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

Summary

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.

Abstract

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.

Introduction

Noninvasive brain stimulation is a promising assessment tool and treatment for many neurological disorders, such as Parkinson's disease, Alzheimer's disease, and stroke^1,2,3,4. There is accumulating evidence establishing the relationship between the behavioral manifestations of neurological diseases and abnormalities of cortical excitability, neuroplasticity, cortico-cortical and cortico-subcortical connectivity^5,6. Therefore, basic knowledge about brain network dynamics and plasticity in neurological conditions can provide invaluable insight into disease diagnosis, progression, and response to therapy. Functional magnetic resonance imaging (fMRI) is a useful tool to understand the complex relations between brain and behavior in both healthy and diseased brain networks and has the potential to improve treatment based on a network perspective^7,8,9. However, fMRI is correlational in nature and cannot provide a causal link between brain function and behavior, nor manipulate functional connectivity to restore abnormal neural circuits associated with behavioral impairments in patients^10,11,12. Transcranial magnetic stimulation (TMS) can both causally measure and modulate human brain function and behavior in health and disease^3,13,14,15.

TMS is a safe, noninvasive method to stimulate the human brain^16,17 and can be used to induce and measure plasticity¹⁸. This method can advance our understanding of causal relationships between individual brain areas and behavior^10,11,12,19 and their specific functional interactions with other nodes of a brain network^20,21,22,23. To date, most studies have focused on the human motor system, given that TMS to the hand area of the motor cortex (M1) can produce motor evoked potentials (MEPs) as physiological readouts for changes associated with motor behavior²⁴, allowing examination of different inhibitory and excitatory circuits at the system level in the human brain²⁵. Recent advances using a conditioning test TMS approach with two coils show that it is possible to measure functional interactions between different cortical areas. In the motor system, dual-site TMS experiments show that inputs from cortical areas interconnected with M1 can change with task demands, age, or disease^14,26. Seminal work by Ferbert and colleagues has found that applying a conditioning stimulus to M1 prior to a test stimulus of the other M1 can result in inhibition of the MEP amplitude, a phenomenon known as short interval interhemispheric inhibition (SIHI)²⁸. A number of TMS studies using this approach have also shown that M1 is strongly interconnected with the contralateral M1, ventral premotor cortex (PMv), dorsal premotor cortex (PMd), supplementary motor area (SMA), pre-SMA, primary sensory cortex (S1), dorsolateral prefrontal cortex (DLPFC), and posterior parietal cortex (PPC) at rest^{27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42}. Interestingly, the effect of stimulation from these cortical areas on motor cortical excitability are anatomically, temporally, and functionally specific to the ongoing brain activity during the preparation of a movement (state- and context-dependent^{43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,69}). However, very few studies using dual-site TMS have characterized patterns of functional cortico-cortical connectivity with motor and cognitive impairments in patients with brain disorders^70,71,72. This affords opportunities to develop new methods for assessing and treating motor and cognitive disorders.

Using this technique, it also has been found that repeated pairs of cortical TMS applied to cortical areas interconnected with M1 such as contralateral M1^68,69,70, PMv^76,77,78, SMA⁷¹, and PPC^80,81,82 can induce changes in synaptic efficiency in specific neural pathways based on the Hebbian principle of associative plasticity^83,84,85,86 and enhance behavioral performance^72,73,74. Still, few studies have used this approach to study circuit and plasticity dysfunction in neurological disorders^{2,75,76,77,78,79,80,81,82,83,84,90,91,92,93,94,95,96}. It remains to be shown whether strengthening functionally specific neural pathways with TMS can restore activity in dysfunctional circuits, or whether the prospective strengthening of intact circuity can augment resilience⁹⁷ in brain networks supporting motor and cognitive function across the lifespan and in disease. The lack of fundamental understanding of the neural mechanisms underlying neurological disorders and effects of stimulation on interconnected dysfunctional brain networks limits current treatment.

Despite its capability, TMS has yet to become a standard part of the armamentarium of neuroscience and clinical tools for understanding brain-behavior relations, pathophysiology of brain disorders, and the effectiveness of treatment. Therefore, to realize its potential and support its large-scale application, standardizing TMS methods is important because it is more likely to increase the rigor of future TMS experiments and reproducibility across independent laboratories. This article outlines how TMS can be used to both measure and manipulate functional interactions. Here, we describe this technique in the motor system (e.g., parieto-motor pathway⁴⁴) by measuring TMS-based output measures (e.g., MEPs), where the method is best understood. However, it is important to note that this protocol also can be adapted to target functional coupling of other subcortical⁸⁵, cerebellar^86,87, and cortical areas.^73,74,88 In addition, neuroimaging techniques such as EEG^89,90,91 and fMRI^92,93 can be used to assess the TMS-induced changes in activity and connectivity^26,94. We conclude by proposing that the study of the functional involvement of circuit-level cortical connectivity with these TMS methods in both health and disease makes it possible to develop targeted diagnoses and innovative therapies based on more sophisticated network models of brain-behavior relations.

Protocol

The following three TMS methods are described below. First, two methods are described to measure cortico-cortical connectivity using dual-site transcranial magnetic stimulation (dsTMS) while participants are either 1) at rest (resting state) or 2) performing an object-directed reach-to-grasp movement (task-dependent). Second, a cortical paired associative stimulation (cPAS) method is described to modulate the interplay between two brain areas in a controlled manner by pairing cortical stimuli (e.g., posterior parietal and primary motor cortices) to strengthen functional specific neural pathways with TMS and induce changes in cortical excitability. A representative data set is provided for each method. All the methods described in this protocol were approved by the University of Michigan Institutional Review Board in accordance with the Declaration of Helsinki.

1. Participant Recruitment

1. Screen all participants for any contraindications to TMS^{95,96,97,98,99,100} and magnetic resonance imaging (MRI) prior to recruitment. Recruit right-handed participants¹⁰¹ for experiments investigating functional connectivity in the motor system.
2. Inform each participant about the study objectives, procedures, and risks approved by the local institutional review board. Obtain written consent before allowing the individual to participate in the study.

2. Electromyography (EMG) Electrode Placement

1. Instruct the participant to sit comfortably in the experimental chair with both arms supported in a relaxed position. Provide a chin rest for participants during TMS to keep head movement to a minimum during stimulation.
2. Clean the skin over the muscle of interest with a mild abrasive. Using a belly-tendon electrode arrangement, place one disposable Ag-AgCl electrode on the belly muscle and another on a bony landmark nearby for a reference site on both hands of the participant. Repeat this step for each muscle of interest.
3. Connect a ground electrode to the ulnar styloid process. It is important to inspect the level of surface contact of the electrodes with the skin throughout the duration of the experiment, because this precludes the impedance quality of the EMG signal. Placing tape over the surface electrode can improve the degree of contact with the skin surface.
   NOTE: For reach-to-grasp actions common muscles studied are 1) the first dorsal interosseous (FDI), 2) abductor pollicis brevis (APB), and 3) abductor digiti minimi (ADM) muscles of the hand.
4. Connect surface electrodes with an EMG amplifier and a data acquisition system. Record and store the EMG signals from the amplifier to the data collection computer with EMG software for online monitoring and offline analysis of the EMG signal. Optionally, amplify the EMG signal 1,000x, and use a band-pass filter between 2 Hz and 2.5 kHz, digitized at 5 kHz by an analog-to-digital interface.

3. Localizing Brain Areas for Targeted TMS

1. Method 1: Localizing without an MRI scan
   1. Using the 10–20 EEG system mark C3, located approximately over the left primary motor cortex (M1), and P3, located approximately over a part of the angular gyrus in the left posterior parietal cortex (PPC), on the participant's scalp. Refer to methods previously described¹⁰² for specific steps to localize brain areas with the 10–20 EEG system (see Figures 3 and 4 from Villamar et al.¹⁰²).
   2. Alternatively, an electroencephalography (EEG) head cap can be used to approximate the brain areas on the scalp. Place an appropriately sized EEG cap on the participant's head and align the Cz position on the cap with the marked Cz position on the participant's scalp. Mark C3 and P3 using the cap.
      NOTE: Localization without an individual's MRI scan has the potential to be inaccurate¹⁰³. Therefore, MRI-based neuronavigation is strongly recommended to increase the accuracy and reliability of targeting the TMS. This can potentially lead to less variability in the TMS-induced aftereffects.
2. Method 2: Using an MRI scan
   1. Before the TMS session, obtain the participant's structural MRI (T1). Upload the scan to a neuronavigation system.
   2. Create a three-dimensional reconstruction of the brain and skin overlay using the neuronavigation software. Place markers on the anatomical landmarks at the tip of the nose, nasion, inion, and the preauricular notches of both ears. Do not use the tragus as it can shift when ear plugs are inserted.
   3. Locate the hand knob, the anatomical landmark that corresponds to M1¹⁰⁴, in the left precentral gyrus. Place a trajectory marker at this point with the neuronavigation system. This point should be aligned 45° from the midsagittal line and approximately perpendicular to the central sulcus. Record and name the anatomical landmark with the neuronavigation system (Figure 1).
   4. Locate the nonmotor area of interest (e.g., over the anterior intraparietal sulcus area in PPC). Place a second trajectory marker over this anatomical landmark. Record and name the location with the neuronavigation system (Figure 1).
3. Perform coil and head registration with the tracking system
   1. Calibrate both TMS coils with the calibration block separately using the neuronavigation system.
   2. Place the head tracker securely on the participant's head so that the tracker is in view throughout the duration of the experiment.
   3. Coregister the anatomical landmarks on the participant's head to the neuronavigation system. If an MRI was not obtained from the participant, use a template MRI from the Montreal Neurological Institute.
      NOTE: It is important to not apply too much force with the pointer on the participant's skin to avoid discomfort and inaccuracies when performing registration. It may be valuable to check regularly throughout the course of the experiment that the head tracker has not shifted. These procedures ensure precision when applying the TMS coil to a target area for stimulation during the experiment.

4. Localizing Optimal TMS Coil Position and Determining Thresholds

NOTE: In this experiment, Coil_M1 refers to the coil used to deliver stimulation to M1, while Coil_Two refers to the coil used to deliver stimulation to the other cortical area of interest (e.g., posterior parietal cortex). Thresholding over M1 must be determined for Coil_Two to calculate the maximum stimulator output (MSO) used over nonmotor areas. Motor threshold values should be reported to allow for comparisons and reproducibility across experiments.

1. Localizing and thresholding with Coil_Two
   1. Position the center of Coil_Two over the target M1 location identified in the previous section to induce a posterior-anterior current direction in the brain.
   2. To find the optimal location for activation of the target muscle, deliver pulses to M1 at 30% of the machine's MSO. Observe whether the delivered stimulation produces a muscle twitch and determine the amplitude of the motor evoked potential (MEP) recorded with the EMG electrodes from the muscle activity displayed by the data acquisition system.
   3. If an MEP or a visible muscle twitch is not observed, continue to increase the stimulator output by 5% increments. The position, rotation, pitch, and yaw of the TMS coil may need to be adjusted to optimize the amplitude of the MEP. Repeat this until a response is observed.
   4. Lower the intensity in a stepwise manner to the lowest intensity that produces at least 5 out of 10 MEP responses with an amplitude of ≥50 μV while the participant is at rest^97,98,105. This is defined as the resting motor threshold (RMT).
   5. Ensure for the duration of the thresholding session that both hands are in a resting position with both arms and hands supported with pillows.
   6. Provide real-time visual or auditory feedback of muscle activity from EMG (e.g., on a monitor or speaker) throughout the session, especially if there is excessive muscle activity (e.g., older adult populations).
   7. Continuously ask participant about levels of comfort.
      NOTE: It is important that all procedures described above are performed separately and repeated for each TMS coil to determine the specific parameters used in the experiment for the different sized coils (e.g., localizing optimal TMS coil position and determining stimulation intensities for motor thresholding). It also is important that the interval between the TMS pulses is >5 s to avoid inducing changes in cortical excitability.
2. Localizing and thresholding with Coil_M1
   1. Repeat the steps described above to find the optimal stimulation location with the Coil_M1.
   2. Determine the lowest stimulator intensity needed to generate MEPs of ≥1 mV in 5 of 10 trials in the target hand muscle when the muscle is completely relaxed. Mark and record the position of Coil_M1 using the neuronavigation system.

5. Dual-site TMS (Resting State)

1. Use two figure-8 shaped coils (e.g., Coil_M1 and Coil_Two) connected to two individual TMS stimulators (e.g., two Magstim 200² units). Deliver the test stimuli (TS) over M1 with Coil_M1 (e.g., D70² figure-8 shaped coil, outside diameter of loop is 7 cm) and the conditioning stimuli (CS) to the other area of interest with Coil_Two. (e.g., D50 Alpha B.I., outside diameter of each loop is 5 cm).
2. Determine the percentage of the MSO intensity for the conditioning stimulus (CS) for Coil_Two.
   NOTE: The percentage of the MSO intensity is often between 70–140 of RMT and will depend on the specific parameters and objectives of the experiment (see Table 3 from Lafleur et al.¹⁴). For this experiment, the CS was set at 90% of RMT, similar to parameters used elsewhere^35,44,60.
3. For the test stimulus (TS), use the previously determined intensity that elicits MEP amplitudes of ~1 mV in the targeted quiescent hand muscle.
4. Set the precise interstimulus interval (ISI) between the CS and TS.
5. Use the supplied control software or external control via TTL pulses to control the ISI for the two pulses. The ISI often ranges from 4–20 ms (see Table 1 from Lafleur et al.¹⁴). For this experiment, the CS to PPC preceded the TS to M1 by an ISI of 5 ms.
6. Using a custom-made coding script, generate in random order the single-pulse TMS trials (TS alone) and paired-pulse TMS trials (CS-TS) at the specified ISI.
7. Position Coil_M1 over the left M1 and position Coil_Two over the other area of interest.
8. Deliver the TS alone trials with Coil_M1. For the paired-pulse (CS-TS) trials, deliver the CS with Coil_Two followed by the TS to Coil_M1 at the predetermined ISIs. This is illustrated in Figure 2. Repeat a minimum of 12 trials for each condition. Deliver the TS at least 1 s after the start of the trial to collect prestimulus EMG activity. Use a 4 s data acquisition sweep for each trial followed by a 1 s intertrial interval.
9. If necessary, adjust the TMS coil positions slightly to accommodate the placement of both coils over the selected targeted locations on the participant's head. Adjust and record the new location of Coil_M1 and Coil_Two using the neuronavigation system accordingly.
10. Use the trigger button on the TMS machine for the supplied control software or the custom-made coding script from the external controller to deliver the programmed TMS pulses.
    NOTE: For this experiment, a data acquisition system (e.g., CED Micro 1401) and software package (e.g., Signal version 7) were used to generate stimuli, capture data, control the external equipment, and run the analysis. The custom-made coding scripts used to run and analyze data from the experiments are available from the corresponding author.

6. Dual-site TMS (Task Context)

NOTE: Dual-site TMS also can be used to test whether functional connectivity at rest can be modulated by different task contexts.

1. Follow the same method described in the section above to examine functional interactions between different cortical areas interconnected to M1, but during the preparatory phase of a task that engages the network (e.g., during the action plan for a grasp).
2. Determine the time course and a cortical area of interest (e.g., PPC) to study functional interactions with M1 during the preparation of a complex movement plan (e.g., object-driven precision grip or whole-hand grasp^{43,44,45,46,47,48,49,106}) for selective hand muscles.
3. Using a custom-made coding script, generate in random order the timing of TS alone trials and paired-pulse trials (CS-TS) at a given ISI after the 'GO' cue during the reaction time period (plan phase) such that the MEP recordings are collected before the movement initiation (premovement period) for the task.
4. Deliver single-pulse TMS (TS alone) or paired-pulse TMS (CS-TS) probes between 50 and 800 ms after the 'GO' cue^47,49 during the action plan of complex hand movements. See Figure 3 for timing of an event-related trial for this experiment. The custom-made coding scripts used to run the timing of event-related trials are available from the corresponding author.
   1. Before the testing session with TMS, have the participant perform the task for a minimum of 50 practice trials to establish a consistent reaction time. Encourage the participant to ask questions about the task to ensure reliable performance during the testing session with TMS.
   2. Use the custom-made coding script to deliver all combinations of single-pulse TMS (TS alone) or paired-pulse TMS (CS-TS) and task (e.g., grasp a smaller top or grasp a larger bottom object) during the reaction time period (plan phase) such that the MEP recordings are collected before actual movement initiation.

7. Cortical Paired Associative Stimulation (cPAS)

NOTE: This protocol involves delivering pairs of monophasic pulses to two different cortical areas over short periods to induce pathway-specific changes in synaptic strength between connections within the human brain. This approach is based on Hebbian principles of spike timing dependent plasticity^{107,108,109,110}. Similar to dual-site TMS methods, cPAS is delivered with two TMS machines connected to two individual TMS coils over two different cortical areas (e.g., PPC and M1).

1. Using a custom-made coding script, generate 100 pairs of stimuli at 0.2 Hz (8.3 min duration each). For the experimental cPAS_Two→M1 condition, deliver the first stimuli over the nonmotor area (e.g., PPC) with Coil_Two with a specified pulse intensity (e.g., 90% RMT) for 5 ms before the second stimuli over M1 with Coil_M1 with a pulse intensity that elicits a MEP amplitude of ~1 mV in the targeted hand muscle.
2. It is important to control for: 1) directionality of the connectivity (CTRL_M1→Two); 2) timing (CTRL_ISI=500ms); and 3) stimulation site (CTRL _{Control site→M1}) in separate sessions. For examples see^{72,74,111,112}. The custom-made coding scripts for each cPAS condition are available from the corresponding author. The stimulation parameters (e.g., intensities and ISI) can be adjusted for different cortical areas. Refer to Table 2 from Lafleur et al.¹⁴ for a summary of plasticity protocols.
3. Use the procedures described in previous sections to guide the precise location of the TMS coils.
4. Obtain baseline corticospinal measurements with Coil_M1 (e.g., ~24 MEPs).
5. Randomize the participants to one of four intervention groups: 1) cPAS _Two→M1; 2) CTRL_M1→Two; 3) CTRL_ISI=500ms; 4) CTRL _{Control site→M1}.
6. For this experiment only the experimental cPAS _Two→M1 condition was tested and the PPC was used as the area of interest. When performing multiple sessions on the same participant, it is important that each experimental session is separated by at least 48 h in a randomized order to prevent crossover effects. It also is important to repeat sessions within each participant at the same time of day to control for alertness.
7. Use the custom-made coding script to deliver the specified cPAS condition.
8. Monitor the muscle activity of the other (left) hand during the experiment with EMG to ensure the hand is fully relaxed during the protocol.
9. Obtain corticospinal measurements with Coil_M1 (e.g., about 24 MEPs) at different times after cPAS (e.g., 0, 10, 20, 30, 40, 50, 60 min) to examine the time course of the TMS-induced effect on brain excitability.
   NOTE: The experimental protocol used here is shown in Figure 4. Most studies to date have focused on the motor system because the MEP is a reliable outcome measure. However, behavioral measures^72,73,74 and functional connectivity strength with fMRI^92,93 and EEG^89,90 following TMS manipulation of associative plasticity can also be investigated. These methods can also be adopted for different cortical areas that do not include M1 as a cortical target.

8. Data Processing and Analysis

1. Visually inspect EMG data offline and discard any traces showing muscle activity in which the root mean square EMG activity in the muscles exceeded a background level of 10 μV during the 100 ms immediately before the TMS pulse to ensure the muscles were at rest^59,113.
2. Similarly, discard any trials with EMG activity that coincide with the TMS pulse during the movement preparation period (e.g., 800 ms window^47,49) in dual-site TMS task context trials to exclude anticipatory responses.
3. For each MEP trial, measure the peak-to-peak amplitude between the minimum and maximum values in mV in the time window between 50 ms before and 100 ms after the TS¹⁰⁵.
4. Calculate the mean of the MEP amplitudes in millivolts from the TS alone trials and the paired-pulse (CS-TS) trials for each participant. Calculate the mean across all participants. Report these values.
5. Next, normalize the mean MEP amplitude from paired-pulse stimulation (CS-TS) trials from the unconditioned single-pulse (TS alone) trials for each participant and condition. Express the MEP amplitudes as a ratio to the baseline TS condition.

6. Calculate the mean across all participants. Report these values.

Results

Figure 5 shows the size of an exemplar MEP response elicited in the FDI muscle by TMS for an unconditioned test stimuli (TS alone to M1, blue trace) or conditioned stimuli from PPC (CS-TS, red trace) while the participant was at rest (top panel) or planning a goal-directed grasping action to an object (bottom panel). At rest, the PPC exerts an inhibitory influence on ipsilateral M1, as shown by the decrease in MEP amplitudes potentiated by a subthreshold CS delivered over PPC 5 ms before a s...

Discussion

The dual-site TMS method described here can be employed to investigate functional interactions between different cortical areas interconnected with the primary motor cortex while a participant is at rest or planning a goal-directed action. While brain imaging is correlative, basic knowledge from dual-site TMS methods can reveal causal brain-behavior relations associated with changes in cortico-cortical circuits. In addition, cortical paired associative stimulation with two TMS coils applied in areas interconnected with M...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alpha B.I. D50 coil (coated)	Magstim		50mm coil
BrainSight 2.0 Software	Rogue Research		Neuronavigation software
BrainSight frameless Stereotactic System	Rogue Research		Neuronavigation equiptment
D702 Coil	Magstim		70mm coil
Discovery MR750	General Electric		3.0T MRI machine
Disposable Earplugs	3M		Foam earplugs
ECG Electrodes 30mm x 24mm	Coviden-Kendall	H124SG	Disposable electrodes
Four Channel Isolated Amplifier	Intronix Technologies Corporation	2024F	EMG amplifier
gGAMMAcap	g.tec Medical Engineering		EEG head cap
Micro1401-3	Cambridge Electronic Design		Scientific data recorder and processing machine
Nuprep Skin Prep Gel	Weaver and Company		Skin prep abrasive gel
Signal v.7	Cambridge Electronic Design		Data acquisition and analysis software
The Magstim BiStim2	Magstim		Transcranial magnetic stimulator (two 2002 units)