We would like to thank our funders for their support: Academy of Medical Sciences (J.P.D., A.E.P.), Versus Arthritis (J.P.D., A.E.P.), Jean Golding Institute Seedcorn Grant (J.P.D., A.E.P., G.W., A.C.S., M.M.P.), and Biotechnology and Biological Sciences Research Council collaborative training partnership doctoral studentship with Eli Lilly (G.W.T.N.). We would like to extend our thanks to all the contributors to the development of APTrack. We would also like to thank our volunteers who participated in the microneurography experiments and our Patient and Public Involvement and Engagement collaborators for their invaluable contributions.

<ol>
	<li>Dubin, A. E., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors:+The+sensors+of+the+pain+pathway.">Nociceptors: The sensors of the pain pathway.</a> Journal of Clinical Investigation. 120 (11), 3760-3772 (2010).</li><li>Serra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneurographic+identification+of+spontaneous+activity+in+C-nociceptors+in+neuropathic+pain+states+in+humans+and+rats.">Microneurographic identification of spontaneous activity in C-nociceptors in neuropathic pain states in humans and rats.</a> Pain. 153 (1), 42-55 (2012).</li><li>Serra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperexcitable+C+nociceptors+in+fibromyalgia.">Hyperexcitable C nociceptors in fibromyalgia.</a> Annals of Neurology. 75 (2), 196-208 (2014).</li><li>Namer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+changes+in+conduction+velocity+recovery+cycles+of+single+nociceptors+in+a+patient+with+erythromelalgia+with+the+I848T+gain-of-function+mutation+of+Nav1.7.">Specific changes in conduction velocity recovery cycles of single nociceptors in a patient with erythromelalgia with the I848T gain-of-function mutation of Nav1.7.</a> Pain. 156 (9), 1637-1646 (2015).</li><li>Kleggetveit, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+spontaneous+activity+of+C-nociceptors+in+painful+polyneuropathy.">High spontaneous activity of C-nociceptors in painful polyneuropathy.</a> Pain. 153 (10), 2040-2047 (2012).</li><li>Orstavik, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+function+of+C-fibers+in+patients+with+diabetic+neuropathy.">Abnormal function of C-fibers in patients with diabetic neuropathy.</a> Journal of Neuroscience. 26 (44), 11287-11294 (2006).</li><li>Orstavik, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+C-fibres+in+patients+with+a+chronic+painful+condition.">Pathological C-fibres in patients with a chronic painful condition.</a> Brain. 126, 567-578 (2003).</li><li>Raja, S. N., Ringkamp, M., Guan, Y., Campbell, J. N., John, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonica+Award+Lecture:+Peripheral+neuronal+hyperexcitability:+The+&amp;#34;low-hanging&amp;#34;+target+for+safe+therapeutic+strategies+in+neuropathic+pain.">Bonica Award Lecture: Peripheral neuronal hyperexcitability: The &amp;#34;low-hanging&amp;#34; target for safe therapeutic strategies in neuropathic pain.</a> Pain. 161, S14-S26 (2020).</li><li>Middleton, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+human+nociceptors:+From+fundamentals+to+clinic.">Studying human nociceptors: From fundamentals to clinic.</a> Brain. 144 (5), 1312-1335 (2021).</li><li>Marshall, A., Alam, U., Themistocleous, A., Calcutt, N., Marshall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+and+emerging+electrophysiological+biomarkers+of+diabetic+neuropathy+and+painful+diabetic+neuropathy.">Novel and emerging electrophysiological biomarkers of diabetic neuropathy and painful diabetic neuropathy.</a> Clinical Therapeutics. 43 (9), 1441-1456 (2021).</li><li>Themistocleous, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+excitability+does+not+differ+between+painful+and+painless+diabetic+or+chemotherapy-induced+distal+symmetrical+polyneuropathy+in+a+multicenter+observational+study.">Axonal excitability does not differ between painful and painless diabetic or chemotherapy-induced distal symmetrical polyneuropathy in a multicenter observational study.</a> Annals of Neurology. 91 (4), 506-520 (2022).</li><li>Bostock, H., Cikurel, K., Burke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+tracking+techniques+in+the+study+of+human+peripheral+nerve.">Threshold tracking techniques in the study of human peripheral nerve.</a> Muscle Nerve. 21 (2), 137-158 (1998).</li><li>Kiernan, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+axonal+excitability:+Consensus+guidelines.">Measurement of axonal excitability: Consensus guidelines.</a> Clinical Neurophysiology. 131 (1), 308-323 (2020).</li><li>Sauer, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+receptor+potentials+be+detected+with+threshold+tracking+in+rat+cutaneous+nociceptive+terminals.">Can receptor potentials be detected with threshold tracking in rat cutaneous nociceptive terminals.</a> Journal of Neurophysiology. 94 (1), 219-225 (2005).</li><li>Vallbo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneurography:+How+it+started+and+how+it+works.">Microneurography: How it started and how it works.</a> Journal of Neurophysiology. 120 (3), 1415-1427 (2018).</li><li>Torebjork, H., Hallin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+classification+of+C-unit+activity+in+intact+human+skin+nerves.">A new method for classification of C-unit activity in intact human skin nerves.</a> Advances in Pain Research and Therapy. 1, 29-34 (1976).</li><li>Brown, G. L., Holmes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+activity+on+mammalian+nerve+fibres+of+low+conduction+velocity.">The effects of activity on mammalian nerve fibres of low conduction velocity.</a> Proceedings of the Royal Society of London. Series B: Biological Sciences. 144 (918), 1-14 (1956).</li><li>Obreja, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+activity-dependent+conduction+velocity+changes+differentiate+classes+of+unmyelinated+mechano-insensitive+afferents+including+cold+nociceptors,+in+pig+and+in+human.">Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human.</a> Pain. 148 (1), 59-69 (2010).</li><li>Serra, J., Campero, M., Ochoa, J., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+slowing+of+conduction+differentiates+functional+subtypes+of+C+fibres+innervating+human+skin.">Activity-dependent slowing of conduction differentiates functional subtypes of C fibres innervating human skin.</a> Journal of Physiology. 515, 799-811 (1999).</li><li>Weidner, C., Schmidt, R., Schmelz, M., Torebjork, H. E., Handwerker, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+potential+conduction+in+the+terminal+arborisation+of+nociceptive+C-fibre+afferents.">Action potential conduction in the terminal arborisation of nociceptive C-fibre afferents.</a> Journal of Physiology. 547, 931-940 (2003).</li><li>Siegle, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+Ephys:+An+open-source,+plugin-based+platform+for+multichannel+electrophysiology.">Open Ephys: An open-source, plugin-based platform for multichannel electrophysiology.</a> Journal of Neural Engineering. 14 (4), 045003 (2017).</li><li>Sanders, J. I., Kepecs, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+programmable+pulse+generator+for+physiology+and+behavior.">A low-cost programmable pulse generator for physiology and behavior.</a> Frontiers in Neuroengineering. 7, 43 (2014).</li><li>Zimmermann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotyping+sensory+nerve+endings+in+vitro+in+the+mouse.">Phenotyping sensory nerve endings in vitro in the mouse.</a> Nature Protocols. 4 (2), 174-196 (2009).</li><li>Dunham, J. P., Sales, A. C., Pickering, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided,+open-source+microneurography:+Approaches+to+improve+recordings+from+peripheral+nerves+in+man.">Ultrasound-guided, open-source microneurography: Approaches to improve recordings from peripheral nerves in man.</a> Clinical Neurophysiology. 129 (11), 2475-2481 (2018).</li><li>Levitt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformed+up-down+methods+in+psychoacoustics.">Transformed up-down methods in psychoacoustics.</a> Journal of the Acoustical Society of America. 49 (2), 467 (1971).</li><li>Turnquist, B., RichardWebster, B., Namer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+detection+of+latency+tracks+in+microneurography+recordings+using+track+correlation.">Automated detection of latency tracks in microneurography recordings using track correlation.</a> Journal of Neuroscience Methods. 262, 133-141 (2016).</li><li>Kiernan, M. C., Burke, D., Andersen, K. V., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+measures+of+axonal+excitability:+A+new+approach+in+clinical+testing.">Multiple measures of axonal excitability: A new approach in clinical testing.</a> Muscle Nerve. 23 (3), 399-409 (2000).</li></ol>

G.W.T.N. is a BBSRC Collaborative Training Partnership Doctoral Studentship with the University of Bristol and Eli Lilly and Company (BB/T508342/1). A.P.N. is a current employee of Eli Lilly and Company and may own stock in this company.

APTrack is a software plugin for use with the Open Ephys platform. We have chosen this platform as it is open-source, flexible, and cheap to implement. Not including the cost of the constant current stimulator, all the equipment required to start using the plugin could be purchased for around $5,000 USD at the time of writing. We hope this will enable researchers to implement APTrack in their peripheral nerve electrophysiology studies more easily. Furthermore, researchers can freely modify the software to fit their experimental needs. Importantly, this tool has allowed the electrical threshold tracking of single C-fiber nociceptors, for the first time, in humans.
The higher the signal-to-noise ratio, the better the algorithms can identify action potentials. The signal-to-noise ratio during microneurography was sufficient in the majority of our recordings, but users must be alert to the risk of signal degradation over time. This is particularly important for longer experimental protocols, because if the tracked action potential&#39;s amplitude drops below the detection threshold, the stimulation amplitude will be increased mistakenly; this can be mitigated by experimenters monitoring the plugin and then adjusting the settings if required. The signal-to-noise ratio is improved with bandpass filtering, but larger transients may still be misidentified as action potentials should they arrive during the search box time window. The risk of misidentifying transient noise as an action potential can be reduced by narrowing the time window during which the plugin searches for action potentials and by optimizing the threshold settings. However, there are still situations one may encounter that impede the plugin&#39;s performance. Spontaneous activity may cause difficulties if larger-amplitude action potentials fall within the algorithm&#39;s search box window, as they will be misidentified as the target action potential. Additionally, spontaneous activity in the neuron of interest may mean that the electrical stimulation falls during its refractory period, causing failure to generate an action potential. Difficulties using the software can also arise when primary afferent neurons exhibit flip-flop, whereby alternate terminal branches of a single neuron are stimulated, thus causing the evoked action potential to have two (or more) baseline latencies that are mutually exclusive20. During recordings from neurons exhibiting flip-flop with high signal-to-noise ratios, we successfully performed latency and electrical threshold tracking by increasing the search box width to encapsulate all the potential conduction velocities that the neuron exhibited. However, the electrical threshold may vary depending on the terminal branch of the neuron that is being excited, which is likely in part due to differences in the distance from the site of the electrical stimulation to the alternate nociceptor terminals. Additional work on the action potential identification process to include, for example, template matching is feasible and could be integrated into this software. The GUI plugins for band-stop or adaptive noise filtration could also be used upstream of APTrack in the signal chain should they be developed.
We consider the electrical threshold determined to be the current required to elicit an action potential 50% of the time, over a user-defined number of electrical stimuli, typically 2-10. The morphology of electrical stimulation is 0.5 ms and positive, square wave pulses. This is not the same as determining the rheobase, a commonly used measure of neuronal excitability. The plugin could be adapted to determine the rheobase. However, we pursued a simpler measure, as dynamic changes in excitability, such as those hypothesized to occur during heating, would have been more difficult to quantify with rheobase changes than our electrical threshold estimate.
This software can be used in both human and rodent experiments. This is made possible by flexible support for the electrical stimulation systems. The software will work with any stimulator that accepts an analog command voltage or can be manually interfaced with a stepper motor. For microneurography, we used it with a CE-marked constant current stimulator that was designed for use in human research and had its stimulation controlled by a dial. Stimulators that accept analog voltage commands can be noisy as they do not disconnect the circuit between stimuli, meaning any 50/60 Hz hum or noise on the analog input will be transmitted to the recording. A stimulator that requires an additional TLL trigger signal to connect the circuit, permitting a stimulus at a current analogous to the analog voltage input to be generated, is ideal for use with the plugin. This prevents the noise from being transmitted to the recording between stimuli.
The software uses a simple up-down method to estimate the electrical threshold. This has been used in psychophysics tests for many decades25. In line with the up-down method, the electrical threshold tracking algorithm for modulating the stimulation amplitude only considers the previous stimulation&#39;s amplitude and response when calculating the next stimulation&#39;s amplitude. This means that the stimulation amplitude will oscillate around the true electrical threshold, thus producing a 50% firing rate, assuming the threshold is stable. The minimum size of an increment or decrement is 0.01 V; this is equivalent to 0.01 mA assuming the stimulator has a 1 V:1 mA input-to-output ratio and sufficient resolution to achieve step changes this small. The plugin will update the live estimate of the target action potential&#39;s electrical threshold every time it reaches a 50% firing rate over a user-defined number of previous stimuli (2-10). Post hoc, we recommend using a rolling average of the stimulation amplitude over the last 2-10 stimuli to estimate the electrical threshold, and it should be noted that this estimate will only be accurate when the firing rate is relatively stable at 50%. In both the live and post hoc&#160;estimates of the electrical threshold, there is a balance of resolution, reliability, and time to consider. Using smaller increment and decrement steps will increase the accuracy of the electrical threshold estimate but will increase the time taken to find the new electrical threshold initially and following perturbation. Calculating the electrical threshold over a greater number of previous stimuli will provide better reliability but will increase the time required to reach an accurate estimate.
APTrack was designed for use in peripheral nerve recordings, specifically to track the electrical thresholds of C-fibers during experimental and pathological perturbations over periods where the action potential latency may vary depending on the underlying neuronal activity. This method will enable the examination of not only axonal excitability but also of nociceptor generator potentials in healthy volunteers and patients. We anticipate that other fields of electrophysiology may adopt and adapt this tool for use in any experiment that requires the electrical threshold tracking of a stimulus-locked activity. For example, this could as easily be adapted for optogenetic stimulation with light pulses driven from APTrack. The plugin is open-source and available to researchers under a GPLv3 license. It is built on the Open Ephys platform, which is an adaptable, low-cost, open-source data acquisition system. The plugin provides additional hooks for downstream plugins to extract the action potential information and provide additional user interfaces or adaptative paradigms. The plugin provides a simple user interface for the visualization and latency tracking of action potentials in real time. It can also play back previous data and visualize them using the temporal raster plot. Furthermore, it can also perform latency tracking during the playback of previous data. While there are other software packages available for real-time latency tracking, they are not open-source and cannot perform electrical threshold tracking26,27. APTrack has an advantage over traditional methods of identifying constant latency action potentials from voltage traces as it uses a temporal raster plot for the data visualization. Furthermore, our experiences of using it in experiments with low signal-to-noise ratios have indicated that the temporal raster plot visualization method allows the identification of constant latency action potentials that may have been otherwise missed.
Whole-nerve threshold tracking is a widely used method for assessing axonal excitability13. Single-neuron electrical threshold tracking in rodent C-fibers has been used previously to quantify nociceptor excitability14, and its utility in humans is recognized10,11; however, until now, this was not possible. We provide a novel, open-source tool to directly measure single nociceptor excitability in both rodent and human peripheral nerve electrophysiological studies. APTrack enables the real-time, open-source, electrical threshold tracking of single-neuron action potentials in humans, for the first time. We anticipate that it will facilitate translational studies of nociceptors between rodents and humans.

Nociceptors are primary afferent neurons in the peripheral nervous system that are activated by overtly or potentially tissue-damaging events and play a critical protective role in acute pain1. Electrophysiological recordings from C-fiber and A&#948;-fiber nociceptors in animal models, healthy human volunteers, and patients have revealed sensitization and abnormal spontaneous activity in a diverse range of pain conditions2,3,4,5,6,7. Understanding the mechanisms that underlie these changes in nociceptor excitability in patients could enable targeted therapeutic interventions8. However, there are few tools to assess nociceptor excitability directly, particularly in patients9, but the potential for the utility of such tools is well recognized10,11.
Whole-nerve electrical threshold tracking can be used to examine axonal excitability in humans12. However, as large, myelinated, peripheral neurons contribute disproportionally to the amplitude of the sensory compound action potential, whole-nerve electrical threshold tracking does not allow the assessment of C-fiber function11,13. Indeed, in a previous study, whole-nerve electrical threshold tracking in chronic neuropathic pain cohorts with diabetic neuropathy and chemotherapy-induced polyneuropathy showed no differences in axonal excitability11.
In a previous study, electrical threshold tracking at the single-neuron level was used to examine the excitability of C-fiber nociceptors during teased-fiber recordings in an ex vivo rat skin-nerve preparation14. The authors demonstrated that an increased potassium concentration, acidic conditions, and bradykinin all increased C-fiber nociceptor excitability, as reflected by a reduced electrical threshold for action potential generation. Furthermore, heating the receptive field of the heat-sensitive nociceptors reduced their electrical threshold, whereas heat-insensitive nociceptors exhibited an increase in their electrical threshold14. This provides important proof that single-neuron electrical threshold tracking is possible and can be of utility, but there are currently no software and/or hardware solutions available to enable such investigations, particularly for human studies.
In humans, microneurography is the only available method to directly assess the electrophysiological properties of C-fibers15. This approach has been used to demonstrate nociceptor dysfunction in patients with chronic pain2,3,4,5,6,7. Microneurography can detect single-neuron action potentials; however, due to the low signal-to-noise ratios, researchers use the marking technique to characterize C-fiber activity16. In the marking technique, suprathreshold electrical stimulation is applied to C-fiber receptive fields in the skin. This electrical stimulation generates an action potential that occurs at a constant latency, which is determined by the C-fiber&#39;s conduction velocity. C-fibers exhibit activity-dependent slowing, whereby their conduction velocity reduces and, therefore, their conduction latency increases during periods of action potential discharge17. Under basal conditions, C-fibers do not normally generate action potentials in the absence of noxious stimuli, and, therefore, their conduction latency in response to low-frequency electrical stimulation is constant. Mechanical, thermal, or pharmacological stimuli, which evoke firing, induce activity-dependent slowing, which increases the latency of the action potentials evoked by concomitant low-frequency electrical stimulation. This allows the objective identification of responses to the applied non-electrical stimuli in the context of a low signal-to-noise ratio. Therefore, activity-dependent slowing can be used to functionally characterize C-fibers16. Indeed, different functional classes of C-fibers exhibit distinctive patterns of activity-dependent slowing in electrical stimulation paradigms that involve varying the stimulation frequency18,19. This variability in the latency of C-fiber action potentials presents a challenge for algorithms designed to monitor them.
Ongoing activity in a nociceptor leads to increased variability in its latency during low-frequency electrical stimulation, and this is again due to activity-dependent slowing. This increased variability, or jitter, is a quantifiable proxy measure of excitability2. Further causes of variability in action potential latency include flip-flop, where alternate terminal branches of a single neuron are stimulated, which causes the evoked action potential to have two (or more) baseline latencies that are mutually exclusive20. Finally, changes in the temperature of a peripheral neuron&#39;s terminal branches also cause action potential latency changes in a thermodynamic manner, with warming increasing the conduction velocity and cooling slowing the conduction velocity19. Thus, any software seeking to perform closed-loop electrical threshold tracking of nociceptive C-fibers must allow for changes in latency in electrically evoked action potentials.
To achieve our goal of cross-species electrical threshold tracking of C-fiber nociceptors, we developed APTrack, an open-source software plugin for the Open Ephys platform21, to enable real-time, closed-loop, electrical threshold tracking, and latency tracking. We provide proof-of-concept data demonstrating that C-fiber nociceptor electrical threshold tracking during human microneurography is possible. Furthermore, we show that this tool can be used in rodent ex vivo teased-fiber electrophysiology, thus enabling translational studies between humans and rodents. Here, we will describe in detail how researchers can implement and use this tool to aid their study of nociceptor function and excitability.

<table><tbody><tr><td>12V DC Power Supply&amp;nbsp;</td><td>NA</td><td>NA</td><td>To power uStepper S-lite. Required for dial-controlled stimulators.</td></tr><tr><td>36 Pin Electrode Adapter Board</td><td>Intan Technology</td><td>C3410</td><td>APTrack Dependency. For connecting electrode input to headstage. $255 USD as of March 2021.</td></tr><tr><td>APTrack Plugin</td><td>NA</td><td>NA</td><td>https://github.com/Microneurography/APTrack</td></tr><tr><td>Bipolar Ag/AgCl Recording Electrode</td><td>Custom</td><td>NA</td><td>Recording electrode for the skin-nerve preparation. Or equivalent.</td></tr><tr><td>Bipolar Concentric Stimulating Electrode</td><td>World Precision Instruments</td><td>SNE-100</td><td>For electrical stimulation in the mouse skin-nerve preparation. Or equivalent.</td></tr><tr><td>Bipolar Transcutaneous Stimulating Electrode</td><td>Custom</td><td>NA</td><td>For transcutaneous electrical stimulation while searching for single-neuron action potentials during microneurography.</td></tr><tr><td>BNC T Splitter (1+)</td><td>NA</td><td>NA</td><td>APTrack Dependency. Any standard BNC T splitter.</td></tr><tr><td>BNC to BNC cables (3+)</td><td>NA</td><td>NA</td><td>APTrack Dependency. Any standard BNC cables.&amp;nbsp;</td></tr><tr><td>C6H11NaO7</td><td>Merck</td><td>S2054</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>CaCl2</td><td>Merck</td><td>C5670</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>Digitimer DS4 Constant Current Stimulator</td><td>Digitimer</td><td>DS4</td><td>Constant current stiulator for animal research. &amp;pound;1,695 GBP as of September 2022.&amp;nbsp;</td></tr><tr><td>Digitimer DS7 Constant Current Stimulator</td><td>Digitimer</td><td>DS7A</td><td>Constant current stiulator for human research. &amp;pound;3,400 GBP as of September 2022.&amp;nbsp;</td></tr><tr><td>Electroaccupuncture Classic Plus Stimulating Electrodes</td><td>Harmony Medical</td><td>NA</td><td>For fixed position intradermal electrical stimulation of the dorsal aspect of the foot during human microneurography.</td></tr><tr><td>Glucose</td><td>Fisher Scientific</td><td>G/0450/60</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>HDMI Cable</td><td>NA</td><td>NA</td><td>APTrack Dependency. Any standard passive HMDI cable. To connect OE I/O Board to OE Acquisition Board.</td></tr><tr><td>KCl</td><td>Merck</td><td>P9541</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>MgSO4</td><td>Acros Organics</td><td>213115000</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>Mineral Oil</td><td>Merck</td><td>330779</td><td>Electrical insulation for nerve recordings in th skin-nerve preparation. Or equivalent.</td></tr><tr><td>NaCl</td><td>Merck</td><td>S9888</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>NaHCO3</td><td>Merck</td><td>S6014</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>NaHCO3</td><td>Merck</td><td>S0751</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>Open Ephys Acquisition Board</td><td>Open Ephys</td><td>NA</td><td>APTrack Dependency. Includes USB cable to connect to computer and mains socket power supply. &amp;euro;2,955 EUR as of September 2022.</td></tr><tr><td>Open Ephys Graphical User Interface</td><td>Open Ephys</td><td>NA</td><td>https://github.com/open-ephys/plugin-GUI</td></tr><tr><td>Open Ephys I/O Board</td><td>Open Ephys</td><td>NA</td><td>APTrack Dependency. For ADC voltage inputs via BNC cables. &amp;euro;12.5 EUR without connectors, &amp;euro;85 EUR with connectors as of September 2022.</td></tr><tr><td>PulsePal V2</td><td>Sanworks</td><td>1102</td><td>APTrack Dependency. Open-source DAC and train generator. $725 USD pre-assembled as of September 2022. Approx. $275 USD for self-assembly.</td></tr><tr><td>RHD 6ft SPI Cable</td><td>Intan Technology</td><td>C3206</td><td>APTrack Dependency. For connecting headstage to OE Acquisition Board. $295 USD as of March 2021</td></tr><tr><td>RHD2216 16ch Bipolar Headstage</td><td>Intan Technology</td><td>C3313</td><td>APTrack Dependency. For data acquisition and digitization. $725 USD as of March 2021. Or equivalent RHD2000 series headstage.</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S/8560/60</td><td>Skin-nerve preparation synthetic interstitial fluid constituent. Or equivalent.</td></tr><tr><td>TCS-II Thermal Stimulator</td><td>QST.Lab</td><td>NA</td><td>For thermal stimualtion of nociceptor receptive fields during human microneurography.</td></tr><tr><td>Tungsten Microelectrode Pair (Active + Reference)</td><td>FHC</td><td>30085</td><td>For microneurography recordings. 35mm.</td></tr><tr><td>Ultrasound Scanner iQ+&amp;nbsp;</td><td>Butterfly Network</td><td>NA</td><td>For ultrasound-guided electrode insertion during microneurography.</td></tr><tr><td>USB 3.0 5kV RMS Isolation</td><td>Inota Technology</td><td>7055-D</td><td>For isolating human microneuroography participant from computer. &amp;euro;459 EUR as of September 2022.</td></tr><tr><td>USB-A to micro USB-B cable (2)</td><td>NA</td><td>NA</td><td>APTrack Dependency. To connect computer to PulsePal and to uStepper S-lite if using stepper-stimulator interfacing.&amp;nbsp;</td></tr><tr><td>uStepper S-lite + NEMA17 motor</td><td>uStepper</td><td>NA</td><td>To interface with stimulators via a control dial. &amp;euro;50 EUR as of September 2022.</td></tr><tr><td>Von Frey Filaments</td><td>Ugo Basile</td><td>37450-275</td><td>For mechanical stimulation of receptive fields during sensory phenotyping of nociceptors.</td></tr></tbody></table>

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 (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). 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&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-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.

A representative example of the software working to control an experiment is shown in Figure 7. It iteratively adjusts the stimulation amplitude using an up-down method to effectively find the electrical threshold of single nociceptors. For the first time, we demonstrate the feasibility of real-time single-neuron electrical threshold tracking in humans during microneurography (Figure 7A). Additionally, we show electrical threshold tracking in a mouse A&#948;-fiber (Figure 7B). The identification of action potentials by threshold crossing, as used here, is sufficient for tracking electrical thresholds over time. We recommend users take steps to minimize electrical noise during their recordings, such as by using a Faraday cage and bandpass filters to improve the signal-to-noise ratio.
To demonstrate that electrical threshold tracking can be used as a measure of changes in nociceptor excitability in humans, tracking of the electrical threshold during a stepped heating paradigm was performed (Figure 8). Increasing the temperature of the nociceptor terminals decreased the electrical stimulation current required to elicit an action potential, reflecting an increase in nociceptor excitability (Figure 8C). This was likely caused by the generation of receptor potentials by the heat-sensitive ion channels expressed in the C-fiber nociceptor14. At the highest temperature step, 44 &#176;C, thermally evoked action potentials were elicited (Figure 8A, stimulus number 86-96). This causes an increase in the electrical threshold as the nociceptor may be in a refractory state following high-frequency discharge. As expected, the latency of the tracked action potential decreased as the temperature increased. This is thought to occur due to a thermodynamic effect on the conduction machinery, which increases the conduction velocity of the C-fiber. This C-fiber may also be exhibiting flip-flop (Figure 8B, stimulus number 47-54), which can result in the following electrical stimulation being erroneously increased in amplitude if the action potential falls outside of the algorithm search window.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64898/64898fig01.jpg" /> 
Figure 1: A schematic of the equipment setup and cable connections required for nociceptor electrical threshold tracking with APTrack in rodents and humans. Note the two different methods of stimulation amplitude controls: a stepper motor for manually adjusted stimulators in our human setup, and a PulsePal for input voltage-controlled stimulators in our rodent setup. (1) A PC (Windows, Mac, or Linux) running the plugin for the Open Ephys platform. (2) A stepper motor that operates the stimulation amplitude dial on the DS7. (3) A constant current stimulator approved for use in humans; here we used a DS7. (4) A USB 3.0 optoisolator, which isolates the human participant from the PC (optional, only required for human research). (5) A PulsePal V2 Pulse Generator, which generates TTL time stamps (output channel 2) and voltage steps corresponding to the requested stimulation amplitude (output channel 1). (6) A constant current stimulator for use in animals; here, we used a DS4. (7) A DC power supply for the system (mains DC power supply used for the rodent setup and battery DC power supply used for the human setup). (8) An acquisition board. (9) An I/O board to connect the BNC coaxial cables carrying the signals to be recorded, such as the thermocouple outputs and TTL markers. (10) The mouse skin-nerve preparation undergoing nociceptor electrophysiological recordings. (11) A human participant undergoing microneurography recording from C-fibers in the superficial peroneal nerve. (12) An Intan RHD2216 headstage for the acquisition and digitization of the recordings. (13) An Intan Electrode Adapter Board, which the recording electrodes are connected to and which allows the signal to be passed to the RHD2216 headstage. (14) A thermal stimulation system that can output the temperature via a BNC coaxial connection. (15) A 3.3 V battery-powered button/foot pedal that is used for marking the mechanical stimulation events and drug applications. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64898/64898fig02.jpg" /> 
Figure 2: Template signal chain. The red arrow points to the button for enabling the ADC input from the I/O board. The yellow arrow indicates the drop-down menu for selecting the Open Ephys file format. The green arrow indicates the Play and Record buttons. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64898/64898fig03.jpg" /> 
Figure 3: Graphical user interface. The GUI consists of four main components. (1) Temporal Raster Plot panel (green) for data visualization and the settings associated with controlling the plot. A constant latency response showing gradual activity-dependent slowing is indicated by the green arrow. (2) Stimulation Control panel (yellow) for setting the stimulation amplitude parameters and loading the stimulation paradigm scripts. (3) Multi-Unit Tracking Table (blue) for adding the action potentials for tracking and activating the latency and electrical threshold tracking. (4) Options Menu for selecting the color styles and the input channel for the data and TTL triggers. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64898/64898fig04.jpg" /> 
Figure 4: Facilitation of the identification of constant latency action potentials through real-time data visualization on a temporal raster plot using APTrack. This is a high signal-to-noise ratio example. The data presented in the temporal raster plot are from a human C-fiber recording from the superficial peroneal nerve during microneurography. Voltage Trace is the oscilloscope-like LFP Viewer plugin within Open Ephys. The APTrack User Interface is the graphical user interface of the plugin. The tracked action potential is indicated by green arrows, and the circular slider on the border of the temporal raster plot is for controlling the search box position where the algorithms will search for threshold crossing events. The electrical stimulation artifact is marked in blue on the voltage trace. The stimulation amplitude of the analog voltage command is indicated in red; note that this may not be the same as the stimulation current amplitude depending on the scaling factor set on the stimulator. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64898/64898fig05.jpg" /> 
Figure 5: Graphical representation of the latency tracking algorithm. In simple terms, if an action potential is detected by threshold crossing, the search box will adjust its position to center itself at the time of the peak voltage. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64898/64898fig06.jpg" /> 
Figure 6: Graphical representation of the electrical threshold tracking algorithm. In simple terms, if an action potential is detected by threshold crossing, the stimulation amplitude will be decreased by the decrement rate. If no action potential is detected, the stimulation amplitude will be increased by the increment rate. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64898/64898fig07.jpg" /> 
Figure 7: Automated electrical threshold tracking of single-neuron action potentials at a 0.25 Hz stimulation frequency. (A) Sequential traces of a human C-fiber of the superficial peroneal nerve during a microneurography experiment. (B) Sequential traces of a mouse A&#948;-fiber of the saphenous nerve during skin-nerve preparation teased fiber electrophysiology. The traces were colored red when an action potential was identified, resulting in a decrease in the stimulus amplitude. The software algorithm effectively finds the stimulus amplitude required for a 50% likelihood of firing. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64898/64898fig08.jpg" /> 
Figure 8: Electrical threshold tracking at a 0.25 Hz stimulation frequency during the thermal stimulation of a human C-fiber nociceptor. The y-axis encodes the stimulation number from the start of the paradigm. (A) Voltage trace for 4,000 ms following electrical stimulation, with threshold crossing events marked in red. (B) Voltage trace from A zoomed in around the tracked action potential. The traces were colored red when the tracked action potential was detected. The vertical blue line is the baseline latency of the tracked unit. (C) Stimulation current commanded by APTrack. The vertical blue line is the baseline electrical threshold. (D) Receptive field TCS-II thermal stimulating probe temperature. <a href="https://www.jove.com/files/ftp_upload/64898/64898fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>107.8 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>3.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.67 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1.53 mM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>0.69 mM</td>
		</tr>
		<tr>
			<td>Sodium Gluconate</td>
			<td>9.64 mM</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>7.6 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5.55 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Contents of the synthetic interstitial fluid for the mouse skin-nerve preparation23.

Watch this Scientific Journal Video about Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research at JoVE.com

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

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

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1.2K Views.  University of Bristol. Deze tool kan worden gebruikt om verschillende mechanismen van nociceptorsensibilisatie te onderzoeken, en het is deze sensibilisatie die sommige vormen van chronische pijn kan veroorzaken. Geautomatiseerde elektrische drempeltracking biedt een betrouwbare, online, real-time meting van de prikkelbaarheid van nociceptor. Als zodanig biedt deze maatregel een belangrijke translationele brug, waardoor metingen kunnen worden gedaan bij zowel mensen als dieren.Op dezelfde manier, waardoor ef...

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

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Behavior

Psychophysically-anchored, Robust Thresholding in Studying Pain-related Lateralization of Oscillatory Prestimulus Activity

In perceptual studies, it is often important to objectively assess the equality of delivered stimulation across participants or to quantify the intra-individual sensation magnitude that is evoked by stimulation over multiple trials. This requires a robust mapping of stimulus magnitude to perceived intensity and is commonly achieved by psychophysical estimation methods such as the staircase procedure. Newer, more efficient procedures like the QUEST algorithm fit a psychophysical function to the data in real time while at the same time maximizing the efficiency of data collection. A robust estimate of the threshold intensity between painful and nonpainful perceptions can then be used to reduce the influence of variations in sensory input in subsequent analyses of oscillatory brain activity. By stimulating at a constant threshold intensity determined by an adaptive estimation procedure, the variance in the ratings can be directly attributed to perceptual processes. Oscillatory activity can then be contrasted between &#34;pain&#34; and &#34;no-pain&#34; trials directly, yielding activity that closely relates to perceptual classification processes in nociception.

Psychophysical methods such as the QUEST estimation procedure can efficiently yield robust estimates of the stimulation intensity at which nonpainful sensations transition into painful sensations. By stimulating repeatedly at the threshold intensity, the variability in rating responses can directly be attributed to perceptual classifications in subsequent analyses.

In perceptual studies, it is often important to objectively assess the equality of delivered stimulation across participants or to quantify the ...

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Biology

Multi-Modal Signals for Analyzing Pain Responses to Thermal and Electrical Stimuli

The assessment of pain relies mostly on methods that require a person to communicate. However, for people with cognitive and verbal impairments, existing methods are not sufficient as they lack reliability and validity. To approach this problem, recent research focuses on an objective pain assessment facilitated by parameters of responses derived from physiology, and video and audio signals. To develop reliable automated pain recognition systems, efforts have been made in creating multimodal databases in order to analyze pain and detect valid pain patterns. While the results are promising, they only focus on discriminating pain or pain intensities versus no pain. In order to advance this, research should also consider the quality and duration of pain as they provide additional valuable information for more advanced pain management. To complement existing databases and the analysis of pain regarding quality and length, this paper proposes a psychophysiological experiment to elicit, measure, and collect valid pain reactions. Participants are subjected to painful stimuli that differ in intensity (low, medium, and high), duration (5 s / 1 min), and modality (heat / electric pain) while audio, video (e.g., facial expressions, body gestures, facial skin temperature), and physiological signals (e.g., electrocardiogram [ECG], skin conductance level [SCL], facial electromyography [EMG], and EMG of M. trapezius) are being recorded. The study consists of a calibration phase to determine a subject&#8217;s individual pain range (from low to intolerable pain) and a stimulation phase in which pain stimuli, depending on the calibrated range, are applied. The obtained data may allow refining, improving, and evaluating automated recognition systems in terms of an objective pain assessment. For further development of such systems and to investigate pain reactions in more detail, additional pain modalities such as pressure, chemical, or cold pain should be included in future studies. Recorded data of this study will be released as the &#8220;X-ITE Pain Database&#8221;.

This article focuses on the experimental elicitation of pain through heat (thermal) and electrical stimulation while recording physiological, visual, and paralinguistic responses. It aims at collecting valid multimodal data for analyzing pain based on its intensity, quality, and duration.&#160;

The assessment of pain relies mostly on methods that require a person to communicate. However, for people with cognitive and verbal impairments, existing ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Real-Time Mouse Eye Squint Detection for Migraine Pain Tracking Using DeepLabCut

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Author Spotlight: Deciphering Electrical Networks Behind Complex Brain Activities and Disorders

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Medicine

Conventional and Threshold-Tracking Transcranial Magnetic Stimulation Tests for Single-handed Operation

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used to examine corticospinal excitability. Paired-pulse TMS paradigms (e.g., short- and long-interval intracortical inhibition (SICI/LICI), short-interval intracortical facilitation (SICF), and short- and long-latency afferent inhibition (SAI/LAI)) provide information about intracortical inhibitory and facilitatory networks. This has long been done by the conventional TMS method of measuring changes in the size of the motor-evoked potentials (MEPs) in response to stimuli of constant intensity. An alternative threshold-tracking approach has recently been introduced whereby the stimulus intensity for a target amplitude is tracked. The diagnostic utility of threshold-tracking SICI in amyotrophic lateral sclerosis (ALS) has been shown in previous studies. However, threshold-tracking TMS has only been used in a few centers, in part due to the lack of readily available software but also perhaps due to uncertainty over its relationship to conventional single- and paired-pulse TMS measurements.
A menu-driven suite of semi-automatic programs has been developed to facilitate the broader use of threshold-tracking TMS techniques and to enable direct comparisons with conventional amplitude measurements. These have been designed to control three types of magnetic stimulators and allow recording by a single operator of the common single- and paired-pulse TMS protocols.
This paper shows how to record a number of single- and paired-pulse TMS protocols on healthy subjects and analyze the recordings. These TMS protocols are fast and easy to perform and can provide useful biomarkers in different neurological disorders, particularly neurodegenerative diseases such as ALS.

We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator.

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

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

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