Peripheral signaling pathways contributing to non-histaminergic itch in humans.

BACKGROUND: Chronic itch (chronic pruritus) is a major therapeutic challenge that remains poorly understood despite the extensive recent analysis of human pruriceptors. It is unclear how the peripheral nervous system differentiates the signaling of non-histaminergic itch and pain. METHODS: Here we used psychophysical analysis and microneurography (single nerve fiber recordings) in healthy human volunteers to explore the distinct signaling mechanisms of itch, using the pruritogens ß-alanine, BAM 8-22 and cowhage extract. RESULTS: The mode of application (injection or focal application using inactivated cowhage spicules) influenced the itch/pain ratio in sensations induced by BAM 8-22 and cowhage but not ß-alanine. We found that sensitizing pre-injections of prostaglandin E2 increased the pain component of BAM 8-22 but not the other pruritogens. A-fibers contributed only to itch induced by ß-alanine. TRPV1 and TRPA1 were necessary for itch signaling induced by all three pruritogens. In single-fiber recordings, we found that BAM 8-22 and ß-alanine injection activated nearly all CM-fibers (to different extents) but not CMi-fibers, whereas cowhage extract injection activated only 56% of CM-fibers but also 25% of CMi-fibers. A "slow bursting discharge pattern" was evoked in 25% of CM-fibers by ß-alanine, in 35% by BAM 8-22, but in only 10% by cowhage extract. CONCLUSION: Our results indicate that no labeled line exists for these pruritogens in humans. A combination of different mechanisms, specific for each pruritogen, leads to itching sensations rather than pain. Notably, non-receptor-based mechanisms such as spatial contrast or discharge pattern coding seem to be important processes. These findings will facilitate the discovery of therapeutic targets for chronic pruritus, which are unlikely to be treated effectively by single receptor blockade.

Spontaneous activity of specific C-nociceptor subtypes from diabetic patients and mice: Involvement of reactive dicarbonyl compounds and (sensitized) transient receptor potential channel A1.

BACKGROUND: Diabetic metabolism causes changes of the chemical milieu including accumulation of reactive carbonyl species, for example, methylglyoxal (MGO). MGO activates chemosensitive TRPA1 on nociceptors, but the contribution to neuronal pathophysiology causing pain and hyperalgesia in diabetic neuropathy is not fully understood. METHODS: We employed single-nerve-fiber recordings in type 2 diabetes patients with (spDN) and without cutaneous pain (DN) and in streptozotocin-diabetic and healthy mice. In mice, we measured Ca++ transients in cultured DRG neurons and stimulated CGRP release from hairy skin. RESULTS: In diabetic patients, we recorded a large proportion of pathologically altered nerve C-fibers (79%). In spDN patients we found a higher percentage (72%) of spontaneously active C-nociceptors than in DN patients (15%). The proportion of spontaneous activity was highest among pathological fibers with mechanoinsensitive fiber properties which are particularly sensitive to MGO in contrast to mechanosensitive fibers. Mouse polymodal nociceptors, in contrast to purely mechanosensitive C-fibers, showed highest prevalence of TRPA1-related chemosensitivity. In diabetic mice about 37% of polymodal nociceptors developed spontaneous activity and exhibited significantly greater MGO responses, indicating sensitized TRPA1 receptors. Low-threshold mechanosensitive Aδ-fibers were vigorously activated by MGO but independently of TRPA1 activation. INTERPRETATION: Our translational findings suggest that TRPA1-expressing C-nociceptors, which in human correspond to mechanoinsensitive and in mice to polymodal nociceptors, are especially vulnerable to develop spontaneous activity. Those two different nociceptor classes might share the functional role as dicarbonyl-sensitive chemosensors and represent the critical nociceptor population that support the development of pain and hyperalgesia in diabetic neuropathy.

Cyclic changes of sensory parameters in migraine patients.

BACKGROUND: Migraine shows a cyclic pattern with an inter-ictal-, a pre-ictal, an ictal- and a post-ictal phase. We aimed to examine changes in psychophysical parameters during the migraine cycle. METHODS: The perception of nociceptive and non-nociceptive stimuli and an electrically induced axon-reflex-erythema were assessed in 20 healthy controls and 14 migraine patients on five consecutive days according to different phases of the migraine cycle. Pain was rated three times during a 10-second electrical stimulus. The size of the axon-reflex-erythema was determined using laser-Doppler-imaging. Intensity and hedonic estimates of odours presented by Sniffin' Sticks were rated. RESULTS: In healthy controls, no significant changes over the test days were observed. In migraine patients pain thresholds at the head decreased with an ictal minimum. Less habituation after five seconds of stimulation at the head was found pre-ictally, whereas reduced habituation to 10-second electrical stimulation was present in all phases. The axon-reflex-erythema size showed an inter-ictal-specific minimum at the head. odours were perceived ictally as more unpleasant and intense. CONCLUSIONS: Somatosensory functions, pain thresholds and habituation as predominantly central parameters, axon-reflex-erythema as a peripheral function of trigeminal neurons and odour perception as a predominantly extra-thalamic sensation change specifically over the migraine cycle indicating complex variations of neuronal signal processing.

Maximum axonal following frequency separates classes of cutaneous unmyelinated nociceptors in the pig.

KEY POINTS: C-nociceptors are generally assumed to have a low maximum discharge frequency of 10-30 Hz. However, only mechano-insensitive 'silent' C-nociceptors cannot follow electrical stimulation at 5 Hz (75 pulses) whereas polymodal C-nociceptors in the pig follow stimulation at up to 100 Hz without conduction failure. Sensitization by nerve growth factor increases the maximum following frequency of 'silent' nociceptors in pig skin and might thereby contribute in particular to intense pain sensations in chronic inflammation. A distinct class of C-nociceptors with mechanical thresholds >150 mN resembles 'silent' nociceptors at low stimulation frequencies in pigs and humans, but is capable of 100 Hz discharge and thus is suited to encode painfulness of noxious mechanical stimuli. ABSTRACT: Using extracellular single-fibre recordings from the saphenous nerve in pig in vivo, we investigated peak following frequencies (5-100 Hz) in different classes of C-nociceptors and their modulation by nerve growth factor. Classes were defined by sensory (mechano-sensitivity) and axonal characteristics (activity dependent slowing of conduction, ADS). Mechano-insensitive C-nociceptors (CMi) showed the highest ADS (34% ± 8%), followed only 66% ± 27% of 75 pulses at 5 Hz and increasingly blocked conduction at higher frequencies. Three weeks following intradermal injections of nerve growth factor, peak following frequency increased specifically in the sensitized mechano-insensitive nociceptors (20% ± 16% to 38% ± 23% response rate after 72 pulses at 100 Hz). In contrast, untreated polymodal nociceptors with moderate ADS (15.2% ± 10.2%) followed stimulation frequencies of 100 Hz without conduction failure (98.5% ± 6%). A distinct class of C-nociceptors was exclusively sensitive to strong forces above 150 mN. This class had a high ADS (27.2% ± 7.6%), but displayed almost no propagation failure even at 100 Hz stimulation (84.7% ± 17%). Also, among human mechanosensitive nociceptors (n = 153) those with thresholds above 150 mN (n = 5) showed ADS typical of silent nociceptors. C-fibres with particularly high mechanical thresholds and high following frequency form a distinct nociceptor class ideally suited to encode noxious mechanical stimulation under normal conditions when regular silent nociceptors are inactive. Sensitization by nerve growth factor increases maximum discharge frequency of silent nociceptors, thereby increasing the frequency range beyond their physiological limit, which possibly contributes to excruciating pain under inflammatory conditions.

Tuning in C-nociceptors to reveal mechanisms in chronic neuropathic pain.

OBJECTIVE: Develop and validate a low-intensity sinusoidal electrical stimulation paradigm to preferentially activate C-fibers in human skin. METHODS: Sinusoidal transcutaneous stimulation (4Hz) was assessed psychophysically in healthy volunteers (n = 14) and neuropathic pain patients (n = 9). Pursuing laser Doppler imaging and single nociceptor recordings in vivo in humans (microneurography) and pigs confirmed the activation of "silent" C-nociceptors. Synchronized C-fiber compound action potentials were evoked in isolated human nerve fascicles in vitro. Live cell imaging of L4 dorsal root ganglia in anesthetized mice verified the recruitment of small-diameter neurons during transcutaneous 4-Hz stimulation of the hindpaw (0.4mA). RESULTS: Transcutaneous sinusoidal current (0.05-0.4mA, 4Hz) activated "polymodal" C-fibers (50% at ∼0.03mA) and "silent" nociceptors (50% at ∼0.04mA), intensities substantially lower than that required with transcutaneous 1-ms rectangular pulses ("polymodal" ∼3mA, "silent" ∼50mA). The stimulation induced delayed burning (nonpulsating) pain and a pronounced axon-reflex erythema, both indicative of C-nociceptor activation. Pain ratings to repetitive stimulation (1 minute, 4Hz) adapted in healthy volunteers by Numeric Rating Scale (NRS) -3 and nonpainful skin sites of neuropathic pain patients by NRS -0.5, whereas pain even increased in painful neuropathic skin by approximately NRS +2. INTERPRETATION: Sinusoidal electrical stimulation at 4Hz enables preferential activation of C-nociceptors in pig and human skin that accommodates during ongoing (1-minute) stimulation. Absence of such accommodation in neuropathic pain patients suggest axonal hyperexcitability that could be predictive of alterations in peripheral nociceptor encoding and offer a potential therapeutic entry point for topical analgesic treatment. Ann Neurol 2018;83:945-957.

Cyclic changes in sensations to painful stimuli in migraine patients.

INTRODUCTION: Migraine is characterized by cycling phases (interictal, preictal, ictal and postictal) with differing symptoms, while in chronic tension type headache pain phases are fluctuating. The question we asked is whether these phases are associated with changes in parameters of somatosensation and axon-reflex erythema. METHODS: Patients with episodic migraine and chronic tension type headache were examined psychophysically in the interictal, preictal and ictal phase and healthy subjects on five different test days. Thresholds and suprathreshold ratings of pressure and electrical pain were assessed on three different regions of the head. In migraine patients and in healthy controls, electrically induced axon-reflex erythema was measured in the area of the first trigeminal branch. All migraine patients filled out questionnaires about prodromal symptoms at every visit. RESULTS: The axon-reflex erythema was always larger in patients with migraine in contrast to healthy subjects. The pressure pain threshold was lower in migraine patients and chronic tension type headache in comparison to healthy subjects. Electrical pain thresholds did not differ between headache patients and healthy subjects and showed no changes between the phases. However, suprathreshold pain ratings showed less habituation solely in the preictal phase of migraine. The number of prodromal symptoms in migraine patients was increased in the preictal and ictal phase. DISCUSSION: Reduced habituation was the unique sign of the preictal phase in migraine patients, independently of prodromal symptoms, whereas a larger axon-reflex erythema and higher pressure pain sensitivity are constitutional and non-phase dependent properties of migraine. Reduced inhibitory mechanisms in the preictal phase may contribute to trigger headache attacks in migraine.

Etomidate and propylene glycol activate nociceptive TRP ion channels.

BACKGROUND: Etomidate is a preferred drug for the induction of general anesthesia in cardiovascular risk patients. As with propofol and other perioperatively used anesthetics, the application of aqueous etomidate formulations causes an intensive burning pain upon injection. Such algogenic properties of etomidate have been attributed to the solubilizer propylene glycol which represents 35% of the solution administered clinically. The aim of this study was to investigate the underlying molecular mechanisms which lead to injection pain of aqueous etomidate formulations. RESULTS: Activation of the nociceptive transient receptor potential (TRP) ion channels TRPA1 and TRPV1 was studied in a transfected HEK293t cell line by whole-cell voltage clamp recordings of induced inward ion currents. Calcium influx in sensory neurons of wild-type and trp knockout mice was ratiometrically measured by Fura2-AM staining. Stimulated calcitonin gene-related peptide release from mouse sciatic nerves was detected by enzyme immunoassay. Painfulness of different etomidate formulations was tested in a translational human pain model. Etomidate as well as propylene glycol proved to be effective agonists of TRPA1 and TRPV1 ion channels at clinically relevant concentrations. Etomidate consistently activated TRPA1, but there was also evidence for a contribution of TRPV1 in dependence of drug concentration ranges and species specificities. Distinct N-terminal cysteine and lysine residues seemed to mediate gating of TRPA1, although the electrophile scavenger N-acetyl-L-cysteine did not prevent its activation by etomidate. Propylene glycol-induced activation of TRPA1 and TRPV1 appeared independent of the concomitant high osmolarity. Intradermal injections of etomidate as well as propylene glycol evoked severe burning pain in the human pain model that was absent with emulsification of etomidate. CONCLUSIONS: Data in our study provided evidence that pain upon injection of clinical aqueous etomidate formulations is not an unspecific effect of hyperosmolarity but rather due to a specific action mediated by activated nociceptive TRPA1 and TRPV1 ion channels in sensory neurons.

Photosensitization in Porphyrias and Photodynamic Therapy Involves TRPA1 and TRPV1.

UNLABELLED: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. SIGNIFICANCE STATEMENT: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.

C-fiber recovery cycle supernormality depends on ion concentration and ion channel permeability.

Following each action potential, C-fiber nociceptors undergo cyclical changes in excitability, including a period of superexcitability, before recovering their basal excitability state. The increase in superexcitability during this recovery cycle depends upon their immediate firing history of the axon, but also determines the instantaneous firing frequency that encodes pain intensity. To explore the mechanistic underpinnings of the recovery cycle phenomenon a biophysical model of a C-fiber has been developed. The model represents the spatial extent of the axon including its passive properties as well as ion channels and the Na/K-ATPase ion pump. Ionic concentrations were represented inside and outside the membrane. The model was able to replicate the typical transitions in excitability from subnormal to supernormal observed empirically following a conducted action potential. In the model, supernormality depended on the degree of conduction slowing which in turn depends upon the frequency of stimulation, in accordance with experimental findings. In particular, we show that activity-dependent conduction slowing is produced by the accumulation of intraaxonal sodium. We further show that the supernormal phase results from a reduced potassium current Kdr as a result of accumulation of periaxonal potassium in concert with a reduced influx of sodium through Nav1.7 relative to Nav1.8 current. This theoretical prediction was supported by data from an in vitro preparation of small rat dorsal root ganglion somata showing a reduction in the magnitude of tetrodotoxin-sensitive relative to tetrodotoxin -resistant whole cell current. Furthermore, our studies provide support for the role of depolarization in supernormality, as previously suggested, but we suggest that the basic mechanism depends on changes in ionic concentrations inside and outside the axon. The understanding of the mechanisms underlying repetitive discharges in recovery cycles may provide insight into mechanisms of spontaneous activity, which recently has been shown to correlate to a perceived level of pain.

Pathogenesis and Management of Pruritus in PBC and PSC.

Pruritus is a preeminent symptom in patients with chronic cholestatic liver disorders such as primary biliary cirrhosis and primary sclerosing cholangitis. More than two-thirds of these patients experience itching during the course of their disease. This symptom is also frequently observed in patients with other causes of cholestasis such as cholangiocarcinoma, inherited forms of cholestasis and intrahepatic cholestasis of pregnancy, but may accompany almost any other liver disease. The pathogenesis of pruritus of cholestasis remains largely elusive. Increased concentrations of bile salts, histamine, serotonin, progesterone metabolites and endogenous opioids have been controversially discussed as potential pruritogens. However, for these molecules, neither a correlation with itch intensity nor a causative link could be established. The G protein-coupled receptor for bile salts, TGR5, has been shown to be expressed in dorsal root ganglia and give rise to itch in rodents, albeit upon stimuli with suprapathological concentrations of bile salts. The potent neuronal activator lysophosphatidic acid (LPA) and its forming enzyme, autotaxin (ATX), could be identified in the serum of patients with cholestatic pruritus. ATX activity correlated with itch severity and effectiveness of several anti-pruritic therapeutic interventions in cholestatic patients. Thus, the ATX-LPA-axis may represent a key element in the pathogenesis of this agonizing symptom. Treatment options for pruritus of cholestasis remain limited to a few evidence-based and several experimental medical and interventional therapies. The current guideline-based recommendations include the anion exchange resins colestyramine, the pregnane X receptor-agonist and enzyme inducer rifampicin, the µ-opioid antagonist naltrexone, and the selective serotonin reuptake inhibitors sertraline. Still, a considerable part of patients is unresponsive to these drugs and requires experimental approaches including phototherapy, plasmapheresis, albumin dialysis or nasobiliary drainage. This review outlines the current knowledge on pathogenesis of cholestatic pruritus and summarizes evidence-based and experimental therapeutic interventions for cholestatic patients with itch.

Spontaneous activity in peripheral sensory nerves: a systematic review.

ABSTRACT: In the peripheral nervous system, spontaneous activity in sensory neurons is considered to be one of the 2 main drivers of chronic pain states, alongside neuronal sensitization. Despite this, the precise nature and timing of this spontaneous activity in neuropathic pain is not well-established. Here, we have performed a systematic search and data extraction of existing electrophysiological literature to shed light on which fibre types have been shown to maintain spontaneous activity and over what time frame. We examined both in vivo recordings of preclinical models of neuropathic pain, as well as microneurography recordings in humans. Our analyses reveal that there is broad agreement on the presence of spontaneous activity in neuropathic pain conditions, even months after injury or years after onset of neuropathic symptoms in humans. However, because of the highly specialised nature of the electrophysiological methods used to measure spontaneous activity, there is also a high degree of variability and uncertainty around these results. Specifically, there are very few directly controlled experiments, with less directly comparable data between human and animals. Given that spontaneous peripheral neuron activity is considered to be a key mechanistic feature of chronic pain conditions, it may be beneficial to conduct further experiments in this space.

Axon reflex flare and quantitative sudomotor axon reflex contribute in the diagnosis of small fiber neuropathy.

INTRODUCTION: Objective diagnosis of small fiber impairment is difficult. METHODS: We used the quantitative sudomotor axon reflex test (QSART) and axon-reflex-flare-test in the foot and thigh of 46 patients with peripheral neuropathy to assess C-fiber function in addition to conventional neurography and thermal threshold testing. RESULTS: In all patients, small fiber impairment was suspected because of abnormal warmth detection thresholds (76% of all tested) and/or pain in the feet. A total of 83% had reduced axon-reflex flare areas and 17% lower QSART scores. Patients with pure small fiber neuropathy had higher rates of reduced flare areas (87.5%) and sweating rates (25.5%). There was no difference between patients with and without pain regarding thermotesting and axon-reflex testing. CONCLUSIONS: Both axon-reflex tests are helpful to identify objectively patients with small fiber impairment. Afferent and efferent C-fiber classes can be impaired differently. These tests detect small fiber impairment, but they cannot differentiate between painful and nonpainful neuropathy.

Measuring pain and nociception: Through the glasses of a computational scientist. Transdisciplinary overview of methods.

In a healthy state, pain plays an important role in natural biofeedback loops and helps to detect and prevent potentially harmful stimuli and situations. However, pain can become chronic and as such a pathological condition, losing its informative and adaptive function. Efficient pain treatment remains a largely unmet clinical need. One promising route to improve the characterization of pain, and with that the potential for more effective pain therapies, is the integration of different data modalities through cutting edge computational methods. Using these methods, multiscale, complex, and network models of pain signaling can be created and utilized for the benefit of patients. Such models require collaborative work of experts from different research domains such as medicine, biology, physiology, psychology as well as mathematics and data science. Efficient work of collaborative teams requires developing of a common language and common level of understanding as a prerequisite. One of ways to meet this need is to provide easy to comprehend overviews of certain topics within the pain research domain. Here, we propose such an overview on the topic of pain assessment in humans for computational researchers. Quantifications related to pain are necessary for building computational models. However, as defined by the International Association of the Study of Pain (IASP), pain is a sensory and emotional experience and thus, it cannot be measured and quantified objectively. This results in a need for clear distinctions between nociception, pain and correlates of pain. Therefore, here we review methods to assess pain as a percept and nociception as a biological basis for this percept in humans, with the goal of creating a roadmap of modelling options.

odML-Tables as a Metadata Standard in Microneurography.

Metadata standards are well-established for many types of electrophysiological methods but are still lacking for microneurographic recordings of peripheral sensory nerve fibers in humans. Finding a solution for daily work in the laboratory is a complex process. We have designed templates based on odML and odML-tables to structure and capture metadata and provided an extension to the existing GUI to enable database searching.

Corrigendum: Decoding neuropathic pain: Can we predict fluctuations of propagation speed in stimulated peripheral nerve?

[This corrects the article DOI: 10.3389/fncom.2022.899584.].

PyDapsys: an open-source library for accessing electrophysiology data recorded with DAPSYS.

In the field of neuroscience, a considerable number of commercial data acquisition and processing solutions rely on proprietary formats for data storage. This often leads to data being locked up in formats that are only accessible by using the original software, which may lead to interoperability problems. In fact, even the loss of data access is possible if the software becomes unsupported, changed, or otherwise unavailable. To ensure FAIR data management, strategies should be established to enable long-term, independent, and unified access to data in proprietary formats. In this work, we demonstrate PyDapsys, a solution to gain open access to data that was acquired using the proprietary recording system DAPSYS. PyDapsys enables us to open the recorded files directly in Python and saves them as NIX files, commonly used for open research in the electrophysiology domain. Thus, PyDapsys secures efficient and open access to existing and prospective data. The manuscript demonstrates the complete process of reverse engineering a proprietary electrophysiological format on the example of microneurography data collected for studies on pain and itch signaling in peripheral neural fibers.

odML-Tables as a Metadata Standard in Microneurography.

Metadata is essential for handling medical data according to FAIR principles. Standards are well-established for many types of electrophysiological methods but are still lacking for microneurographic recordings of peripheral sensory nerve fibers in humans. Developing a new concept to enhance laboratory workflows is a complex process. We propose a standard for structuring and storing microneurography metadata based on odML and odML-tables. Further, we present an extension to the odML-tables GUI that enables user-friendly search functionality of the database. With our open-source repository, we encourage other microneurography labs to incorporate odML-based metadata into their experimental routines.

A modelling study to dissect the potential role of voltage-gated ion channels in activity-dependent conduction velocity changes as identified in small fiber neuropathy patients.

Objective: Patients with small fiber neuropathy (SFN) suffer from neuropathic pain, which is still a therapeutic problem. Changed activation patterns of mechano-insensitive peripheral nerve fibers (CMi) could cause neuropathic pain. However, there is sparse knowledge about mechanisms leading to CMi dysfunction since it is difficult to dissect specific molecular mechanisms in humans. We used an in-silico model to elucidate molecular causes of CMi dysfunction as observed in single nerve fiber recordings (microneurography) of SFN patients. Approach: We analyzed microneurography data from 97 CMi-fibers from healthy individuals and 34 of SFN patients to identify activity-dependent changes in conduction velocity. Using the NEURON environment, we adapted a biophysical realistic preexisting CMi-fiber model with ion channels described by Hodgkin-Huxley dynamics for identifying molecular mechanisms leading to those changes. Via a grid search optimization, we assessed the interplay between different ion channels, Na-K-pump, and resting membrane potential. Main results: Changing a single ion channel conductance, Na-K-pump or membrane potential individually is not sufficient to reproduce in-silico CMi-fiber dysfunction of unchanged activity-dependent conduction velocity slowing and quicker normalization of conduction velocity after stimulation as observed in microneurography. We identified the best combination of mechanisms: increased conductance of potassium delayed-rectifier and decreased conductance of Na-K-pump and depolarized membrane potential. When the membrane potential is unchanged, opposite changes in Na-K-pump and ion channels generate the same effect. Significance: Our study suggests that not one single mechanism accounts for pain-relevant changes in CMi-fibers, but a combination of mechanisms. A depolarized membrane potential, as previously observed in patients with neuropathic pain, leads to changes in the contribution of ion channels and the Na-K-pump. Thus, when searching for targets for the treatment of neuropathic pain, combinations of several molecules in interplay with the membrane potential should be regarded.

Sea-anemone toxin ATX-II elicits A-fiber-dependent pain and enhances resurgent and persistent sodium currents in large sensory neurons.

BACKGROUND: Gain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations. RESULTS: In large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel ß4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less ß4 mRNA than large sensory neurons. With the ß4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected. CONCLUSION: ATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of ß4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

Decoding Neuropathic Pain: Can We Predict Fluctuations of Propagation Speed in Stimulated Peripheral Nerve?

To understand neural encoding of neuropathic pain, evoked and resting activity of peripheral human C-fibers are studied via microneurography experiments. Before different spiking patterns can be analyzed, spike sorting is necessary to distinguish the activity of particular fibers of a recorded bundle. Due to single-electrode measurements and high noise contamination, standard methods based on spike shapes are insufficient and need to be enhanced with additional information. Such information can be derived from the activity-dependent slowing of the fiber propagation speed, which in turn can be assessed by introducing continuous "background" 0.125-0.25 Hz electrical stimulation and recording the corresponding responses from the fibers. Each fiber's speed propagation remains almost constant in the absence of spontaneous firing or additional stimulation. This way, the responses to the "background stimulation" can be sorted by fiber. In this article, we model the changes in the propagation speed resulting from the history of fiber activity with polynomial regression. This is done to assess the feasibility of using the developed models to enhance the spike shape-based sorting. In addition to human microneurography data, we use animal in-vitro recordings with a similar stimulation protocol as higher signal-to-noise ratio data example for the models.