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, 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⁺ block and allows Ca²⁺ to enter the postsynaptic neuron⁵. This Ca²⁺ influx triggers downstream signaling cascades, including the activation of calcium/calmodulin-stimulated protein kinase II (CaMKII), which promotes the phosphorylation⁶ and insertion of α-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 subjects^8,9,10. 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 excitability^10,12. 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 effects^8,13. 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'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'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.

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

1. 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.
2. 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.

2. Preparation of the head model using a neuronavigation system

1. 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.
2. 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.
3. 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.
4. Coil calibration
   1. 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> 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.
   2. 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.

3. Motor hotspot identification

1. 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.
2. 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).
3. 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.
4. 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.
5. 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 <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.
6. Determine the motor hotspot
   1. 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.
   2. 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.
7. 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.

4. RMT determination

1. 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.

5. Assessment of LTP-like plasticity

1. 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.
2. 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.
3. 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.
4. 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.
5. Calculate the normalized MEP amplitude at each time point as:
   
   Classify individuals with a grand average normalized MEP value >1.1 as facilitated, <0.9 as inhibited, and between 0.9 and 1.1 as unchanged following each iTBS condition³⁸.

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 provided by coil placement guided by neuronavigation. Time-dependent increases in MEP amplitude following iTBS indicate LTP-like plasticity. The data can be analyzed by comparing baseline and post-stimulation raw MEP amplitudes and normalized MEP amplitudes, as well as by classifying individual responses as facilitation, inhibition, or unchanged. Overall, these representative results demonstrate that the described protocol enables accurate motor hotspot localization, reproducible stimulation, and quantitative assessment of stimulation-induced LTP-like plastic changes.

Neuronavigation system setup and localization

The neuronavigation system setup and localization procedure were performed to identify and register individual anatomical landmarks across axial, sagittal, and coronal planes, including the nasion, left supratragic notch, and right supratragic notch. These landmarks served as fiducial references for the subsequent creation of the individualized 3D head model, ensuring accurate coregistration between anatomical structures and stimulation targets (Figure 1). Spatial registration was initialized by identifying the same three anatomical landmarks on the scalp. The system provided real-time visual feedback on the coil position and its alignment relative to the predefined stimulation sites within the M1 contralateral to the target muscle, ensuring that stimulation was delivered accurately to the targeted cortical areas.

Figure 1: Landmark registration. Identification of anatomical landmarks on the participant's cranial using the neuronavigation system to enable accurate spatial registration. Please click here to view a larger version of this figure.

Establishment of 3D head model

An individualized 3D head model of the participant's scalp was generated based on neuronavigation registration and scalp surface sampling. The mean registration error during alignment of anatomical landmarks and head shape was below 1.5 mm, enabling precise coil placement throughout the stimulation session (Figure 2). 

Figure 2: 3D head model construction. Visualization of the reconstructed 3D head model based on neuronavigation registration and scalp surface sampling, allowing precise coil tracking and cortical mapping during stimulation. Please click here to view a larger version of this figure.

Identifying motor hotspot

The motor hotspot was functionally identified based on TMS-evoked MEPs by stimulating the brain with TMS and recording MEPs. The site producing the strongest response was defined as the motor hotspot (Figure 3). 

Figure 3: Motor hotspot localization. Real-time display of the stimulation site over the M1 contralateral to the target muscle corresponding to the motor hotspot for the target APB. Please click here to view a larger version of this figure.

Determining RMT

The RMT was determined using single-pulse TMS. The RMT was the lowest stimulation intensity at which MEPs with peak-to-peak amplitudes >50 µV were observed in at least 5 out of 10 consecutive trials, as per the standard RMT definition28, ensuring that the TMS stimulation was above the threshold for effective motor activation (Figure 4). 

Figure 4: RMT determination. Representative MEP waveform recorded from the target APB during RMT assessment. Numbers 1-10 indicate 10 consecutive single-pulse TMS trials. Please click here to view a larger version of this figure.

Baseline measurements

Prior to iTBS, corticospinal excitability was assessed by delivering 20 single-pulse TMS stimuli at 120% RMT with 5 s intervals over the identified motor hotspot (Figure 5). 

Figure 5: Baseline MEPs. Twenty representative MEPs from the target APB were elicited by single-pulse TMS at 120% RMT under relaxed conditions. Please click here to view a larger version of this figure.

Induction of LTP-like plasticity

The iTBS protocol was delivered at 80% of the individual RMT, using bursts of three pulses at 50 Hz repeated at 5 Hz (600 pulses over 200 s). Stimulator mode logs confirmed that all sessions delivered the planned pulse count without interruption, and the output intensity remained stable throughout.

LTP-like plasticity quantification

Following the application of the iTBS protocol, MEP amplitudes were recorded at multiple time points (e.g., 5 min, 10 min, 15 min, and 30 min) to observe changes in cortical excitability over time (Figure 6). 

Figure 6: Post-iTBS MEPs. Representative MEPs from one participant were recorded from the target APB at 120% RMT at (A) 5 min, (B) 10 min, (C) 15 min, and (D) 30 min after iTBS. Each panel shows 20 waveforms, illustrating time-dependent modulation in amplitude. Please click here to view a larger version of this figure.

Raw MEPs

To quantify excitability changes, the mean peak-to-peak MEP amplitudes were calculated at baseline and at each post-stimulation time point (Figure 7). 

Figure 7: Mean MEP amplitudes. The mean MEP amplitudes are recorded at baseline and at 5 min, 10 min, 15 min, and 30 min following iTBS in a representative participant. Each data point represents the mean of 20 single-pulse TMS stimuli, with error bars indicating the standard deviation (SD). Please click here to view a larger version of this figure.

Normalized MEPs

The MEP amplitudes at each post-stimulation time point were normalized to baseline. The time-dependent increase and subsequent decline in MEP amplitude reflect the characteristic profile of LTP-like plasticity (Figure 8). 

Figure 8: Normalized mean MEP amplitudes. The MEP amplitudes were normalized to baseline values (post/baseline ratio) at 5 min, 10 min, 15 min, and 30 min after iTBS in a representative participant. Please click here to view a larger version of this figure.

A noticeable increase in MEP amplitude was observed within the first few minutes after stimulation, reflecting a transient enhancement in corticospinal excitability. This enhancement gradually declines over time. According to the predefined classification criteria38 (normalized MEP value >1.1 as facilitated, <0.9 as inhibited, and between 0.9 and 1.1 as unchanged), the representative participant was classified as facilitated, with the mean normalized MEP value across all post-stimulation time points (5 min, 10 min, 15 min, and 30 min) exceeding 1.1. This time-dependent modulation is commonly interpreted as a manifestation of LTP-like plasticity.

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 neuronavigation system.

LTP, an established cellular model of synaptic plasticity³⁹, is essential for tuning synaptic efficacy and for learning and memory⁴⁰. Changes in LTP-like plasticity may reflect altered neurophysiological functioning and have been explored as a potential biomarker for early detection, prognosis, and treatment monitoring in neurological conditions such as AD⁴¹, stroke⁴², and PD⁴³. Hippocampal LTP dysfunction emerges at early stages of AD, preceding the onset of cognitive decline⁴⁴. This impairment is thought to be driven by amyloid-β and tau accumulation, indicating that LTP-like changes may serve as sensitive biomarkers for early diagnosis of AD⁴⁵. Similarly, in ischemic stroke, alterations in LTP-like plasticity have been positively correlated with recovery potential. In the acute phase, patients often show a reduced propensity for the induction of LTP-like changes in the affected motor cortex, and stronger LTP-like responses in this region have been associated with more favorable rehabilitation outcomes⁴². In PD, LTP-like plasticity in the primary motor cortex is reduced, regardless of the presence of dyskinesias, highlighting its potential value as a biomarker for disease monitoring⁴⁶. These findings underscore the utility of LTP-like plasticity as a functional biomarker for patient stratification and therapy monitoring in diverse clinical settings.

iTBS, a form of rTMS, can non-invasively and reliably induce LTP-like plasticity in the human cortex³. Compared with traditional high-frequency rTMS, iTBS achieves comparable or stronger induction of LTP-like effects in a shorter duration, reduces participant discomfort, and allows repeated measurements with minimal fatigue or adaptation⁸. Other non-invasive plasticity probes, such as tDCS or rTMS, can also modulate cortical excitability, but they often exhibit greater inter-individual variability or require more complex setups and longer intervention periods^47,48. However, the clinical application of iTBS demands rigorous procedural consistency. Small variations in factors such as coil positioning and stimulation parameters can introduce significant variability. By contrast, neuronavigation-guided iTBS ensures precise coil positioning, consistent stimulation intensity, and reproducible induction of LTP-like plasticity, enhancing both reliability and applicability in clinical and research settings⁴⁹. This approach therefore provides a unique opportunity to observe and measure plasticity-related phenomena in a controlled, efficient, and reproducible manner.

In clinical assessments, even minor inconsistencies in participant posture or coil placement can introduce variability in evaluation outcomes⁵⁰. To minimize this, stable seating with head support is required to ensure head stability and minimize movement during the procedure. This posture not only improves participant comfort but also ensures consistent coil placement across sessions, which is critical for both the efficacy of stimulation and the reproducibility of data. Moreover, the use of a neuronavigation system further enhances procedural precision. The neuronavigation system enables real-time tracking of the coil's position and orientation in 3D space⁴⁹, significantly enhancing targeting accuracy. This system not only ensures consistent placement of the TMS coil over the targeted cortical region but also provides continuous feedback on any positional deviations, including changes in angle and distance from the scalp²⁰. In addition to technical precision, ensuring participant safety is paramount. Although iTBS is generally safe and well-tolerated, it is imperative to adhere to established safety protocols. This includes thorough pre-screening for contraindications such as a history of epilepsy or the presence of metallic implants⁵¹. Furthermore, continuous monitoring of participant status during stimulation is implemented to ensure any potential adverse events are promptly detected and managed.

In practice, several common challenges may arise during iTBS application, for which practical modifications and troubleshooting strategies are available. First, coil placement variability can compromise stimulation accuracy. This can be minimized by using neuronavigation systems to ensure precise targeting. Second, participant movement is a frequent source of variability. To address this, participants should be seated comfortably in a chair with head and arm support and instructed to remain as still as possible. Third, signal quality issues, such as poor EMG recordings, may interfere with outcome assessment. Proper skin preparation and secure electrode placement are essential. During MEP evaluation, trials with artifacts are repeated or excluded. Finally, hardware and runtime parameters require verification. Calibration errors greater than 3 mm should prompt immediate recalibration. Confirm delivered intensity (%RMT) and pulse counts match the protocol specifications. Together, these modifications and quality checkpoints enhance reproducibility and ensure that the iTBS protocol is executed as intended.

Despite the standardized workflow, several inherent limitations of the technique should be acknowledged. First, the effects of iTBS are highly state-dependent and can vary with baseline cortical excitability, time of day, or ongoing neural activity, which may limit reproducibility across individuals. Second, motor-evoked potentials (MEPs), although commonly used as an index of corticospinal excitability, capture only a fraction of the underlying neurophysiological processes and provide limited insight into network-level changes. Finally, while iTBS can reliably induce transient increases in cortical excitability, evidence for its long-term effects and clinical translatability remains insufficient.

In summary, this study provides a structured workflow for inducing and measuring LTP-like plasticity using image-guided TMS. By integrating technical accuracy, standard positioning, and real-time neuronavigation feedback, this approach serves as a valuable instructional model for researchers and clinicians seeking to apply TMS in neurophysiological investigations.