Investigating Transcranial Alternating Current Stimulation (tACS) Preconditioning Effects on Resting and Active Motor Threshold
Phase: NA
Conditions: Understand the Effects of tACS on the Brain
Sponsor: University of Manitoba

 
 
 
Study Protocol –  Version 5, March 30, 2023 
 
 
NCT05503823 
 
 
Project Title: Investigating Transcranial Alternating Current Stimulation (tACS) 
Preconditioning Effects on Resting and Active Motor Threshold 
 
 

1. Summary of the Project 
Transcranial Magnetic Stimulation (TMS) is a non-invasive brain stimulation technique which 
uses a large and rapidly changing magnetic field pulse for stimulation. Exposure to TMS has 
been associated with the improvement of the brain’s plasticity and memory loss [1]. These 
after-effects of TMS are temporal and short-term. However, repetitive sessions of 
preconditioning the brain before TMS has the potential to extend the duration of these after-
effects while also reducing the amount of exposure necessary for observable results. The 
proposed study focuses on providing some insight on the effects of priming the brain via 
Transcranial Alternating Current Stimulation (tACS) and Transcranial Random Noise Stimulation 
(tRNS) before applying TMS.  
In this study, single pulse TMS is applied over the primary motor cortex, specifically for the 
hand area to find the resting and active motor thresholds. The Resting Motor Threshold (RMT) 
is the lowest intensity to produce involuntary hand twitches while the hand is at a relaxed 
position or when the motor evoked potential has a peak-to-peak amplitude of greater than 
50μV for 5 of 10 trials [2][3]. The active motor threshold (AMT) is similarly the lowest intensity 
at which the participant shows involuntary hand twitches, but this time while the hand is 
contracted. AMT is also measured similarly as the lowest intensity at which the motor evoked 
potential has a peak-to-peak amplitude of greater than 100μV for 5 of 10 trials [4][5]. The 
electromyography (EMG) of the First Dorsal Interosseous muscle (FDI) is recorded before the 
application of tACS as a baseline for both RMT and AMT. Then the tACS/tRNS is applied at 40Hz 
for 10 minutes with the active electrode over the exact location of the TMS application (the 
hand area of the primary motor cortex) and the reference over the right supraorbital area. Then 
the EMG is recorded immediately after tACS/tRNS, 30 minutes after, and 1 hour after exposure 
to tACS/tRNS. These recordings are done both in resting and active states of the muscle. The 
objective is to observe a change in the motor evoked potentials (MEPs) after exposure to tACS 
or tRNS and analyze the time of the sustained MEP change.  
 
The proposed study will be tested on 50 cognitively healthy participants from a diverse age 
group (18-95).  
An increase in the peak-to-peak value of MEP post-tACS or post-tRNS could prove to indicate as 
a new technique to lower the RMT, resulting in lower intensity exposure to TMS. This will be 
helpful when TMS is used for treatments such as depression disorders. The goal of this study is 
to eventually find a preconditioning technique to increase the duration of TMS sessions to help 
in treatment of initial stages of Alzheimer's disease. 
 
2. Background 
Previous studies have investigated preconditioning the brain through electrical stimulation of 
the brain mainly using direct current application [6][7]. These studies show promising 
improvement of the motor evoked potential (MEP) when transcranial electric stimulation (tES) 

techniques such as tDCS (transcranial direct current stimulation) are applied before a session of 
TMS [6][7]. This study attempts to extend the previous findings by investigating the effects of 
alternating current and random noise stimulations on the brain known as tACS and tRNS 
respectively. 
tDCS 
tDCS is electrical stimulation of the brain using direct current. The study by Lang et al [6] shows 
a prominent increase of motor evoked potential peak-to-peak amplitude caused by TMS after 
stimulation of the brain with tDCS over the motor cortex. This study concludes that 
preconditioning the brain with tDCS effectively improves the cortical plasticity during rapid TMS 
while also controlling the direction of TMS induced after-effects by changing the placement of 
the cathode and anode during application of electrical stimulation [6].  
A second study conducted by Cosentino et al [7] also investigates the effects of priming the 
brain using tDCS in a similar setting as the previous study. Contrary to the previous study, this 
study shows that neither cathodal nor anodal stimulation caused a higher MEP than the 
baseline [7]. 
tACS 
tACS is electrical stimulation of the brain using alternating current. This stimulation is bipolar 
therefore, it is not expected to see any difference between the cathodal and anodal after-
effects of the priming. In this study we will extend the previous studies to investigate the 
effects of pre-conditioning the brain using tACS. 
tRNS 
tRNS is electrical stimulation of the brain using random noise. This stimulation is also bipolar 
therefore, it is not expected to see any difference between the cathodal and anodal after-
effects of the priming. In the second part of this study, we will investigate the effects of pre-
conditioning the brain using tRNS. 
3. Research Instruments 
The following instruments are used for the research: 
1) Position sensor 
2) Transcranial Magnetic Stimulation (TMS) 
3) Electromyography (EMG) 
4) Transcranial altering current stimulation (tACS) 
5) Transcranial random noise stimulation (tRNS) 
6) Sphygmomanometer 
7) Oximeter 
8) Two questionnaires: 
a. Montreal Cognitive Assessment (MoCA) for screening the participants of no 
cognitive impairment. 

b. Questionnaire to exclude any participant using the exclusion criteria and gather 
information about possible confounding variables. This will be provided upon 
both visits. 
Both questionnaires are attached to this document. 
Position Sensor Camera 
Vicra position sensor camera will be used to record the exact position, direction, and tilt of the 
TMS coil during each pulse. This is done by carefully determining facial landmarks and 
measuring the participant’s scalp using a headband and a pointer equipped with position 
sensors. While the headband is around the participant’s head, the RA will use the pointer to 
identify the nasion, notch above the tragus of the left and right ear, forehead, top of the head, 
back of the head, and the left and right sides of the head. The Brainsight software uses these 
positions marked by the pointer to track the position of all the stimulation pulses to accurately 
stimulate the exact same position for each RMT and AMT recording. 
Transcranial Magnetic Stimulation (TMS) 
TMS will be applied on each participant to measure RMT: 
 
1) Before tACS/tRNS 
2) Immediately after tACS/tRNS 
3) 30 minutes after tACS/tRNS 
4) 1 hour after tACS/tRNS 
The following parameters will be applied for all eight applications of the TMS: 
PARAMETER  
DESCRIPTION/VALUE 
DEVICE 
Magstim Rapid2 Stimulator on single TMS pulse setting with 
the figure-of-eight AirFilm Coil 
APPLICATION LOCATION 
On C3 over the hand area of primary motor cortex 
 
Electromyography (EMG) 
EMG will be recorded during all TMS sessions using the following parameters: 
PARAMETER  
DESCRIPTION/VALUE 
DEVICE 
EMG Recording Pod, Brainsight 2 
APPLICATION LOCATION 
Two electrodes on First Dorsal Interosseous (FDI) muscle and 
the reference electrode on top of the wrist (head of ulna)  
SAMPLING FREQUENCY 
3 kHz 
DURATION FOR EACH 
PULSE 
50 ms before the pulse, 200 ms after the pulse 
 

Transcranial altering current stimulation (tACS) 
The following parameters will be applied during the tACS sessions: 
PARAMETER  
DESCRIPTION/VALUE 
DEVICE 
tES, Soterix Medical Inc. (Model: 2001) 
FREQUENCY 
40 Hz 
APPLICATION LOCATION 
Active electrode on C3 over the right-hand area of the motor 
cortex and reference on the contralateral motor cortex 
INTENSITY 
0.75 mA (1.5 mA peak-to-peak) 
Waveform 
Sinusoidal (bipolar) 
DURATION 
10 minutes 
 
Transcranial random noise stimulation (tRNS) 
The following parameters will be applied during the tRNS sessions: 
PARAMETER  
DESCRIPTION/VALUE 
DEVICE 
tES, Soterix Medical Inc. (Model: 2001) 
APPLICATION LOCATION 
Active electrode on C3 over the right-hand area of the motor 
cortex and reference on the contralateral motor cortex  
INTENSITY 
0.75 mA (1.5 mA peak-to-peak) 
Waveform 
Random Noise (bipolar) 
DURATION 
10 minutes 
4. Participants 
This research requires a total of 50 healthy participants who must meet the following inclusion 
criteria: 
 Between the ages of 18 years and 95 years 
 MoCA score ≥ 26 
 Not diagnosed with any restricting conditions such as history of seizures, epilepsy, 
having pacemakers, being pregnant, having a diagnosed major psychotic disorder, 
having pain relief medication on a daily basis, or having any metallic implants that might 
cause a conflict while using TMS. 
 Not being on any regular nervous system medication for depression, anxiety, sleep, etc. 
 Not have had any brain injury  
All participants will be assessed by MoCA and will fill out the questionnaire after signing the 
consent form. 
5. Recruitment 
Potential participants will be recruited by providing a poster on the supervisor’s webpage 
https://home.cc.umanitoba.ca/~mousavi/ and Biomedical Engineering webpage of University of 
Manitoba at https://umanitoba.ca/engineering/biomedical-engineering. Furthermore, posters 
will be placed around the University of Manitoba campus with permission to recruit 

participants. The posters will have information about the project and an email to contact the 
team to participate in the study. 
6. Data Analysis 
Motor Evoked Potential (MEP) 
Motor evoked potential, as shown in Figure 1, is the involuntary response of the muscle due to 
the magnetic stimulation to the corresponding area of the brain, hand area for the primary 
motor cortex. In this study, we are collecting the FDI (first dorsal interosseous) EMG to observe 
the change in the peak-to-peak value of the MEP. A noticeable increase in the peak-to-peak 
value of the MEP, while a constant amount of TMS intensity is applied to the brain before and 
after priming via tACS/tRNS is used, suggests that the priming has been in fact successful, and 
the motor threshold has been reduced due to the priming. 
The EMG recorded before and after tACS/tRNS will be compared to observe any change in the 
MEP. 
 
Figure 1. Active FDI EMG with MEP and cSP 
Cortical Silent Period (cSP) 
Cortical silent period (cSP) refers to the silent period that is followed immediately after MEP. 
This period is only observable in an active muscle EMG. By measuring the duration of cSP we 
can estimate the intracortical inhibition of pyramidal neurons caused by activation of GABAB 
(gamma-aminobutyric acidB) [8]. A noticeable decrease in the cSP potentially indicates that the 
preconditioning stimulation via tACS/tRNS has been effective. In this study we will be collecting 
the active muscle EMG during TMS sessions before and after application of tACS/tRNS. 
7. Informed Consent   
A consent form will be provided to the volunteers at the beginning of the two-hour session to 
inform them about the requirements of the study, risks and benefits, withdrawal process, 
confidentiality policies, and debriefing procedure. The volunteers who wish to participate in the 

study will sign the consent form and will be provided with a copy of the form for safekeeping. 
The original from will be locked along with the participants assigned code in a locked cabinet in 
the research supervisor’s office. 
8. Procedure 
All volunteers will be contacted by the research assistant (RA) via phone or email. The RA will 
briefly explain the study while also setting a time slot for the volunteers to come in and read 
the consent form and discuss it. If the participant wishes to continue the study and signs the 
consent form, the RA can start the initial assessments by taking the MoCA test and having the 
participant fill out the questionnaire. Once the volunteers meet the criteria of the study, they 
will be assigned a code and enrolled to the study and all their data will be recorded under the 
assigned code. The consent forms which contain the participants code and contact information 
will be locked in a cabinet in the PI’s office. 
Once enrolled, participants will be examined to achieve their RMT (resting motor threshold). To 
do this, first the Vicra camera is turned on and the scalp of the participant is measured and 
saved on the computer. Then single TMS pulses are applied with the coil positioned over the 
right-hand area of the primary motor cortex on the left hemisphere (C3) with initial intensity of 
50% of the maximum intensity. The coil position will be adjusted over the primary motor cortex 
until the “hotspot” is found and an involuntary right-thumb twitch is observed. If no twitch was 
observed after few pulses, the intensity will be increased by increments of 5% until the hotspot 
is found. Once the “hotspot” is found, it will be marked as a target using the coordinating 
camera and the intensity will be reduced by decrements of 1% to the minimum intensity where 
a twitch is observed. The coordinating camera will be used to record the exact position and 
direction of the coil while a hotspot was achieved to help us accurately stimulate the hotspot 
once it is found. Similar procedure is applied to determine RMT for the right side of the brain, 
i.e. left hand movement. 
Once the minimum intensity is recorded for the RMT, the participant gets a 10-minute break 
from the stimulation. During this time, the RA will measure the blood pressure and oxygen level 
and connect the electrodes over the FDI muscle of the right hand to record EMG and prepare 
the participant for the TMS session. During the first TMS session, 10 single pulses are applied to 
the participant over the hotspot recorded by the coordinating camera. The resting EMG of the 
corresponding muscle in a relaxed position will be recorded for further investigation under the 
participant code name. Immediately after, the participant will be asked to gently hold the arm 
rest of the chair and the active EMG will be recorded over 10 single pulses of TMS.  
The participant will be given a 5-minute break, during which the RA place the tACS active 
electrode on the hotspot marked by the coordinating camera and the reference on the 
contralateral motor cortex. When the electrodes are all set up, tACS will be applied gradually 
from lower current intensity to higher for 1 minute to allow the participant to adjust to the 
current. The current is applied at 40 Hz frequency for the duration of 10 minutes. Immediately 
after the application of tACS the participant’s resting and active EMG will be recorded similar to 
the pre-tACS procedure, with 10 pulses of TMS for each of the resting and active experiments. 

Also, the blood pressure and oxygen level will be remeasured. A similar procedure will be 
followed to record the resting and active EMG after 30 minutes and after 1 hour from the initial 
application of tACS. 
During the second session, the participant will be monitored by the exclusion questionnaire. If 
the participant passes the criteria, the RMT will be measured again and a similar procedure to 
the first session will be followed while tRNS is used instead of tACS. The second session can 
begin after 4 hours following the first session and may occur on a separate day depending on 
the participant’s availability. 
Each of these procedures will be applied once on each participant and the experiment for each 
participant will be done after one or two visits to the laboratory. 
Data collection for the study will take place for a period of one year.  
All the data will be deleted and destroyed 3 years after completion of the study. 
9. Deception 
There is no deception in this study. 
10. Feedback/Debriefing 
Participants can ask about their own performance and result of the study once the study is 
completed and published. 
11. Risks and Benefits 
Exposing the brain to magnetic energy via TMS might have some mild risks such as tingling in 
the jaw and the arm and mild headaches. However, participants who are pregnant, use 
pacemakers, or have any metallic implants (e.g., dental implants) will be excluded from the 
study to avoid any conflict while exposed to magnetic energy. 
There are also some potential mild risks while preconditioning the brain using tACS/tRNS. These 
risks include mild pain, headaches, or fatigue. Participants may experience tingling, itching, 
burning sensation, or skin redness where electrodes are connected. Participants that have 
history of seizure or epilepsy will be excluded from the study as tACS and tRNS have limited 
information on the serious adverse effects.  
There are no direct benefits concerning the participants from this study. However, the result of 
the study has the potential to provide sufficient means to treat early stages of memory loss for 
Alzheimer Dementia (AD) patients. 
 
 
 

12. Anonymizing/Confidentiality 
All participants’ personal information, including their name and contact number will be 
remained confidential. Each participant will be assigned with a de-identified code and the 
master file containing the names and codes will be saved on a secure server, ECE server. The 
assessment data will be saved on the PI’s BME server which is physically separate from the ECE 
server. 
13. Compensation 
This study does not provide any compensation for the participants. 
14. Dissemination 
The study result will be presented in relevant seminars, conferences, and journal paper 
publications. 
15. References 
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