Name: Standardized Induction and Assessment of Long-term Potentiation-like Cortical Plasticity Using Transcranial Magnetic Stimulation (Video) | JoVE Journal
Uploaded: 2025-11-07T14:45:00.000+00:00
Duration: 8 min 29 s
Description: Learn standardized neuronavigation-guided iTBS protocols for reliable LTP-like cortical plasticity assessment using transcranial magnetic stimulation. This method improves reproducibility in synaptic plasticity research.

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5 minutes

This study was supported by the National Natural Science Foundation of China (No.82372582, 82503067), the Competitive Project of Jiangsu Province&#39;s Key Research and Development Program (No.BE2023034).

The Ethics Committee of the First Affiliated Hospital with Nanjing Medical University approved the protocols (number 2023-SR-789), and the protocol was registered with the Chinese Clinical Trial Registry (number ChiCTR2400082549). All procedures were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained prior to enrollment in the study.
1. Consent process
<ol>
	<li>Prior to any assessment, explain the aim(s) of the assessment, the main experimental procedures, and any potential risk factors associated with the study. Answer any questions or concerns. Request acknowledgment of the consent process and signature on the informed consent form.</li>
	<li>Failure to meet TMS safety criteria constitutes grounds for exclusion. Ask the following questions with responses documented. A yes response to any criterion constitutes grounds for exclusion: 
	Do you have any metal implants or devices incompatible with TMS (e.g., pacemakers, cochlear implants, aneurysm clips)? 
	Do you have a history of epilepsy or seizures, or a family history of epilepsy? 
	Are you currently taking any medications that may induce seizures? 
	Do you have severe cognitive or communication deficits that would prevent participation in the study? 
	Do you have severe cervical spine disorders (e.g., cervical stenosis or spinal instability)? 
	Do you have any direct injuries or skull defects in the stimulation area? 
	If you are a female of childbearing potential: Are you pregnant or possibly pregnant? If pregnancy is suspected, perform a blood or urine HCG test and exclude positive cases.</li>
</ol>
2. Preparation of the head model using a neuronavigation system
<ol>
	<li>Use the neuronavigation system (version 2.5.3.50294) throughout the protocol to guide coil positioning, register anatomical landmarks, calibrate the coil, and maintain consistent coil placement during RMT determination and iTBS stimulation.</li>
	<li>Neuronavigation system setup: Use the system to obtain real-time visual feedback on the TMS coil position and its alignment relative to anatomical landmarks, ensuring accurate targeting during functional identification of the motor hotspot in the primary motor cortex (M1) corresponding to the contralateral target muscle20.</li>
	<li>Establishment of three-dimensional (3D) head model: Generate the 3D head model either using the standard brain templates provided within the neuronavigation software or constructed from individual magnetic resonance imaging (MRI) scans. By selecting either, the individual MRI or standard template mode, register anatomical landmarks across the axial, sagittal, and coronal planes. Perform scalp surface segmentation to generate a three-dimensional head model.</li>
	<li>Coil calibration
	<ol>
		<li>After completion of head model registration, perform coil calibration to ensure precise real-time tracking within the neuronavigation system. Navigate through TMS Settings, click on Coil Selection&gt; Calibrate Tool Faces. Place the TMS coil, equipped with reflective markers, on a designated calibration block. Align fiducial points on the physical coil with the corresponding points on the calibration block under software guidance.</li>
		<li>After initial calibration, follow the system prompt to position the coil over the calibration block to confirm accuracy. Click Calibrate to initiate the automatic calibration. Accept only calibrations with a spatial error below 3 mm21. Otherwise, perform recalibration. Ensure the coil's position, orientation, and tilt angle can be reliably tracked throughout the entire TMS session22.</li>
	</ol>
	</li>
</ol>
3. Motor hotspot identification
<ol>
	<li>Participant positioning: Ask the participant to sit in a comfortable chair with back and arm support to minimize head and body movement. Keep both hands completely relaxed and still during the procedure. Avoid movements of the non-targeted hand that could confound measurements.</li>
	<li>Head tracking setup: Attach the infrared reflective markers to the forehead to enable real-time tracking of head position during the session. Secure the markers with adhesive patches and ensure they are recognized by the neuronavigation system, indicated by green markers (unrecognized markers appear red).</li>
	<li>Landmark registration: Use a tracked pointer to sequentially mark three anatomical landmarks on the scalp: the nasion, left supratragic notch, and right supratragic notch. Enable the neuronavigation system to spatially register the actual head position with the 3D head model23.</li>
	<li>Head shape sampling: Use the pointer to collect approximately 200 to 300 additional points on the scalp to enhance registration accuracy24. Allow the software to automatically fit an individualized head shape model based on these points to optimize the alignment between MRI images and the actual coordinate system25.</li>
	<li>EMG recording: Place the recording electrode over the belly of the target abductor pollicis brevis (APB) muscle and the reference electrode near the interphalangeal joint of the thumb, approximately 2 cm apart26. Record MEPs at a sampling rate of 100 kHz with a minimum resolution of &lt;0.2 µV and a frequency response range of 1-25 kHz, while electrode impedances were maintained below 5 kΩ. Ensure that the baseline EMG signal shows minimal noise, a stable waveform, and no obvious drift or electrical interference before proceeding27.</li>
	<li>Determine the motor hotspot
	<ol>
		<li>Determine M1 coordinates by administering single-pulse TMS (figure-of-eight coil, 70 mm) approximately 5 cm lateral and 0-1cm anterior relative to the vertex28, contralateral to the target muscle to elicit maximal MEP amplitude, facilitating precise localization of the motor hotspot. Keep the hand completely relaxed to avoid voluntary muscle contraction that could interfere with MEP measurements.</li>
		<li>Orient the handle of the coil 45° posterior to the midline to transmit the electromagnetic current perpendicular to the central sulcus29. Move the coil systematically in 1 cm steps around the marked M1 region in all directions, including anterior, posterior, medial, and lateral30, at 5 s intervals31. Define the motor hotspot as the site producing the largest MEP amplitude in the relaxed APB32.</li>
	</ol>
	</li>
	<li>Motor hotspot marking: Once the functionally defined motor hotspot is identified, mark it immediately within the neuronavigation system. Upon marking, allow the system to automatically record key spatial parameters, including the distance between the coil and the target point, the tilt deviation, and the rotation deviation. Use these metrics to ensure precise and reproducible coil placement throughout the stimulation procedure33.</li>
</ol>
4. RMT determination
<ol>
	<li>Measure the peak-to-peak amplitude using surface EMG data to obtain the MEP, applying the same EMG setup and parameters as described in step 3.5. Define RMT as the minimum stimulus intensity that elicits an MEP with peak-to-peak amplitudes greater than 50 µV in at least five out of ten consecutive single-pulse TMS trials28.</li>
</ol>
5. Assessment of LTP-like plasticity
<ol>
	<li>Baseline assessments: Record 20 consecutive TMS-evoked MEPs at 5 s intervals at the motor hotspot. Set the stimulus intensity of MEPs to 120% RMT, which evokes MEPs of approximately 1 mV34.</li>
	<li>Induction of LTP-like plasticity: Deliver stimulation using a TMS device (figure-of-eight coil, 70 mm). Apply iTBS over the motor hotspot at an intensity of 80% RMT to induce LTP-like plasticity. Use the iTBS protocol consisting of bursts of three stimuli at 50 Hz repeated at 5 Hz. Repeat a 2 s train of this pulse stimulus followed by 8 s of rest for a total of 200 s (600 pulses)10.</li>
	<li>Plasticity assessment: Record 20 MEPs at 5 min, 10 min, 15 min, and 30 min after the iTBS intervention using the same stimulation intensity (120% RMT) to assess plasticity35.</li>
	<li>LTP-like plasticity quantification: Calculate the mean peak-to-peak amplitude of the 20 MEPs recorded at each time point (baseline, 5 min, 10 min, 15 min, and 30 min post-iTBS) to quantitatively reflect cortical excitability36. Post-stimulation MEP amplitudes are expressed as a normalized ratio relative to baseline to standardize excitability measures across sessions and individuals. The outcome is the raw MEP amplitude and normalized MEP amplitude at the final post-iTBS time point, representing the treatment effect37.</li>
	<li>Calculate the normalized MEP amplitude at each time point as: 
	<img alt="Normalized MEP equation showing MEPpost divided by MEPbaseline for data analysis in experiments." src="/files/ftp_upload/69425/69425eq1.jpg" data-altupdatedat="1784001604000" data-alt="Equation 1"> 
	Classify individuals with a grand average normalized MEP value &gt;1.1 as facilitated, &lt;0.9 as inhibited, and between 0.9 and 1.1 as unchanged following each iTBS condition38.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest pursuant to this work.

The application of TMS to investigate synaptic plasticity has great potential for advancing neurophysiological research. Specifically, single-pulse TMS can be used to assess LTP-like plasticity, while rTMS offers a practical, non-invasive method for inducing it. However, the accuracy and reproducibility of these findings depend heavily on the standardization of procedural parameters. Accordingly, this protocol provides a reliable and standardized approach for assessing LTP-like plasticity using TMS guided by a neuronavig...

In recent years, transcranial magnetic stimulation (TMS) has emerged as a non-invasive, cost-effective, and efficient technique for probing and modulating neural activity in the human brain1. Among various stimulation paradigms, intermittent theta burst stimulation (iTBS) has attracted significant attention for its ability to induce long-term potentiation (LTP)-like plasticity in the human motor cortex2. Specifically, iTBS delivers high-frequency bursts at theta intervals, mimicking endogenous theta-gamma coupling patterns associated with synaptic plasticity3. It induces LTP-like plasticity by activating N-methyl-D-aspartate receptors (NMDARs)4, which relieves the Mg2+&#160;block and allows Ca2+&#160;to enter the postsynaptic neuron5. This Ca2+&#160;influx triggers downstream signaling cascades, including the activation of calcium/calmodulin-stimulated protein kinase II (CaMKII), which promotes the phosphorylation6 and insertion of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), thereby enhancing synaptic transmission7. Compared with other non-invasive approaches, such as repeated transcranial magnetic stimulation (rTMS) or transcranial direct current stimulation (tDCS), iTBS can induce LTP-like cortical plasticity with shorter stimulation duration and lower intensity, making it a better-tolerated option in subjects8,9,10. To assess the neuroplastic effects induced by iTBS, researchers commonly measure changes in motor-evoked potential (MEP) amplitudes recorded through electromyography (EMG), which reflect enhanced corticospinal excitability11. Studies have shown that these MEP enhancements can persist for up to 60 min post-stimulation, indicating transient but robust modulation of cortical excitability10,12. Due to its brief administration time and well-established safety profile, iTBS is particularly suitable for repeated applications in both experimental and clinical contexts10. Specifically, a standard iTBS protocol (600 pulses, 192 s), as well as conventional 10-Hz rTMS protocols (1,200-1,500 pulses, 15-20 min), reliably induce comparable LTP-like plasticity effects8,13. As such, it has been increasingly used to probe synaptic plasticity in healthy individuals and patient populations, providing valuable insights into plasticity-related deficits in neurological disorders such as Alzheimer&#39;s disease (AD), stroke, and depression.
Synaptic plasticity, a fundamental mechanism of neural plasticity, underlies critical processes such as learning and memory. It reflects the brain&#39;s ability to modify the strength and efficacy of synaptic connections in response to experience or environmental stimuli14. Among various forms of synaptic plasticity, LTP is a well-established model for learning and memory through the enhancement of synaptic transmission15. Accumulating evidence indicates that impairments in LTP-like plasticity are closely associated with cognitive and behavioral deficits in neurological disorders such as AD16. These impairments may reflect disease-specific disruptions in synaptic signaling and plasticity-related molecular pathways, including alterations in the induction, expression, or maintenance of LTP17. Hence, understanding and quantifying synaptic plasticity is essential for advancing therapeutic strategies to restore cognitive function, motor control, sensory integration, and emotional regulation, and to facilitate effective neurorehabilitation.
While techniques such as iTBS for inducing LTP-like plasticity and single-pulse TMS for assessing cortical plasticity offer exciting potential, their application requires strict adherence to standardized protocols to ensure accuracy and reproducibility. Inconsistent methods can lead to variability, which may hinder the reliability of findings. Moreover, methodological inconsistencies across studies, including differences in stimulation intensity, coil positioning, and the timing of outcome measurements, limit the reproducibility of TMS-induced plasticity findings. In practice, iTBS is typically administered at 80% of the resting motor threshold (RMT)18, and reliable induction of LTP-like plasticity further depends on precise coil positioning, most commonly achieved through neuronavigation guidance19. Accordingly, this article aims to demonstrate a standardized, neuronavigation-guided protocol for inducing LTP-like plasticity through iTBS, followed by the assessment of cortical plasticity using single-pulse TMS. The focus of this article will be on the essential technical procedures and operational considerations necessary to achieve precise and reliable measurements of cortical plasticity.

<table><tbody><tr><td>70 mm TMS coil</td><td>Yiruide Co., Wuhan, China</td><td></td><td></td></tr><tr><td>Electromyography surface electrodes</td><td>Cathay, Shanghai, China</td><td>CM25R</td><td></td></tr><tr><td>Electromyography system</td><td>Yiruide Co., Wuhan, China</td><td></td><td></td></tr><tr><td>Neuronavigation pointer</td><td>ANT Neuro, Germany</td><td></td><td></td></tr><tr><td>Neuronavigation reflective markers</td><td>ANT Neuro, Germany</td><td></td><td></td></tr><tr><td>Neuronavigation system</td><td>ANT Neuro, Germany</td><td>Visor2, version 2.5.3.50294</td><td></td></tr><tr><td>TMS stimulator</td><td>Yiruide Co., Wuhan, China</td><td>NS5000</td><td></td></tr></tbody></table>

standardized induction and assessment of long-term potentiation-like cortical plasticity using transcranial magnetic stimulation

Standardized Induction and Assessment of Long-term Potentiation-like Cortical Plasticity Using Transcranial Magnetic Stimulation

In recent years, transcranial magnetic stimulation (TMS) has emerged as a non-invasive, low-cost, and effective technique for assessing synaptic changes in the brain. Synaptic plasticity is a fundamental mechanism of neural plasticity that reflects the brain&#39;s ability to modify the strength and efficacy of synaptic connections in response to experience or environmental stimuli. Long-term potentiation (LTP) serves as the predominant experimental model for studying synaptic plasticity mechanisms, playing a critical role in understanding synaptic plasticity and overall brain function. Specific stimulation patterns, especially repetitive or burst patterns of pulses such as intermittent theta burst stimulation (iTBS), could induce LTP-like cortical plasticity, providing a feasible non-invasive approach to evaluate synaptic plasticity. However, the application of these techniques requires strict adherence to standardized protocols to ensure the accuracy and reliability of the results. Therefore, this article aims to demonstrate a standardized approach for inducing LTP-like plasticity using iTBS, followed by assessment of synaptic plasticity with single-pulse TMS, with both procedures guided by a neuronavigation system. The focus of this article will be on the essential technical procedures and operational considerations necessary to achieve precise and reliable measurements of cortical plasticity.

During the demonstration, a neuronavigation system was used to guide the accurate positioning of the TMS coil over the motor hotspot, providing real-time spatial feedback and minimizing coil-placement variability. A TMS device (figure-of-eight coil, 70 mm) delivered stimulation throughout the session. To illustrate the procedure, representative results from one participant are presented below. The recorded MEP amplitudes exhibited stable and consistent responses across single-pulse trials, reflecting the stability provid...

This paper describes the standardized induction of Long-term Potentiation-like cortical plasticity using repetitive stimulation protocols, followed by applying single-pulse transcranial magnetic stimulation guided by a neuronavigation system to evaluate synaptic plasticity.

In recent years, transcranial magnetic stimulation (TMS) has emerged as a non-invasive, low-cost, and effective technique for assessing synaptic changes ...

We study corticoplasticity to understand and measure synaptic changes in health and neurological disorders. Variability in stimulation, coil positioning, and the measurement timing makes reproducible iTBS plasticity studies challenging. To begin, explain the aim of the assessment, the main experimental procedures, and any potential risk factors associated with the study.Request an acknowledgement of the consent process and obtain a signature on the informed consent form. Use the neuronavigation system to identify the functional motor hotspot in the primary motor cortex corresponding to the contralateral target muscle. Now, generate a three-dimensional head model.Using the individual MRI scans, select and register anatomical landmarks across the axial, sagittal, and coronal planes. Perform scalp surface segmentation to complete the generation of the three-dimensional head model. After completing head model registration, perform coil calibration to ensure precise real-time tracking within the neuronavigation system.In the software, navigate to Settings, click on TMS Coil, and select Calibrate Tool Faces. Calibrate fiducial points on the coil face, as well as the coils control point marker. Place the TMS coil with reflective markers on the designated calibration block.After initial calibration, follow the system prompt to place the coil over the calibration block and confirm its position for accuracy. Then, click on Neuronavigation to validate single coil. Accept only those calibrations with a spatial error less than three millimeters.Otherwise, repeat the calibration process. Ask the participant to sit in a comfortable chair with both back and arm support to minimize head and body movement. Instruct the participant to keep both hands completely relaxed and still throughout the procedure.Attach infrared reflective markers to the participant's forehead to enable real-time head tracking during the session, and secure the markers using adhesive patches. Using a tract pointer, mark three anatomical landmarks on the participant's scalp:the nasion, the left supratragic notch, and the right supratragic notch. Confirm that the markers are recognized by the neuronavigation system, indicated by their green appearance on screen.Using the same pointer, collect approximately 200 to 300 additional points on the scalp to improve registration accuracy. Allow the neuronavigation software to automatically generate an individualized head shape model from these points to optimize alignment between the MRI scans and the actual coordinate system. Next, place surface electrodes two centimeters apart on the abductor pollicis brevis muscle in a belly-tendon montage.Position the reference electrode distally, near the interphalangeal joint of the thumb. Next, administer single-pulse TMS approximately five centimeters lateral and zero to one centimeters anterior to the vertex, contralateral to the target muscle. Use this to determine the primary motor cortex coordinates that elicit the maximal motor-evoked potential amplitude for motor hotspot localization.Instruct the participant to keep their hand completely relaxed to prevent voluntary muscle contractions that may affect measurements. Orient the handle of the coil 45 degrees posterior to the midline to direct the electromagnetic current perpendicularly to the central sulcus. Systematically move the coil in one-centimeter steps around the motor cortex region in all directions, anterior, posterior, medial, and lateral, at five-second intervals.Identify the motor hotspot as the site that produces the largest motor-evoked potential amplitude and the shortest latency in the relaxed abductor pollicis brevis muscle. Allow the system to automatically record key spatial parameters, including the distance between the coil and the target point, the tilt deviation, and the rotation deviation. Use these values to ensure accurate and reproducible coil placement during stimulation.Using the same surface electromyography setup and parameters, measure the peak-to-peak amplitude of the motor-evoked potential. Define the resting motor threshold, or RMT, as the minimum TMS stimulus intensity that produces motor-evoked potentials over 50 microvolts in at least 5 of 10 single-pulse trials. Record 20 consecutive motor-evoked potentials at five-second intervals from the motor hotspot as a baseline measurement.Set the stimulation intensity to 120%of the RMT to evoke approximately one-millivolt motor-evoked potentials. Deliver stimulation using a TMS device. Set the stimulation intensity to 80%of the RMT.Apply bursts of three pulses at 50 hertz, repeated at five hertz. At 5, 10, 15, and 30 minutes after the iTBS, record 20 motor-evoked potentials using the same stimulation intensity to assess plasticity. Calculate the mean peak-to-peak amplitude of the 20 motor-evoked potentials recorded at baseline and at each post-stimulation time Point to quantify cortical excitability.Express the post-stimulation amplitudes as normalized ratios relative to baseline to standardized measurements across sessions and individuals. Following iTBS stimulation, motor-evoked potential amplitudes were recorded at multiple time points to assess cortical excitability over time. Mean raw motor-evoked potential amplitude increased after stimulation, peaking at 15 minutes and declining by 30 minutes.Normalized motor-evoked potential amplitude showed a similar trend, increasing above baseline and gradually declining by 30 minutes. The mean normalized motor-evoked potential amplitude across all post-stimulation time points exceeded 1.1, classifying the participant as facilitated. We offer standardized neuronavigation-guided iTBS protocol for reliable and reproducible plasticity assessment.Our protocol uses neuronavigation, ensuring precise, reliable iTBS delivery and robust LTP-like plasticity with better consistency. They enable reproducible studies of synaptic plasticity, supporting therapeutic development and neurorehabilitation strategies.

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Learn standardized neuronavigation-guided iTBS protocols for reliable LTP-like cortical plasticity assessment using transcranial magnetic stimulation. This method improves reproducibility in synaptic plasticity research.

Standardized Induction and Assessment of Long-term Potentiation-like Cortical Plasticity Using Transcranial Magnetic Stimulation (Video) | JoVE Journal

Standardized Induction and Assessment of Long-term Potentiation-like Cortical Plasticity Using Transcranial Magnetic Stimulation

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