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.

Evaluating the association of TRPA1 gene polymorphisms with pain sensitivity: a protocol for an adaptive recall by genotype study.

BACKGROUND: Pain is a complex polygenic trait whose common genetic underpinnings are relatively ill-defined due in part to challenges in measuring pain as a phenotype. Pain sensitivity can be quantified, but this is difficult to perform at the scale required for genome wide association studies (GWAS). Existing GWAS of pain have identified surprisingly few loci involved in nociceptor function which contrasts strongly with rare monogenic pain states. This suggests a lack of resolution with current techniques. We propose an adaptive methodology within a recall-by-genotype (RbG) framework using detailed phenotyping to screen minor alleles in a candidate 'nociceptor' gene in an attempt to estimate their genetic contribution to pain. METHODS/DESIGN: Participants of the Avon Longitudinal Study of Parents and Children will be recalled on the basis of genotype at five common non-synonomous SNPs in the 'nociceptor' gene transient receptor potential ankylin 1 (TRPA1). Those homozygous for the common alleles at each of the five SNPs will represent a control group. Individuals homozygous for the minor alleles will then be recruited in a series of three sequential test groups. The outcome of a pre-planned early assessment (interim) of the current test group will determine whether to continue recruitment or switch to the next test group. Pain sensitivity will be assessed using quantitative sensory testing (QST) before and after topical application of 10% cinnamaldehyde (a TRPA1 agonist). DISCUSSION: The design of this adaptive RbG study offers efficiency in the assessment of associations between genetic variation at TRPA1 and detailed pain phenotypes. The possibility to change the test group in response to preliminary data increases the likelihood to observe smaller effect sizes relative to a conventional multi-armed design, as well as reducing futile testing of participants where an effect is unlikely to be observed. This specific adaptive RbG design aims to uncover the influence of common TRPA1 variants on pain sensation but can be applied to any hypothesis-led genotype study where costly and time intensive investigation is required and / or where there is large uncertainty around the expected effect size. TRIAL REGISTRATION: ISRCTN, ISRCTN16294731. Retrospectively registered 25th November 2021.

Ultrasound-guided, open-source microneurography: Approaches to improve recordings from peripheral nerves in man.

OBJECTIVE: Microneurography is the only method for recording from single neurons in intact human nerves. It is challenging - requiring technical expertise, investment in specialised equipment and has sparse data yields. METHODS: We assessed whether ultrasound guidance in combination with an 'open access' amplifier and data capture system (Open-Ephys) would simplify and expand the scope of microneurographic recordings in humans. RESULTS: In 32 healthy consenting volunteers, ultrasound-guidance improved success rates for obtaining cutaneous C-fibres and reduced "Skin to Nerve" times from 28.5 min to 4.5 min for recordings of the peroneal nerve (P < 0.0001). We illustrate the potential utility of ultrasound-guided microneurography for difficult to access nerves with phrenic nerve recording during a Valsalva manoeuvre. We show that Open Ephys is a viable alternative to commercially available recording systems and offers advantages in terms of cost and software customisability. CONCLUSIONS: Ultrasound guidance for microneurography with Open Ephys facilitates cutaneous C nociceptor recordings and allows recordings to be made from nerves previously considered inaccessible. SIGNIFICANCE: We anticipate that the adoption of these techniques will improve microneurography experimental efficiency, adds an important visual learning aid and increases the generalisability of the approach.

Optimal delineation of single C-tactile and C-nociceptive afferents in humans by latency slowing.

C-mechanoreceptors in humans comprise a population of unmyelinated afferents exhibiting a wide range of mechanical sensitivities. C-mechanoreceptors are putatively divided into those signaling gentle touch (C-tactile afferents, CTs) and nociception (C-mechanosensitive nociceptors, CMs), giving rise to positive and negative affect, respectively. We sought to distinguish, compare, and contrast the properties of a population of human C-mechanoreceptors to see how fundamental the divisions between these putative subpopulations are. We used microneurography to record from individual afferents in humans and applied electrical and mechanical stimulation to their receptive fields. We show that C-mechanoreceptors can be distinguished unequivocally into two putative populations, comprising CTs and CMs, by electrically evoked spike latency changes (slowing). After both natural mechanical stimulation and repetitive electrical stimulation there was markedly less latency slowing in CTs compared with CMs. Electrical receptive field stimulation, which bypasses the receptor end organ, was most effective in classifying C-mechanoreceptors, as responses to mechanical receptive field stimulation overlapped somewhat, which may lead to misclassification. Furthermore, we report a subclass of low-threshold CM responding to gentle mechanical stimulation and a potential subclass of CT afferent displaying burst firing. We show that substantial differences exist in the mechanisms governing axonal conduction between CTs and CMs. We provide clear electrophysiological "signatures" (extent of latency slowing) that can be used in unequivocally identifying populations of C-mechanoreceptors in single-unit and multiunit microneurography studies and in translational animal research into affective touch. Additionally, these differential mechanisms may be pharmacologically targetable for separate modulation of positive and negative affective touch information.NEW & NOTEWORTHY Human skin encodes a plethora of touch interactions, and affective tactile information is primarily signaled by slowly conducting C-mechanoreceptive afferents. We show that electrical stimulation of low-threshold C-tactile afferents produces markedly different patterns of activity compared with high-threshold C-mechanoreceptive nociceptors, although the populations overlap in their responses to mechanical stimulation. This fundamental distinction demonstrates a divergence in affective touch signaling from the first stage of sensory processing, having implications for the processing of interpersonal touch.

A novel method for delivering ramped cooling reveals rat behaviours at innocuous and noxious temperatures: A comparative study of human psychophysics and rat behaviour.

BACKGROUND: Thermal sensory testing in rodents informs human pain research. There are important differences in the methodology for delivering thermal stimuli to humans and rodents. This is particularly true in cold pain research. These differences confound extrapolation and de-value nociceptive tests in rodents. NEW METHOD: We investigated cooling-induced behaviours in rats and psychophysical thresholds in humans using ramped cooling stimulation protocols. A Peltier device mounted upon force transducers simultaneously applied a ramped cooling stimulus whilst measuring contact with rat hind paw or human finger pad. Rat withdrawals and human detection, discomfort and pain thresholds were measured. RESULTS: Ramped cooling of a rat hind paw revealed two distinct responses: Brief paw removal followed by paw replacement, usually with more weight borne than prior to the removal (temperature inter-quartile range: 19.1 °C to 2.8 °C). Full withdrawal was evoked at colder temperatures (inter quartile range: -11.3 °C to -11.8 °C). The profile of human cool detection threshold and cold pain threshold were remarkably similar to that of the rat withdrawals behaviours. COMPARISON: Previous rat cold evoked behaviours utilise static temperature stimuli. By utilising ramped cold stimuli this novel methodology better reflects thermal testing in patients. CONCLUSION: Brief paw removal in the rat is driven by non-nociceptive afferents, as is the perception of cooling in humans. This is in contrast to the nociceptor-driven withdrawal from colder temperatures. These findings have important implications for the interpretation of data generated in older cold pain models and consequently our understanding of cold perception and pain.

Inflammation reduces mechanical thresholds in a population of transient receptor potential channel A1-expressing nociceptors in the rat.

Inflammatory hypersensitivity is characterized by behavioural reductions in withdrawal thresholds to noxious stimuli. Although cutaneous primary afferent neurones are known to have lowered thermal thresholds in inflammation, whether their mechanical thresholds are altered remains controversial. The transient receptor potential channel A1 (TRPA1) is a receptor localized to putative nociceptive neurones and is implicated in mechanical and thermal nociception. Herein, we examined changes in the properties of single primary afferents in normal and acutely inflamed rats and determined whether specific nociceptive properties, particularly mechanical thresholds, are altered in the subpopulation of afferents that responded to the TRPA1 agonist cinnamaldehyde (TRPA1-positive afferents). TRPA1-positive afferents in normal animals belonged to the mechanonociceptive populations, many of which also responded to heat or capsaicin but only a few of which responded to cold. In acute inflammation, a greater proportion of afferents responded to cinnamaldehyde and an increased proportion of dorsal root ganglion neurones expressed TRPA1 protein. Functionally, in inflammation, TRPA1-positive afferents showed significantly reduced mechanical thresholds and enhanced activity to agonist stimulation. Inflammation altered thermal thresholds in both TRPA1-positive and TRPA1-negative afferents. Our data show that a subset of afferents is sensitized to mechanical stimulation by inflammation and that these afferents are defined by expression of TRPA1.

Molecular mechanism of voltage sensor movements in a potassium channel.

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization--this motion is accompanied by a reorientation of S4 charges to the extracellular phase.