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

This tool can be used to investigate different mechanisms of nociceptor sensitization, and it is this sensitization that can drive some forms of chronic pain. Automated electrical threshold tracking provides a reliable, online, real-time measure of nociceptor excitability. As such, this measure provides an important translational bridge, allowing measurements to be made in both humans and in animals.

In the same way, allowing effective pathology and treatments to be assessed. In the future, AP Track could be used in chronic pain patients to confirm whether a therapeutic agent is normalizing the excitability of their sensitized nociceptors. This would represent a crucial biomarker of efficacy.

We anticipate that our open-source toolkit, AP Track, will be useful to electrophysiologists studying time loop stimuli of differing magnitudes. For example, we also think it'll be useful for studying optogenetics. Managing an experiment and software simultaneously is challenging at first, so I recommend users load prerecorded data into AP Track to familiarize themselves with its use before attempting an experiment.

We have provided demo data. To begin, connect the acquisition board to the computer using the manufacturer-supplied cable, and power them on. Then, connect the IO board to the analog import on the acquisition board and connect an Intan RHD Recording Headstage to the acquisition board using a Serial Peripheral Interface cable.

Next, connect the Pulse Pal to the computer. Split the signal of the Pulse Pal output channel one using a BNC T Splitter, and then connect it to the constant current stimulator input and the IO board, so that the analog voltage command can be recorded. Connect the Pulse Pal output channel two to the IO board to record the stimulation TTL event markers.

For assembling with a dial-controlled constant current stimulator, power on a constant current stimulator and connect the stepper motor control board to the stepper motor using the manufacturer-supplied cable and magnetic mount. Connect the control board to the computer directly using any standard USB-A to USB micro B cable. Connect the control board and the stepper motor to a custom mounting bracket, and set the stimulation amplitude dial on the constant current stimulator to zero milliamperes.

Then, connect a custom barrel adapter to the stepper motor barrel. Attach the stepper motor and custom mount apparatus to the stimulation amplitude dial on the constant current stimulator using the barrel adapter, and power it on. Open the AP Track GUI and establish a stable peripheral nerve electrophysiological recording.

Identify the receptive field on the skin and position the stimulating electrode there. In the Options menu, select Trigger Channel and choose the ADC channel containing the electrical stimulation TTL marker from Pulse Pal output channel two. Then, select the data channel and choose the channel containing the electrophysiological data.

Click Connect to connect AP Track to the Pulse Pal and stepper motor apparatus. This may take a moment. Once connected, the stepper motor control board will set itself to position zero.

In the stimulation control panel, define the initial minimum and maximum stimulation amplitudes using the slider. Ensure the current stimulation is set above zero, so that TTL markers are generated. Click F to load a file containing the stimulation instructions, and then click on the right arrow to begin the loaded stimulation paradigm.

The temporal raster plot will begin updating with the response to electrical stimulation, with each new stimulation response being displayed as a new column on the right. To successfully detect the single neuron action potentials, go to the temporal raster plot panel and adjust the low, detection, and high image threshold values. With suitable image thresholds set, the threshold crossing events detected by the algorithms will be encoded in green.

Systematically, move the stimulating electrode around the skin area innervated by the nerve. Monitor the temporal raster plot for three threshold crossing events that appear in a row at the same latency while the electrode is in the same stimulation position. This indicates the identification of a constant latency peripheral neuron action potential.

After identifying the single neuron action potential on the temporal raster plot, move the gray linear slider on the right side of the plot to adjust the position of the search box. Then, adjust the search box with rotary slider to an appropriate width. Make the search box width narrow.

To begin tracking the targeted action potential, click on the plus sign below the multi-unit tracking table. A new row will be added to the table containing the details of the target action potential, including the latency location, the percentage firing over two to 10 stimuli, and the peak amplitude detected. The latency tracking algorithm will automatically be executed on it upon every subsequent electrical stimulation.

Check the track spike box in the table to move the search box to the appropriate position for that particular action potential. Calculate the conduction velocity of the peripheral neuron by dividing the distance between the stimulation and recording sites by the latency displayed in the table. To perform the electrical threshold tracking, adjust the increment and decrement rates in the stimulation control panel to the desired rate.

Keep these values equal. Ensure the stimulation frequency is set to an appropriate rate, typically 0.25 to 0.5 Hertz. Manually adjust the stimulation amplitude approximately to the electrical threshold of the neuron.

Then, check the track threshold box in the multi-unit tracking table, which will initiate the electrical threshold tracking algorithm. In the multi-unit tracking table, monitor the firing rate. A firing rate of 50%indicates that the approximate electrical threshold has been determined and the threshold value will be updated.

Finally, apply an experimental manipulation to the receptive field and continue tracking the electrical threshold. This will quantify changes in the peripheral neuron excitability. The sequential traces of a human C fiber of the superficial perennial nerve during a micro neurography experiment and the sequential traces of a mouse A-delta fiber of the saphenous nerve during skin-nerve preparation teased fiber electrophysiology are shown in this figure.

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. The electrical threshold tracking at a 0.25 Hertz stimulation frequency during the thermal stimulation of a human C fiber nociceptor is presented in this figure.

The y-axis encodes the stimulation number from the start of the paradigm. The voltage traces for 4, 000 milliseconds following the electrical stimulation with the threshold crossing events are marked in red. The voltage trace zoomed in around the tracked action potential is shown here.

The vertical blue line is the baseline latency of the tracked unit. The stimulation current commanded by AP Track is shown in this figure. The vertical blue line is the baseline electrical threshold.

The receptive field TCS-II thermal stimulating probe temperature is presented here. As the receptive field of this heat-sensitive C fiber is warmed by the thermal stimulator, the electrical threshold decreases. Choosing the appropriate values for the search box width and detection threshold is important, as they significantly improve AP Track's performance by reducing the impact of electrical noise.

Quantifying the impact of therapeutic agents on hyperexcitability in nociceptors may help us better understand the mechanisms underlying chronic pain. We hope other researchers will utilize this freely available tool to better understand nociceptive biology and the changes that occurred during nociceptor sensitization.