No acknowledgments.

This protocol involves research on human subjects and is in alliance with institutional and ethical guidelines of local ethical committees and the Declaration of Helsinki. Informed consent was obtained from subjects for using their data in the study.
1. Pre-experimental procedures
<ol>
	<li>Screening of the subject. Screen the subject for intracranial implants, epilepsy, history of seizures, and pregnancy. Use questionnaire guidelines to ensure compliance with up-to-date safety precautions32.
	<ol>
		<li>The delivery of electromagnetic pulses with TMS is contraindicated for individuals with intracranial implants of ferromagnetic material, such as shrapnel, aneurysm clips, or fragments from welding. Take precautions with individuals at increased likelihood of seizures.</li>
		<li>TMS assessment poses no fetal risk for pregnant women who are advised to have a conservative stance when dealing with this population. It is safe to apply TMS in pediatric populations, proceed cautiously in certain developmental stages (i.e., closure of the fontanelle, maturation of cortical excitability, and growth of the external auditory canal)33.</li>
	</ol>
	</li>
	<li>Preparation of materials. For this procedure, other than the TMS and EMG devices, have at your disposal a swim cap, alcohol pads (with the preparation of 70% isopropyl alcohol), conductive gel, and a computer turned on with the EMG software setup and a dynamometer appropriate for the investigated muscle (see Table of materials). 
	&#8203;NOTE: Swim caps have the advantage of being the cheapest and most accessible option that still allows for reliable and reproducible TMS assessments without causing the discomfort of marking the subjects&#39; head.</li>
</ol>
2. Appropriate instructions to the patients
<ol>
	<li>Explain the basic steps of the procedure and how much time it will take.</li>
	<li>Instruct the participant to remain awake but not to perform cognitive activities that require extra attention and/or focus (e.g., mathematical calculations, meditation, etc.) and anticipate that they might experience hand/jaw twitching or plausible side effects. Such events might seem unexpected for an inexperienced subject and thus jeopardize the procedure. 
	&#8203;NOTE: Single- and paired-pulse TMS have only been associated with mild, transient, adverse events, including headache, local pain, neck pain, toothache, and paresthesia. Seizures are rare, and no other serious adverse events have been associated33. For extra safety, it is recommended to offer earplugs, due to the possibility of harmful sounds, and bite-blocks for possible masseter contraction34.</li>
</ol>
3. Experimental procedures (Figure 1)
<ol>
	<li>Select the muscle for positioning the electrodes.
	<ol>
		<li>Ask the subject to put their hand over the table, in a prone position. Select the FDI muscle, localized between the first and second metacarpal osseous. To identify the FDI, ask the subject to abduct their index finger against resistance, keeping the rest of the hand still and laying on the table, while you are palpating the area.</li>
		<li>Expose the selected area. Use a disposable razor to shave the area to improve electrode contact with the skin, if necessary, and clean the area with alcohol pads to remove skin oils and other factors that could increase impedance. Certify that there is free skin to ensure contact with the electrode. 
		NOTE: If evaluating lower limb activity, use the TA muscle for electrode placement. It is localized on the lateral side of the tibia and lies near the superficies of the skin. It can be identified by ankle dorsiflexion.</li>
	</ol>
	</li>
	<li>Place the surface EMG electrodes
	<ol>
		<li>With the area exposed and cleaned, apply the conductive gel to each electrode of the channel to ensure good impedance.</li>
		<li>Place the negative electrode on the belly of the FDI muscle (the center or the most prominent bulge of the muscle belly) and the positive on the distal interphalangeal joint, with an inter-electrode distance of at least 1.5 cm. Place the reference electrode (neutral) on the wrist, over the ulnar styloid process. 
		NOTE: The presence of motor endpoints, muscle tendons, or other active muscles can impact the stability of the recordings, so it is important to avoid these locations35. For the TA muscle, the electrodes should be placed at one-third on the line that connects the tip of the fibula and the tip of the medial malleolus. Provide a 20 mm distance between each electrode&#39;s pole and place the reference electrode in the ankle.</li>
	</ol>
	</li>
	<li>Determine the required muscle contraction force
	<ol>
		<li>Use a digital pinch dynamometer and a quadrangular pyramid support to minimize mechanical distortions and elevate the sensitivity for minimal contractions.</li>
		<li>Place the dynamometer between the first and second fingers with the help of the pyramidal support. Ensure that the third, fourth, and fifth fingers lay still on the table, while the 1st and 2nd generate the forces of the pinching movement.</li>
		<li>With the fixed position, ask the participant to press the dynamometer with the first finger and the side of the pyramid with the index finger, squeezing the dynamometer-pyramid system with their maximum force and creating a strong contraction of the FDI muscle.</li>
		<li>Using that value as reference, determine the 20% of maximum force. The participant must practice maintaining the target at 20% of sustained contraction. Allow for variations from 15%-25% of MVC. 
		NOTE: Alternatively, in case of unavailability of a dynamometer that can catch the isolated muscle activity being investigated, use EMG feedback to standardize force. The recording software will measure the maximum peak-to-peak amplitude that corresponds to the subject&#39;s maximum force, and using that value as reference, will determine the 20% MVC. Subjects can receive visual and/or auditory clues of when 20% is achieved.</li>
	</ol>
	</li>
	<li>Identification of the initial location for hotspot searching
	<ol>
		<li>Put a swim cap on the subject&#39;s head. All the reference points will be marked on it.</li>
		<li>Measure the sagittal circumference of the head from the nasion (the point between the forehead and the nose) to the inion (the most prominent point in the occipital region). Divide that value by two and mark that middle spot on the head.</li>
		<li>Mark the location of the patient&#39;s nasion, inion, the helix of both right and left external ears, and right and left supraorbital ridge. This is to certify that the cap has not slipped during the procedure, and/or that in future experiments it will be equally positioned on the patient&#39;s head.</li>
		<li>As described above, measure the tragus-to-tragus distance and add a mark halfway. Mark the intersection between them, a point identified as the vertex (Cz).</li>
		<li>From the vertex, move 5 cm laterally in parallel to the midsagittal line, on the contralateral side to the selected muscle. This mark approximately identifies the (M1), on the same coronal level as the hand motor cortex. Use this as the first spot to initiate the search for the hotspot.</li>
		<li>The hotspot is the area of the motor cortex where the lowest motor threshold is detectable. Set up a low intensity (e.g., 30% of maximum stimulator output [MSO]) and initiate the search by delivering multiple pulses to the first spot.</li>
		<li>Pursue with small intensity increments until identifying the lowest stimulus that detects an EMG-indexed response (i.e., MEP). For the delivery of the stimuli, angle the figure-of-eight coil at 45&#176; in relation to the midsagittal line with the handle pointed toward the posterior of the patient.</li>
		<li>To ensure that the best spot was identified, move around the first spot and test the subsequent ~3 MEPs at 1 cm anterior, 1 cm lateral, 1 cm medial, and 1 cm posterior to it. Repeat this procedure as many times as needed for a consistent response; stick to the spot that elicits the largest MEP36.</li>
		<li>Once the hotspot is found, mark that spot in the patient&#39;s head (swim cap). Use this location during this experiment and the potential follow-up visits. Be cautious as not to cause discomfort to the subject due to extra pressure. Use both hands to support the coil on the subject&#39;s head.</li>
	</ol>
	</li>
	<li>Determine resting motor threshold (RMT)
	<ol>
		<li>Estimate the motor threshold as the minimum intensity required to promote an MEP of a minimal detectable amplitude (usually at least 50-100 &#181;V).</li>
		<li>To determine the motor threshold, apply ten consecutive stimuli at the hotspot and select the lowest intensity that produced an MEP with a peak-to-peak amplitude of at least 50 &#181;V on the target muscle, in 50% of the trials. 
		NOTE: This protocol can be done with the target muscle at rest (resting motor threshold [RMT]) or during active contraction (active motor threshold [AMT]). Both can further be used as references for the suprathreshold TMS pulses. The acquisition of the AMT is more prone to variability because it relies on the standardization of MVC, which can be an issue for longitudinal studies with multiple assessments.</li>
	</ol>
	</li>
	<li>CSP protocol
	<ol>
		<li>Deliver suprathreshold stimuli to elicit MEPs during tonic voluntary contraction of the target muscle.</li>
		<li>Deliver 10 stimuli with the stimulation intensity (SI) of 120% of the RMT with 10 s period in between them. During the application of the stimuli, ask the patient to maintain 20% of the maximum motor contraction of the target muscle, as practiced with the dynamometer.</li>
		<li>To ensure capturing the whole SP, certify that the EMG time window is long enough to capture up to 400 ms of EMG activity. Not infrequently - depending on the disease being studied - subjects might require higher SIs for a successful cSP to be obtained.</li>
	</ol>
	</li>
</ol>

Abhishek Datta is CEO, Co-founder and CTO of Soterix Medical Inc., and Kamran Nazin is Chief Product Officer of the same company. Soterix Medical Inc. provided the material used in the making of this video publication. The remaining authors declare having no competing financial interests.

The default SI to elicit MEP and SPs can vary according to the population. Intensities as low as 80% RMT have been shown to elicit cSP in healthy individuals39, still studies on both healthy and diseased populations have used intensities as high as 150% RMT49,50,51. Although this source of heterogeneity can be inherent to the nature of the target population, it should not be neglected as different SIs have shown to independently (regardless of muscle contraction force) dictate the period of silent EMG activity following the MEP39,49,52. RMTs from 110% to 120% have&#160;successfully elicited SPs in a broad range of populations, while still being tolerable for participants53,54. However, 110% RMT might be borderline, since SIs lower than 110% have failed to elicit SPs or elicited SPs with duration shorter than 50 ms39, which might represent solely spinal rather than cortical or corticospinal components of M1 inhibition. Moreover, higher SIs are associated with diminished stimulation focality and increased patient discomfort-especially in diseased populations with higher RMTs55, in which high percentages of suprathreshold stimuli could correspond to close-to-maximum stimulator output. This can jeopardize participants&#39; adherence to the used protocol56. Even though using 120% RMT seems to be the safest and most suitable SI setup overall, researchers should standardize the SI by checking previous successful experiments done in the population of interest. Standardization across similar populations is important to facilitate further pooled statistics.
The experiment is typically done using a single SI, but some studies have investigated cSP responses at more than one stimulus setup53,57,58,59,60. In the context of conditions with no clear pathophysiology or previous literature, or in which the understanding of the SP behavior is the objective of the study, it is recommended to plot cSPs against subsequent increasing stimulus intensities (i.e., 10% increment in a sigmoid stimulus-response curve)42. In that case, the researcher should consider adding to the protocol resting breaks to avoid muscle fatigue. Even though still contradictory, there is a considerable amount of evidence that cSPs are not influenced by the level of muscle contraction39,61,62; however, it is impacted by muscle fatigue63,64,65,66. A value of 20% of maximum voluntary contraction (MVC) has consistently shown to successfully elicit SPs with being less likely to induce fatigue60,67,68,69.
Another significant parameter that may contribute to the heterogeneity of cSP results in the literature is the selected muscle to evaluate cSP after the TMS stimuli. Studies have found that different muscles may recruit distinctive motor neuron networks, which in turn will have diverging cSP effects. This is true not only for upper versus lower limb musculature, but also for proximal and distal muscles of the same limb. For instance, in two separate studies, Van Kuijk and colleagues have conveyed a more significant sensitivity to TMS parameters, such as cSP, in distal upper-extremity muscles as compared to proximal muscles70,71. And although this difference has not always been statistically significant71, it is still noteworthy and may contribute to heterogeneous results. Moreover, a significant difference in cSP responses in upper and lower limb muscles has also been denoted in studies about fatigue, with upper limbs conveying 30% longer SPs than lower limbs72. Thus, to reduce heterogeneity in cSP results, it is important to standardize the muscle in which the cSP assessment will be evaluated as some are more sensitive to TMS stimuli than others. Therefore, different muscles can drastically change the procedure specificities and interpretation. To illustrate, cSP is also used to evaluate cortical excitability in deeper muscles, such as the laryngeal motor units. Applying the cSP protocol to these structures comes with unique challenges. An example is that of the laryngeal motor cortex; the stimulation site of this protocol is near the EMG electrode, which can increase the number of artifacts requiring adjustments to the EMG amplifier73. Also, needle electrodes that penetrate the skin are needed to measure the EMG activity of these muscles, making the placement, and if needed, reallocation of the electrodes difficult, as well as changing the interpretation of results. Therefore, a limitation of this methodological paper is that its scope is restricted to illustrating a protocol for upper and lower limbs, and that does encompass, for example, the field that explores cSP as a marker of corticobulbar inhibition or psychiatry conditions.
In that matter, literature search supports that the FDI is the most commonly used target muscle for studying upper limb M1 corticospinal inhibition. Reasons include but are not limited to its superficial and large cortical representation in the motor cortex, the lowest motor threshold for stimulation, and the simplicity to perform its isolated and sustained contraction as well as the positioning of the electrodes73,74. For the lower limbs, the use of the TA muscle is most frequent-likely due to its larger cortical representation in comparison to other leg muscles75. Also, the easiness to be isolated from the activity of large muscle groups that compose the lower limbs&#39; musculature plays a role. Despite the importance of lower limb (LL) rehabilitation in the field, fewer studies use the LL MEP given its particular challenges. The brain anatomical region of the LL is more medial and deeper in the inter-hemispheric fissure compared to the upper limbs. However, the use of Neuronavigation has improved the accuracy of the stimuli36, while the use of a double cone coil has successfully targeted LL regions, including the TA muscle, showing lower LL MT than other coil types76,77,78,79, and currently being the standard recommendation to target LL36,44. However, the usage of modern navigation technologies should be contemplated parallel to the feasibility of the protocol. Jung et al. (2010) revealed no significant difference in MEP variability and reproducibility between non-navigated TMS and TMS navigation, which reach a comparable performance level80. Using non-navigated TMS can be more cost-effective in specific circumstances (i.e., limited resources), and therefore was the preferred approach for this protocol that aims to demonstrate a feasible, easy, and reproducible cSP assessment.
Given the promising and versatile use of cSP as a corticospinal inhibition biomarker in different neurological disorders, it is essential to provide researchers with a feasible, reproducible, and still reliable cSP protocol for upper and lower limbs. We highlight that only a few muscles could be represented in the experiment, leading to the absence of investigation of cSP for corticobulbar inhibition. Moreover, the results of the non-exhaustive search provided in Table 1 are not an attempt to summarize existing data but rather to illustrate part of the rationale behind the choice of parameters and insights, therefore, conducted with no scientific rigor. Hopefully, this methodological paper will help researchers advance the investigation of cSP as a biomarker for M1 corticospinal inhibition.
Table 1: Different parameters used on cSP protocols. We extracted data of cSP experiments from 117 different articles. Results are reported if the paradigm was used in &#8805;2 experiments, otherwise they were gathered within others. &#8224;Includes articles that either did not report the method of standardization or that reported not applying standardization.&#160;Abbreviations: MVC = maximum voluntary contraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64231/Table1_CSP.xls" target="_blank">Please click here to download this Table.</a>

The silent period (SP) is a period of electromyographic (EMG) silence that follows a motor-evoked potential (MEP) induced by transcranial magnetic stimulation (TMS) applied during sustained muscle contraction. The suprathreshold TMS pulse can either be applied to the contralateral or ipsilateral primary motor cortex (M1) of the target muscle from which the EMG activity is being recorded yielding two phenomena: contralateral silent period (cSP) and ipsilateral silent period (iSP).
Even though iSP and cSP share similar features, they may reflect slightly different components. The first is thought to reflect transcallosal inhibition and thus be entirely of cortical origin1,2. Conversely, cSP is investigated as a possible surrogate of corticospinal inhibition, most likely mediated by gamma-aminobutyric acid (GABA) B receptors within M13,4,5.
Supporting the role of cSP in GABA-mediated pathways, previous works have found an increase in cSP duration after oral administration of GABA-enhancing components5,6,7,8. Still, spinal processes are also involved in altering its duration. The earlier phase (&#60;50 ms) of the cSP is associated with decreased H-reflex values3-a reflex that is a product of peripheral neurocircuitry and that quantifies the excitability of spinal neurons9. Spinal processing is thought to be mediated through the activation of Renshaw cells, motoneuron after-hyperpolarization, and postsynaptic inhibition by spinal interneurons10,11,12,13,14.
Despite spinal contribution, cSP results mainly from the activation of cortical inhibitory neurons, which are responsible for generating the later part of the cSP (50-200 ms)3,10,13,15,16. In that respect, the early part of cSP duration has been associated with spinal inhibition mechanisms, whereas long cSPs request larger cortical inhibitory mechanisms3,13,17,18.
Therefore, cSP is a promising biomarker candidate for corticospinal maladaptation due to neurological disorders, whereas more significant cSP durations potentially reflect an increase in corticospinal inhibition and vice versa5,11. Accordingly, previous works have found an association between cSP duration and pathologies such as dystonia, Parkinson&#39;s Disease, chronic pain, stroke, and other neurodegenerative and psychiatric conditions19,20,21,22. To illustrate, in a knee osteoarthritis cohort, a higher intracortical inhibition (as indexed by cSP) was associated with younger age, greater cartilage degeneration, and less cognitive performance in the Montreal cognitive assessment scale23. Moreover, cSP changes could also longitudinally index treatment response and motor recovery24,25,26,27,28,29,30.
As promising as the role of cSP in the neuropsychiatry field is, a challenging aspect of its assessment is that it can be too sensitive to protocol variations. For instance, the cSP duration (~100-300 ms)11&#160;is distinguishable between upper and lower limbs. Salerno et al. found an average cSP duration of 121.2 ms (&#177; 32.5) for the first dorsal interosseous muscle (FDI) and 75.5 ms (&#177; 21) for the tibialis anterior muscle (TA), in a sample of fibromyalgia patients31. Thus, the literature conveys a myriad of divergences in the parameters used to elicit cSPs, which in turn jeopardizes comparability across studies and delays the translation to clinical practice. Within a similar population, protocols have been heterogeneous regarding the suprathreshold TMS pulse setting used to stimulate M1 and the target muscle, for example. On top of that, researchers have failed to properly report the parameters used in their protocols.
Therefore, the goal is to provide a visual guideline on how to apply a feasible, reliable, and easily reproducible cSP protocol for evaluating M1 corticospinal excitability of upper and lower limbs and to discuss the practical methodological challenges of that procedure. Also, to help illustrate the reasoning for the choice of parameters, we conducted a non-exhaustive literature review on Pubmed/MEDLINE to identify published papers on cSP in chronic pain and rehabilitation populations, using the search term: Rehabilitation (Mesh) or rehabilitation or chronic pain&#160;or stroke and terms such as transcranial magnetic stimulation and single pulse or cortical silent period. No inclusion criteria were defined for the extraction, and pooled results are displayed in Table 1 for illustrative purposes only.

<table><tbody><tr><td>Alcohol pads</td><td>Medline</td><td></td><td>Preparation with 70% isopropyl alcohol</td></tr><tr><td>Conductive gel</td><td>Weaver and Company</td><td></td><td>Used on the electrode</td></tr><tr><td>Echo Pinch</td><td>JTECH medical</td><td>0902A302</td><td>Digital dynamometer.</td></tr><tr><td>Mega-EMG</td><td>Soterix Medical</td><td>NS006201</td><td>Digital multiple channel EMG with built in software.</td></tr><tr><td>MEGA-TMS coil</td><td>Soterix Medical</td><td>NS063201</td><td>8 shaped TMS coil</td></tr><tr><td>Mega-TMS stimulator</td><td>Soterix Medical</td><td>6990061</td><td>Single Pulse TMS</td></tr><tr><td>Neuro-MEP.NET</td><td>Soterix Medical</td><td></td><td>EMG software used to analyse the muscles eletrical activity.</td></tr><tr><td>Swim cap</td><td>Kiefer</td><td></td><td></td></tr></tbody></table>

measuring contralateral silent period induced by single-pulse transcranial magnetic stimulation to investigate m1 corticospinal inhibition

Contralateral silent period (cSP) is a period of suppression in the background electrical muscle activity captured by electromyography (EMG) after a motor evoked potential (MEP). To obtain this, an MEP is elicited by a suprathreshold transcranial magnetic stimulation (TMS) pulse delivered to the primary motor cortex (M1) of the target muscle selected, while the participant provides a standardized voluntary target muscle contraction. The cSP is a result of inhibitory mechanisms that occur after the MEP; it provides a broad temporal assessment of spinal inhibition in its initial ~50 ms, and cortical inhibition after. Researchers have tried to better understand the neurobiological mechanism behind the cSP to validate it as a potential diagnostic, surrogate, and predictive biomarker for different neuropsychiatric diseases. Therefore, this article describes a method to measure M1 cSP of lower and upper limbs, including a selection of target muscle, electrode placement, coil positioning, method of measuring voluntary contraction stimulation, intensity setup, and data analysis to obtain a representative result. It has the educational objective of giving a visual guideline in performing a feasible, reliable, and reproducible cSP protocol for lower and upper limbs and discussing practical challenges of this technique.

After following the step-by-step procedure, the delivery of a suprathreshold TMS pulse (120% of the RMT) will elicit an observable MEP in the EMG recording of the target muscle, and a subsequent period of background EMG activity suppression of approximately 150 ms to 300 ms (Figure 2). From that EMG pattern, it&#39;s possible to calculate the cSP metrics. The most reported outcomes are the duration (in the range of ms) of the relative and absolute SP. The relative SP is measured from the MEP start to the reappearance of EMG activity. One alternative is using the amplified motor stimulated output (MSO = 120% of RMT, as per the protocol) to establish the onset of relative SP. Since the true onset at a network level cannot be known, select the MEP onset as the initial start point to increase experimental reliability13. On the other hand, the absolute SP can be measured from the end of the MEP to the onset of the reemergence of voluntary EMG activity. For instance, using a recording of the resting EMG activity of the subject as reference for qualitative comparison. Those temporal parameters can be identified manually or using automated software37.
A fundamental methodological question for accurate cSP calculation is the definition of the reemergence of EMG background activity. Two approaches can be explored here: The first is using individual trial calculation. In this case, the calculation is based on trial-by-trial measure, using each recording to calculate the cSP duration. Then, a mean (or median) of the individual trials can be calculated and reported. The second approach is using rectified multiple trials. For this approach, all trials will be rectified, and then have to be averaged and overlapped with each other. Then, using the rectified and averaged traces, calculate the cSP duration using average temporal marks. The main advantage of this method is its precision and easier identification of the reappearance of voluntary EMG activity relative to the tonic baseline EMG level36. Using rectified average is advantageous because it is more comparable and reduces the between-subject variability.
It is important to mention that the cSP duration can be prolonged as a sigmoid function of stimulus intensity38, but it is barely affected by the degree of intentional contraction of the target muscle39. Moreover, MEP amplitudes increase with increases in stimulus intensities. Kojima et al. demonstrated that these increases in MEP amplitude (secondary to increases in intensity) is also accompanied by increases in cSP duration40. This behavior is expected since the MEP and cSP duration are thought to be influenced by common factors38. These common factors seem to be present throughout the cortical spinal tract and not in the motor unit; since the increases in stimulus intensity increases both, but increases in muscles contraction do not affect the cSP duration.
With this discussion, it is possible to conclude that stimulus intensity and muscle contraction must be carefully considered during the analysis and interpretation of findings. The cSP is characterized by a linear increase due to the SI, but then a plateau is reached at high intensities; this pattern is highly variable across subjects39, since they could have unique slopes and different plateau intensities. One alternative analysis could include evaluating the cSP during progressively increased intensities to perform an input-output (I/O) curve, and then the cSP can be obtained using the intensity at which the I/O curve reaches the plateau41,42. Finally, since cSP is affected by any activity or exposure that can produce cortical excitability and inhibition changes, it is recommended to assess and record general confounders in the analysis. For instance, using a reporting checklist for TMS experiments43.
cSP interpretation 
The TMS test in the current study was used to show the implementation of a feasible and versatile biomarker of M1 inhibition. In general, the longer the duration of the SP, a higher corticospinal M1 inhibition is observed44. However, several factors need to be considered for its interpretation. First, the cSP is defined by both spinal and cortico-subcortical processes45. The spinal components account for approximately the first 50 ms46. The remaining duration is highly influenced by cortical mechanisms such as M1 interneurons inhibition and other inhibitory afferences within M1 (from subcortical regions and other cortices), mediated mainly by GABAergic B neurons after an important cortical activation eliciting MEPs6. It has been suggested that the role of this inhibition is to prevent unwanted movements and sustain motor control47. Second, behavioral and cognitive factors can impact the CSP duration, as well as motor and non-motor neuropsychiatric disorders45,48. Due to this dual nature of cSP, its values need to be interpreted within the experiment context (target population and the use of concomitant motor control tasks).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64231/64231fig01.jpg" /> 
Figure 1: Experimental steps. 1. Electrode placement on the belly of the FDI muscle 2. Positioning of the dynamometer between fingers. 3. Voluntary contraction of the target muscle to test the standardization of 20% MVC 4.&#160;Head measurements and TMS pulses for identification of the hotspot and the RMT (lowest stimuli that elicits an MEP of at least 50 mV in five out of ten trials) 5. CSP protocol, consistent of 10 pulses with 120% RMT spaced out by 10s, during sustained muscle contraction. In the bottom center figure, the small red rectangle represents a single TMS pulse and divides pre-TMS stimulus (sustained muscle contraction and background EMG activity) and the cSP recording. CSP is considered from the start of the MEP until the reemergence of EMG baseline activity, represented inside the blue rectangle. In the yellow rectangle, the MEP latency is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64231/64231fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64231/64231fig03.jpg" /> 
Figure 2: MEP in the EMG recording of the target muscle. On the X-axis, milliseconds (ms), and the Y axis, millivolts (mV) of the EMG signal. From left to right: the red line indicates the background electrical muscle activity before the MEP, subsequently, after the electric effect of the TMS pulse is observed it is followed by the motor-evoked potential. After the MEP, there is a suppression of the EMG signal known as the SP. It can be relative, counting the interval from the beginning of the MEP wave to the return of EMG background activity or absolute SP, counting the interval from the end of the MEP to the return of the background wave. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64231/64231fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition at JoVE.com

Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition

Contralateral silent period (cSP) assessment is a promising biomarker to index cortical excitability and treatment response. We demonstrate a protocol to assess cSP intended for studying M1 corticospinal inhibition of upper and lower limbs.

Contralateral silent period (cSP) is a period of suppression in the background electrical muscle activity captured by electromyography (EMG) after a motor ...

measuring-contralateral-silent-period-induced-single-pulse

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.5K Views.  Harvard Medical School. In dit protocol demonstreren we een haalbare, gemakkelijk reproduceerbare en betrouwbare methode voor de beoordeling van corticospinale remming van de bovenste en onderste ledematen. Contralaterale stille periode is een metriek die informatie verschaft uit complexe corticospinale remmende circuits. Deze methode is daarom een veelbelovende kandidaat van een diagnostische, surrogaat- en voorspellende biomarker voor verschillende neurologische aandoeningen.Danielle Pimenta, Anne Pessotto ...

Video: Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition

Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily disrupt information processing in a brain region, generating a short-lived &#8220;virtual lesion.&#8221; Stimulation that interferes with task performance indicates that the affected brain region is necessary to perform the task normally. In other words, unlike neuroimaging methods such as functional magnetic resonance imaging (fMRI) that indicate correlations between brain and behavior, TMS can be used to demonstrate causal brain-behavior relations. Furthermore, by varying the duration and onset of the virtual lesion, TMS can also reveal the time course of normal processing. As a result, TMS has become an important tool in cognitive neuroscience. Advantages of the technique over lesion-deficit studies include better spatial-temporal precision of the disruption effect, the ability to use participants as their own control subjects, and the accessibility of participants. Limitations include concurrent auditory and somatosensory stimulation that may influence task performance, limited access to structures more than a few centimeters from the surface of the scalp, and the relatively large space of free parameters that need to be optimized in order for the experiment to work. Experimental designs that give careful consideration to appropriate control conditions help to address these concerns. This article illustrates these issues with TMS results that investigate the spatial and temporal contributions of the left supramarginal gyrus (SMG) to reading.

Transcranial magnetic stimulation (TMS) is a technique for non-invasively disrupting neural information processing and measuring its effect on behavior. When TMS interferes with a task, it indicates that the stimulated brain region is necessary for normal task performance, allowing one to systematically relate brain regions to cognitive functions.

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily ...

Behavior

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

Intracortical Inhibition Within the Primary Motor Cortex Can Be Modulated by Changing the Focus of Attention

It is well recognized that an external focus (EF) compared with an internal focus (IF) of attention improves motor learning and performance. Studies have indicated benefits in accuracy, balance, force production, jumping performance, movement speed, oxygen consumption, and fatiguing task. Although behavioral outcomes of using an EF strategy are well explored, the underlying neural mechanisms remain unknown. A recent TMS study compared the activity of the primary motor cortex (M1) between an EF and an IF. More precisely, this study showed that, when adopting an EF, the activity of intracortical inhibitory circuits is enhanced.
On the behavioral level, the present protocol tests the influence of attentional foci on the time to task failure (TTF) when performing submaximal contractions of the first dorsal interosseous (FDI). Additionally, the current paper describes two TMS protocols to assess the influence of attentional conditions on the activity of cortical inhibitory circuits within the M1. Thus, the present article describes how to use single-pulse TMS at intensities below the motor threshold (subTMS) and paired-pulse TMS, inducing short-interval intracortical inhibition (SICI) when applied to the M1. As these methods are assumed to reflect the responsiveness of GABAergic inhibitory neurons, without being affected by spinal reflex circuitries, they are well suited to measuring the activity of intracortical inhibitory circuits within the M1.
The results show that directing attention externally improves motor performance, as participants were able to prolong the time to task failure. Moreover, the results were accompanied by a larger subTMS-induced electromyography suppression and SICI when adopting an EF compared to an IF. As the level of cortical inhibition within the M1 was previously demonstrated to influence motor performance, the enhanced inhibition with an EF might contribute to the better movement efficiency observed in the behavioral task, indicated by a prolonged TTF with an EF.

Using two different transcranial magnetic stimulation (TMS) protocols, this manuscript describes how to measure and compare cortical inhibition within the primary motor cortex when adopting different attentional foci.

It is well recognized that an external focus (EF) compared with an internal focus (IF) of attention improves motor learning and performance. Studies have ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Conventional and Threshold-Tracking Transcranial Magnetic Stimulation Tests for Single-handed Operation

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used to examine corticospinal excitability. Paired-pulse TMS paradigms (e.g., short- and long-interval intracortical inhibition (SICI/LICI), short-interval intracortical facilitation (SICF), and short- and long-latency afferent inhibition (SAI/LAI)) provide information about intracortical inhibitory and facilitatory networks. This has long been done by the conventional TMS method of measuring changes in the size of the motor-evoked potentials (MEPs) in response to stimuli of constant intensity. An alternative threshold-tracking approach has recently been introduced whereby the stimulus intensity for a target amplitude is tracked. The diagnostic utility of threshold-tracking SICI in amyotrophic lateral sclerosis (ALS) has been shown in previous studies. However, threshold-tracking TMS has only been used in a few centers, in part due to the lack of readily available software but also perhaps due to uncertainty over its relationship to conventional single- and paired-pulse TMS measurements.
A menu-driven suite of semi-automatic programs has been developed to facilitate the broader use of threshold-tracking TMS techniques and to enable direct comparisons with conventional amplitude measurements. These have been designed to control three types of magnetic stimulators and allow recording by a single operator of the common single- and paired-pulse TMS protocols.
This paper shows how to record a number of single- and paired-pulse TMS protocols on healthy subjects and analyze the recordings. These TMS protocols are fast and easy to perform and can provide useful biomarkers in different neurological disorders, particularly neurodegenerative diseases such as ALS.

We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator.

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used ...

Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter TMS

Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable tool to probe the procedural and declarative influence over sensorimotor integration during skilled motor actions. This manuscript describes the methodology and contributions of short-latency afferent inhibition (SAI) for understanding sensorimotor integration. SAI quantifies the effect of a convergent afferent volley on the corticospinal motor output evoked by transcranial magnetic stimulation (TMS). The afferent volley is triggered by the electrical stimulation of a peripheral nerve. The TMS stimulus is delivered to a location over the primary motor cortex that elicits a reliable motor-evoked response in a muscle served by that afferent nerve. The extent of inhibition in the motor-evoked response reflects the magnitude of the afferent volley converging on the motor cortex and involves central GABAergic and cholinergic contributions. The cholinergic involvement in SAI makes SAI a possible marker of declarative-procedural interactions in sensorimotor performance and learning. More recently, studies have begun manipulating the TMS current direction in SAI to tease apart the functional significance of distinct sensorimotor circuits in the primary motor cortex for skilled motor actions. The ability to control additional pulse parameters (e.g., the pulse width) with state-of-the-art controllable pulse parameter TMS (cTMS) has enhanced the selectivity of the sensorimotor circuits probed by the TMS stimulus and provided an opportunity to create more refined models of sensorimotor control and learning. Therefore, the current manuscript focuses on SAI assessment using cTMS. However, the principles outlined here also apply to SAI assessed using conventional fixed pulse width TMS stimulators and other forms of afferent inhibition, such as long-latency afferent inhibition (LAI).

Author Spotlight: Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning  

Short-latency afferent inhibition (SAI) is a transcranial magnetic stimulation protocol to probe sensorimotor integration. This article describes how SAI can be used to study the convergent sensorimotor loops in the motor cortex during sensorimotor behavior.

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable ...

Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video