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

Summary

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.

Abstract

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.

Introduction

Transcranial Electrical Stimulation (tES) is a neuromodulatory technique which allows the modification of neuronal states through different current waveforms¹. Among different types of tES, transcranial Alternating Current Stimulation (tACS) enables the delivery of sinusoidal external oscillatory potentials in a specific frequency range and the modulation of physiological neural activity underlying perceptual, motor and cognitive processes². Using tACS, it is possible to investigate potential causal links between endogenous oscillatory activity and brain processes.

In vivo, it has been shown that spiking neural activity is synchronized at different driving frequencies, suggesting that neuronal firing can be entrained by electrically applied fields³. In animal models, weak sinusoidal tACS entrains the discharged frequency of the widespread cortical neuronal pool⁴. In humans, tACS combined with online Electroencephalography (EEG) allows the induction of the so-called "Entrainment" effect on endogenous oscillatory activity by interacting with brain oscillations in a frequency-specific manner⁵. However, combining tACS with neuroimaging methods for a better understanding of the online mechanisms is still questionable because of AC-induced artifacts⁶. In addition, it is not possible to directly record the EEG signal over the stimulated target area without using a ring-like electrode which is a questionable solution⁷. Thus, there is a lack of systematic studies on this topic.

So far, there is no clear evidence about the lasting effects of tACS after stimulation cessation. Only a few studies have shown weak and unclear after-effects of tACS on the motor system⁸. Moreover, EEG evidence is still not clear about the after-effects of tACS⁹. On the other hand, most tACS studies showed prominent online effects^{10,11,12,13,14,15,16,17,18}, which are difficult to measure at a physiological level because of technical constraints. Thus, the overall goal of our method is to provide an alternative approach to test online and frequency-dependent effects of tACS on the motor cortex (M1) by delivering single pulse Transcranial Magnetic Stimulation (TMS). TMS allows researchers to "probe" the physiological state of the human motor cortex¹⁹. Moreover, by recording Motor Evoked Potentials (MEP) on the subject's contralateral hand, we can investigate the effects of the ongoing tACS¹¹. This approach lets us accurately monitor changes in corticospinal excitability by measuring MEP amplitude during online electrical stimulation delivered at different frequencies in an artifact-free fashion. In addition, this approach can also test online effects of any other waveform of tES.

To demonstrate the combined tACS-TMS effects, we will show the protocol by applying 20 Hz AC stimulation over the primary motor cortex (M1) while online neuronavigated single pulse TMS is delivered interspersed by random intervals from 3 to 5 s in order to test M1 cortical excitability.

Protocol

All procedures were approved by the local research ethics committee of the Higher School of Economics (HSE), Moscow, with consent from all participants.

NOTE: Participants must report no history of implanted metal devices, neurological or psychiatric disease, drug abuse or alcoholism. TMS is used according to the most recent safety guidelines²⁰. Subjects must be fully informed of the nature of the research and sign an informed consent form before starting the experiment. We show an entire set of equipment needed to run the online-combined tACS-TMS protocol by stimulation of the dominant M1 ( Figure 1; Table of Materials).

1. Place Electromyography (EMG) Electrodes in a Bipolar Belly-tendon Montage

1. Clean the skin using a cleaning scrub under all the electrodes to achieve low skin impedance (below 10 kOhm).
2. Place the active EMG electrode on the first dorsal interosseous (FDI) muscle, reference electrode on the bone 2 cm distally and the ground electrode more proximally on the arm.

2. Identifying the Target for the Stimulation Protocol

NOTE: Here, we use the frameless TMS navigation system to achieve a proper positioning of the TMS coil.

1. Place the tracking sensors over the glabella between the eyebrows and above the nose of the participant.
2. Open the navigation system software. Use individual participants' structural T1 Magnetic Resonance Imaging (MRI) data and perform a co-registration of the participant's head and a 3D MRI head via the navigation system.
3. Accurately, place the coil over the primary motor hand-area, the so called "motor knob" region (Figure 2).
4. Start applying single pulse TMS and test MEPs; TMS is delivered by a stimulator (see Table of Materials) connected to a standard figure-of-eight 75-mm coil. To localize the "hotspot" of the left M1, hold the coil tangential to the scalp, with the handle pointing backward and laterally, angled at 45° from the midline sagittal axis of the participant's head.
5. Once the hotspot (i.e., the scalp point eliciting MEPs at threshold from the contralateral examined hand muscles) is found, mark it with a pencil to facilitate the application of the tACS target electrode.

3. tACS Electrodes Preparation

1. Connect 2 surface saline-soaked sponge electrodes (size: 5 cm x 7 cm) to the stimulation device, which can generate electrical alternating current (e.g., Brainstim).
2. In order to minimize skin sensation, constantly saturate the electrodes with a saline solution to keep impedances below 10 kOhm throughout the whole stimulation session.

4. tACS Protocol Set Up

1. To set up the tACS protocol using the stimulator device, first check the battery status.
2. Using the software, open a new session and manage a new stimulation protocol.
   1. Name the protocol (e.g., "Beta").
   2. Set the frequency of the stimulation (e.g., 20 Hz).
   3. Choose the waveform (e.g., sinusoidal).
   4. Set the total duration of the stimulation protocol (e.g., 600 s).
   5. Finally, set the intensity of stimulation (e.g., 1 mA), set offset, fade in, fade out, and phase at "0".
      NOTE: A little timing to fade in and out the stimulation (around 30 s) can be suggested, in order to avoid any adverse or uncomfortable neurosensory effects for the subject.
   6. Activate the device's "Bluetooth" function and upload the protocol from the software to the stimulator.

5. tACS Electrodes Montage

1. Place the "target" electrode over the scalp corresponding to the marked point. Place the "reference" electrode over the ipsilateral shoulder by using specific sticky tape, in a "monopolar montage"²¹.
2. Carefully adjust the first elastic strap on the head with respect to the neuro-navigation head-sensors position. Then, by using the second strap, fix the target electrode position.
3. Once tACS electrodes are placed both on the scalp and on the ipsilateral shoulder, connect them to the stimulator.
4. Before the start of the stimulation session, ensure by visual inspection that position of the target electrode is centered over the marked hotspot.

6. Identifying the Resting Motor Threshold (RMT)

1. Place the TMS coil over the target tACS electrode and carefully adjust the coil position over the hotspot (Figure 3) by using the neuro-navigation system.
2. Measure the RMT accordingly to the combined tACS-TMS setup (i.e., TMS coil over the electrode). Specifically, adjust the TMS intensity with respect to the thickness of the tACS electrode in order to check for a reliable RMT.
   1. Measure the RMT individually, it is defined as the minimum intensity required to induce an MEP in the FDI muscle with an amplitude of 50 mV (peak-to-peak) in 5 out of 10 trials²².
3. Set the intensity of the TMS stimulation at 110% of the RMT in order to start the experimental session.

7. Experimental Procedure

1. Open the EMG software and start EMG recording.
2. Start the tACS stimulation.
3. During the stimulation, deliver TMS single pulses interspersed by random intervals from 3 to 5 seconds.
4. Ensure that each session of the stimulation (e.g., 20 Hz tACS stimulation followed by a sham/another control frequency) lasts no more than 90 seconds with an inter-session interval around 3 minutes, in order to avoid possible carryover effect of the preceding stimulation frequency/condition^11,13.

Results

The first evidence of a tACS/TMS combined approach was shown by Kanai et al. in 2010. In that study, the authors applied tACS over the primary visual cortex (V1) and demonstrated a frequency-specific modulation of the visual cortical excitability measured by online TMS-induced phosphene perception¹⁵. A more refined version of the protocol was adopted to investigate a physiological modulation of the motor cortex excitability by Feurra et al. in 201...

Discussion

This approach represents a unique opportunity to directly test online effects of tACS of the primary motor cortex by measuring corticospinal output through MEPs recording. However, the placement of the TMS coil over the tACS electrode represents a critical step that should be accurately performed. Therefore, we would firstly suggest experimenters find a target point by single pulse TMS, then mark it on the scalp and, only after that, place the tACS electrode over the hotspot. Moreover, the availability of a neuronavigati...

Materials

Name	Company	Catalog Number	Comments
BrainStim, high-resolution transcranial stimulator	E.M.S., Bologna, Italy	EMS-BRAINSTIM
Pair of 1,5m cables for connection of conductive silicone electrodes	E.M.S., Bologna, Italy	EMS-CVBS15
Reusable conductive silicone electrodes 50x50mm	E.M.S., Bologna, Italy	FIA-PG970/2
Reusable spontex sponge for electrode 50x100mm	E.M.S., Bologna, Italy	FIA-PG916S
Rubber belts – 75 cm	E.M.S., Bologna, Italy	FIA-ER-PG905/8
Plastic non traumatic button	E.M.S., Bologna, Italy	FIA-PG905/99
Brainstim	E.M.S., Bologna, Italy
MagPro X100 MagOption - transcranial magnetic stimulator	MagVenture, Farum, Denmark	9016E0731
8-shaped coil MC-B65-HO-2	MagVenture, Farum, Denmark	9016E0462
Chair with neckrest	MagVenture, Farum, Denmark	9016B0081
Localite TMS Navigator - Navigation platform, Premium edition	Localite, GmbH, Germany	21223
Localite TMS Navigator - MR-based software, import data for morphological MRI (DICOM, NifTi)	Localite, GmbH, Germany	10226
MagVenture 24.8 coil tracker, Geom 1	Localite, GmbH, Germany	5221
Electrode wires for surface EMG	EBNeuro, Italy	6515
Surface Electrodes for EEG/EMG	EBNeuro, Italy	6515
BrainAmp ExG amplifier - bipolar amplifier	Brain Products, GmbH, Germany
BrainVision Recorder 1.21.0004	Brain Products, GmbH, Germany
Nuprep Skin Prep Gel	Weaver and Company, USA
Syringes
Sticky tape
NaCl solution