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

Summary

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.

Abstract

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'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.

Introduction

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.

Protocol

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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70 mm TMS coil	Yiruide Co., Wuhan, China
Electromyography surface electrodes	Cathay, Shanghai, China	CM25R
Electromyography system	Yiruide Co., Wuhan, China
Neuronavigation pointer	ANT Neuro, Germany
Neuronavigation reflective markers	ANT Neuro, Germany
Neuronavigation system	ANT Neuro, Germany	Visor2, version 2.5.3.50294
TMS stimulator	Yiruide Co., Wuhan, China	NS5000