN, and at least 7 genes involved in genetically inherited systemic diseases associated with SFN. This systematic review aims to consolidate and provide an updated overview on the genetic variants of SFN to date---SCN genes and beyond. Awareness of these genetic causes of SFN is imperative for providing treatment directions, prognostication, and management of expectations for patients and their health-care providers.