Assessing Primary Motor Cortex Excitability and Excitability Modulation by Pairing Transcranial Magnetic Stimulation with Electromyography

Our research investigates our standardized TMS-EMG protocols can provide stable and clinically meaningful markers of neuroplasticity, ultimately supporting their potential use as reliable biomarkers in neuropsychiatric research and clinical practice, The main challenges are methodological inconsistencies in the literature, including heterogeneous stimulation parameters, limited standardization of EMG protocols, and small sample sizes, reducing reliability and reproducibility of experimental results. We demonstrated that structured and comprehensive protocol offers a replicable framework for investigating neuroplasticity-like phenomena in the human brain, providing strong foundations for translational research and potential future clinical applications. Our protocol was built by combining neuro navigation, validated EMG procedures, and optimized simulation parameters, ensuring precise code placement, stable recordings, and reliable results across participants and sessions.

Future research should test whether excitability modulation reliably distinguishes health individuals from those with neuropsychiatric conditions, such as major depressive disorder, exploring group specific neuroplasticity differences, diagnostic sensitivity, and prognostic value. To begin, plug the medical grade silver or silver chloride electrodes into the corresponding contacts to prepare three electrodes. Connect the cables with the electrodes to the EMG board.

Connect a cable from the TMS machine to the EMG board to output the trigger signal for each TMS pulse. On one of the computer monitors, open the Bonsai software and load the EMG data acquisition script. Click the Start button to start data acquisition only when ready.

Attach paper strips over all areas of the TMS machine screen where the maximum stimulation output percentage is displayed, to blind the technician during resting motor threshold, and active motor threshold assessment. Position a tripod mounted camera to maintain a clear view of all neuro navigation components throughout the entire session. Then, place the coil calibration tracker and the calibration board on top of the TMS coil.

Now, launch the neuro navigation system software, input the participant identification number, and proceed to calibrate all neuronavigation components. After positioning the participant, use water sandpaper to gently scrub the skin over the first dorsal interosseous. To improve the electrode skin interface.

Rinse the area with gauze embedded in alcohol and repeat the same procedure over the contralateral elbow. Once the skin is dry, place the recording electrode on the hand aligning the center approximately 2.5 centimeters along the direction of the muscle tendon over the first dorsal interosseous. Then place the ground electrode over the contralateral elbow to serve as a zero voltage reference point.

Place a swimming cap on the participant's head in an anterior posterior orientation. Align the cap with the eyebrow line at the front, and the root of the helix of each ear leaving the ears exposed. Offer the participant earplugs, and explain how to use them.

Draw the medial sagittal line and the inter tragus line on the cap to identify their intersection point at the vertex. From the vertex point, measure five centimeters forward along the sagittal line, and five centimeters to the left along the inter tragus line to define two new points. Next, draw a diagonal line connecting the lateral point to the medial anterior point.

From the lateral point, measure 2.5 centimeters in the antero medial direction along the diagonal line to mark the initial estimated motor hotspot. Around the initial motor hotspot estimate, mark four additional points spaced within a 0.5 centimeter radius to serve as alternative testing sites. Next, place the elastic headband onto the participant's head, making sure that the marker strip is facing the camera and that the band fits comfortably.

Using the pointer tool, define key anatomical landmarks on the participant's head, including the nasion, left and right tragus, and head shape points. This allows the system to align, and track stimulation targets based on head and brain anatomy. Position the TMS coil tangentially on the participant's scalp, with the handle angled posteriorly at 45 degrees to the midsagittal line.

Deliver pulses at 30%of the maximum stimulation output over the initial motor hotspot estimate. Then, increase stimulation intensity in five to 10%increments of maximum stimulation output until a consistent motor response is observed. Deliver single pulses at the initial estimate and surrounding marked locations to determine which site most consistently produces high amplitude motor evoked potentials and isolated first dorsal interosseous contractions.

Highlight the overall region being considered for the motor hotspot using a red marker line. In the neuronavigation software, select the pulse that produced the most reliable response to define the motor hotspot. To establish the resting motor threshold, or RMT, use the neuro navigation system to guide the coil back to the identified motor hotspot.

Deliver 10 single pulse stimulations spaced five seconds apart, and identify the minimum percentage of maximum stimulation output that produces at least five motor evoked potentials exceeding 50 microvolts in the contralateral first dorsal interosseous. Starting from the previously defined motor hotspot intensity, reduce the stimulation level in 2%increments until fewer than five out of 10 pulses result in a motor evoked potential of 50 microvolts or more in the contralateral first dorsal interosseous. Next, increase the maximum stimulation output in 1%increments until five or more out of 10 pulses generate a 50 microvolt or higher response in the contralateral first dorsal interosseous.

Record the percentage of maximum stimulation output that defines the resting motor threshold. Instruct the participant to generate maximum voluntary activation of the first dorsal interosseous muscle by forcefully pressing the index fingernail against the base of the thumb, to form a tight circle shape. Approximately two seconds after the muscle contraction begins, press the space bar on the computer running the Bonsai Script to record the time point while avoiding the initial movement artifact.

Now, ask the participant to maintain a sub maximal muscle contraction between 10 and 20%of their maximum voluntary isometric contraction. Use EMG feedback to monitor, and guide the participant to maintain the correct contraction level. For active motor threshold, use the neuro navigation system to guide the coil to the previously identified motor hotspot.

Determine the active motor threshold as the minimum percentage of maximum stimulation output that elicits motor evoked potentials of at least 200 microvolts, in five out of 10 pulses during slight voluntary contraction. From the resting motor threshold intensity, reduce the stimulation level by 2%decrements until fewer than five out of 10 pulses produce a 200 microvolt response. Then increase the intensity in 1%steps until at least five out of 10 pulses result in motor evoked potentials of 200 microvolts or more.

Record the percentage of maximum stimulation output that defines the active motor threshold. Accurately position the coil over the defined motor hotspot using the neuro navigation system. Check that surface EMG electrodes are correctly positioned over the first dorsal interosseous and verify that the signal quality shows minimal noise.

Load the single pulse stimulation protocol on the TMS device. And enter the resting motor threshold value previously recorded. Now, begin running the stimulation protocol.

Then load the intermittent theta burst stimulation protocol onto the TMS device. Enter the previously recorded active motor threshold and start the intermittent theta burst stimulation protocol. Immediately after the intermittent theta burst stimulation protocol ends, start a stopwatch to monitor post stimulation intervals.

At time 0 immediately after the end of the cortical excitability modulation protocol, reapply the single pulse stimulation protocol using 120%of the resting motor threshold. Validate the neuronavigation accuracy by placing the pointer on the nasion, and checking alignment in the neuro navigation system software. Ensure that the spatial deviation is less than five millimeters and record the measured value.

Finally, after completing the final stimulation block, stop the Bonsai Script and finalize the neuro navigation session in the software. Confirm that a binary data file is generated from the Bonsai acquisition session. Significant increases in motor evoked potential amplitude were observed at T0, T10, and T20, following intermittent theta burst stimulation during the baseline session.

But not at T30. At follow-up, significant increases in motor evoked potential amplitude were again observed at T0, T10, T20, and T30. Test retest reliability of motor evoked potential changes at T0 showed moderate to good consistency, with an intra class correlation coefficient of 0.67.