Mechanism of Action for Transcranial Alternating Current (tACS) Stimulation for the Treatment of Major Depressive Disorder (MDD)
Phase: NA
Conditions: Major Depressive Disorder, MDD
Sponsor: University of North Carolina, Chapel Hill

 
 
 Mechanism of Action of Transcranial Alternating Current Stimulation 
for the Treatment of Major Depressive Disorder 
 
 
 
NCT number NCT03994081 
Document Date 05/02/2022 
 

Mechanism of Action of Transcranial Alternating 
Current Stimulation for the Treatment of Major 
Depressive Disorder 
Protocol Number: 20-1822 
Principal Investigator: David Rubinow, MD 
Co-Investigator: Flavio Frohlich, PhD 
Co-Investigator: Crystal Schiller, PhD 
Funded by: Foundation of Hope 
Version Number:  2 
02 May 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

NIH-FDA Phase 2 and 3 IND/IDE Clinical Trial Protocol Template   
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Summary of Changes from Previous Version: 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
1.2 
Updating study schema 
We deleted unnecessary administration of questionnaire to increase feasibility and reduce burden for the 
participant.  
1.3 
Updating of Schedule of Activities (SoA) 
Now, the SoA provides accurate and clear information over all study procedures.  
8.2 
No YMRS ratings during the stimulation week 
Signs of Mania are assessed during Screening, at Day 1, Day 5 and follow-up to ensure safety. The 
assessment during D2-D4 prolongs the experimental session without a significant gain in safety. 
1.1, 4.1 
We deleted the MRI study visit 
With regards to feasibility, we opted against the use of MRI and deleted it from the Master Protocol. The 
MRI was initially not included for safety purposes but as potential correlative research outcome.  
5.5 
Use of non-encrypted emails 
Potential participants often did not respond to encrypted email. We hope to foster recruitment by using 
standard emails. No medical data is inquired in any outgoing email and all medical data is collected within a 
HIPAA conform Zoom call or via RedCap.  
5.5 
Recruitment procedure, RedCap Screening, Pre-
Screening (remotely) 
We slightly restructured out recruitment process to facilitate recruitment and simply the process for our 
participants.  
6.3, 9.4.6 
Updated Randomization Personnel 
Addressing personnel changes in our lab. 
1.1, 4.1 
Adjusted total hours for study 
The Master Protocol is now matching the actual study duration. 
1.1 
Electronic informed consent / HIPAA consent 
We like to cover all parts of our study optimally by informed consent. Since the first two sessions are held 
remotely via phone/Zoom, we opt for an electronic consent obtained during the first session (pre-
screening). A paper copy of the consent is handed to the participant within the first stimulation session.  
9.4.6 
We do not plan an interim analysis 
Results would be flawed by low number of participants 
5.2 
We reduced the necessary duration of stable 
antidepressant medication from 6 to 4 weeks.  
A clinical benefit after initiating an antidepressant treatment is expected after 2-4 weeks. We therefore 
demand a stable (or none) antidepressant medication for at least 4 weeks. Note that this does not demand 
the patient to initiate a new pharmacological treatment but requires a stable dose on his current 
medication. 
5.2 
We specified the exclusion criteria related to alcohol and 
substance use. Now, moderate and severe 
alcohol/substance use disorder are defined as 
exclusion criteria. 
We specified the diagnosis auf AUD and substance use disorder based on DSM-V and present now a clear 
cut off to exclude participants.  
10.1.6, 8.3.5 
Since the inclusion of the DSMB is not necessary for this 
study, we deleted the phrases related to that institution. 
Adapting the Master Protocol to reflect the actual experiment.  
7.1. 
We specified what happens if a subject withdraws from 
the study during the stimulation period.  
We will try to schedule the 2-week follow-up to administer the respective questionnaires. This is assessment 
is related to research purpose only. 
5.1. 
Inclusion age changed to 18-70 years 
Represent a broader spectrum of society.  

  
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Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. 1 
1. PROTOCOL SUMMARY ....................................................................................................................................... 2 
1.1 Synopsis ............................................................................................................................................................ 2 
1.2 Schema .............................................................................................................................................................. 4 
1.3 Schedule of Activities (SoA) ........................................................................................................................... 5 
2. INTRODUCTION .................................................................................................................................................... 6 
2.1 Study Rationale ................................................................................................................................................ 6 
2.2 Background ....................................................................................................................................................... 6 
2.3 Risk/Benefit Assessment ................................................................................................................................ 6 
2.3.1 Known Potential Risks 6 
2.3.2 Known Potential Benefits ......................................................................................................................... 8 
2.3.3 Assessment of Potential Risks and Benefits ........................................................................................ 8 
3. Objective and Outcome Measures....................................................................................................................... 8 
4. STUDY DESIGN ..................................................................................................................................................... 8 
4.1 Overall Design .................................................................................................................................................. 8 
4.2 Scientific Rationale for Study Design ............................................................................................................ 9 
4.3 Justification for Dose........................................................................................................................................ 9 
4.4 End of Study Definition .................................................................................................................................. 10 
5. STUDY POPULATION ........................................................................................................................................ 10 
5.1 Inclusion Criteria ............................................................................................................................................. 10 
5.2 Exclusion Criteria............................................................................................................................................ 10 
5.3 Lifestyle Considerations ................................................................................................................................ 11 
5.4 Screen Failures ............................................................................................................................................... 11 
5.5 Strategies for Recruitment and Retention .................................................................................................. 11 
6. STUDY INTERVENTION .................................................................................................................................... 12 
6.1 Study Intervention(s) Administration............................................................................................................ 12 
6.1.1 Study Intervention Description .............................................................................................................. 12 
6.1.2 Dosing and Administration..................................................................................................................... 14 
6.3 Measures to Minimize Bias: Randomization and Blinding ....................................................................... 14 
6.4 Study Intervention Compliance .................................................................................................................... 15 
6.5 Concomitant Therapy .................................................................................................................................... 15 
   6.5.1 Rescue Medicine 
15 
7. STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 15 
7.1 Discontinuation of Study Intervention .......................................................................................................... 15 
7.2 Participant Discontinuation/Withdrawal from the Study ............................................................................ 16 
7.3 Lost to Follow-Up............................................................................................................................................ 16 
8. STUDY ASSESSMENTS AND PROCEDURES .............................................................................................. 16 
8.1 Efficacy Assessments .................................................................................................................................... 16 
8.1.1 
Physiological Changes 
17 
8.1.2 
Clinical Evaluations 
17 
8.1.3 
Self-Report Assessments 
17 
8.2 Safety and Other Assessments .................................................................................................................... 18 
8.3 Adverse Events and Serious Adverse Events ........................................................................................... 19 
8.3.1 Definition of Adverse Events (AE) ........................................................................................................ 19 
8.3.2 Definition of Serious Adverse Events (SAE) ....................................................................................... 19 

  
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8.3.3 Classification of an Adverse Event ....................................................................................................... 20 
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up................................................ 21 
8.3.5 Adverse Event Reporting ....................................................................................................................... 22 
8.3.6 Reporting of Pregnancy ......................................................................................................................... 22 
8.4 Unanticipated Problems ................................................................................................................................ 22 
8.4.1 Definition of Unanticipated Problems (UP) ......................................................................................... 22 
8.4.2 Unanticipated Problem Reporting ........................................................................................................ 23 
8.4.3 Reporting Unanticipated Problems to Participants ............................................................................ 23 
9. STATISTICAL CONSIDERATIONS ................................................................................................................... 23 
9.1 Statistical Hypotheses ................................................................................................................................... 23 
9.2 Sample Size Determination .......................................................................................................................... 23 
9.3 Populations for Analyses ............................................................................................................................... 23 
9.4 Statistical Analyses ........................................................................................................................................ 24 
9.4.1 General Approach 
24 
9.4.2 Analysis of the Primary Efficacy Endpoint(s) (EEG) .......................................................................... 24 
9.4.3 Analysis of the Secondary Endpoint(s)(Correlation between EEG Changes and Symptoms of 
Depression) 
24 
9.4.4 Safety Analyses 
25 
9.4.5 Baseline Descriptive Statistics .............................................................................................................. 25 
9.4.6 Planned Interim Analyses ...................................................................................................................... 25 
9.4.7 Sub-Group Analyses 
25 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ..................................... 26 
10.1 Regulatory, Ethical, and Study Oversight Considerations ..................................................................... 26 
10.1.1 Informed Consent Process .................................................................................................................. 26 
10.1.2 Study Discontinuation and Closure .................................................................................................... 26 
10.1.3 Confidentiality and Privacy .................................................................................................................. 27 
10.1.4 Future Use of Stored Specimens and Data ...................................................................................... 27 
10.1.5 Key Roles and Study Governance ..................................................................................................... 27 
10.1.6 Safety Oversight 
28 
10.1.7 Clinical Monitoring 
28 
10.1.8 Quality Assurance and Quality Control ............................................................................................. 29 
10.1.9 
Data Handling and Record Keeping ........................................................................... 29 
10.1.10 
Protocol Deviations 
30 
10.1.11 
Publication and Data Sharing Policy ........................................................................... 30 
10.1.12 
Conflict of Interest Policy .............................................................................................. 30 
10.2 
Additional Considerations ................................................................................................................. 30 
10.3 
Abbreviations ...................................................................................................................................... 31 
10.4 
Protocol Amendment History ........................................................................................................... 33 
11 
REFERENCES ............................................................................................................................................. 34 
 

Mechanism of Action of tACS for the Treatment of MDD  
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STATEMENT OF COMPLIANCE 
 
The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP) and the following:    
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 
46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
 National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible 
for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects 
Protection and ICH GCP Training.  
 
The protocol, informed consent form(s), recruitment materials, and all subject materials will be 
submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both the 
protocol and the consent form must be obtained before any subject is enrolled.  Any amendment to the 
protocol will require review and approval by the IRB before the changes are implemented to the study.  
In addition, all changes to the consent form will be IRB-approved; a determination will be made 
regarding whether a new consent needs to be obtained from subjects who provided consent, using a 
previously approved consent form. 
 
 

Mechanism of Action of tACS for the Treatment of MDD  
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1. PROTOCOL SUMMARY 
1.1 SYNOPSIS  
Title: 
Mechanism of Action of Transcranial Alternating Current Stimulation for the 
Treatment of Major Depressive Disorder 
Study Description: 
The purpose of this clinical trial is to investigate the mechanism of action of 
non-invasive transcranial alternating current stimulation (tACS) in subjects 
with major depressive disorder (MDD). We will recruit 20 males and females 
with unipolar, non-psychotic MDD. Eligible subjects will have 5 consecutive 
40-minute stimulation sessions. Subjects will be randomly assigned to one 
of two groups: sham stimulation or 10Hz (alpha) tACS. Participation will 
include 8 visits, two of them remotely. Participants will give electronic 
informed consent during the first remote screening sessions. At the initial 
stimulation session (D1), the electronic consent is printed out and handed 
to the participant. Eligible subjects will undergo 5 consecutive stimulation 
sessions with ongoing assessment of suicidality and manic symptoms. 
Subjects will fill in a questionnaire about stimulation side effects. 
Neurophysiological measures with high-density EEG will be taken before 
and after every stimulation session, as well as at the 2-week follow-up visit. 
Clinical assessments will be performed at baseline (D1), Day 5 of 
stimulation, and at the follow-up visit using the Hamilton Depression Rating 
Scale (HDRS-17). Additional assessments of symptomology, quality of life, 
sleep, and arousal will be completed per the research schedule and are 
mostly implemented as electronic surveys.  
Objectives: 
 
Primary Objective: To investigate the physiological changes in subjects with 
MDD over the course of a 5-day, 40-minute stimulation protocol, specifically 
changes in alpha oscillation power from resting state EEG recordings over 
the course of the intervention (D1 to D5 of stimulation, to the follow-up 
visit).  
Secondary Objectives: To elucidate the relationship between changes in 
EEG and changes in depressive symptoms, by comparing the changes in 
clinical assessments (e.g., HDRS) and the change in alpha oscillation power 
over the course of the intervention (baseline to day 5 of stimulation, to the 
follow-up visit).  
Endpoints: 
Primary Endpoint: Immediate effects on resting state EEG recordings on 
each day of stimulation as well as long term effects 2-weeks after 
stimulation.  
Secondary Endpoints: Relationship between symptoms of depression per 
self-report and clinician rated and physiological changes. 
Study Population: 
We will recruit 20 males and non-pregnant females (age 18-70) with a 
diagnosis of unipolar, non-psychotic MDD, free of benzodiazepines and 
anticonvulsant medications. Eligible subjects will have a Hamilton 
Depression Rating Scale (HDRS-17) > 8 and low suicide risk (determined by 
scoring less than 3 on the Suicide Item on the HDRS as well as having no 
active intent as determined by the Columbia Suicide Severity Rating Scale 

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(C-SSRS). Subjects will be recruited from the Chapel Hill, Durham and 
Raleigh areas. 
Phase: 
N/A 
Description of 
Sites/Facilities Enrolling 
Participants: 
University of North Carolina at Chapel Hill.   
Description of Study 
Intervention: 
We will the XCSITE100 stimulator designed by PulvinarNeuro for 
investigational purposes to deliver either active sham or 10Hz transcranial 
alternating current stimulation.  Active sham treatment will include 20 
seconds of ramp in to 40 seconds of 10Hz tACS with a ramp out of 20 
seconds for a total of 80 seconds of stimulation.  The choice of an active 
sham is motivated to enhance success of subject blinding by mimicking skin 
sensations associated with tACS. 10Hz tACS will also have a 20 second ramp 
in and ramp out with 40-minutes of stimulation for a total of 2440 seconds. 
Stimulation waveforms are sine-waves with a zero-to-peak amplitude of 
1mA.  Subjects will stay in a relaxed yet experimentally controlled state by 
watching a nature movie such as “Reefscape” during stimulation. 
 
Study Duration: 
2 years 
Participant Duration: 
Participation for each subject will last no longer than 2-3 weeks total. 
Completion will involve a maximum number of 8 appointments. The initial 
session is a remote (via Zoom) pre-screening. Participants are asked general 
exclusion criteria regarding brain stimulation studies and describe their 
mental health history. The meeting takes approx. 30 minutes. If no exclusion 
criteria are present, an appointment for the screening session is scheduled. 
This session – in person or remotely – involves the clinical assessment of 
depression severity (HDRS), psychiatric diagnose (M.I.N.I.) and suicidality (C-
SSRS). After consultation with the study PI Dr. Rubinow, treatment 
resistance is determined (Maudsley Staging Method) and participants are 
randomized and start the five-day stimulation procedure.  
The initial session (D1) and D5 will take approximately 4 hours. D2-D4 will 
take 90 minutes. The 2-week follow-up will take approximately 3 hours. We 
estimate total participation time to be approximately 17 hours. 
 
 

Mechanism of Action of tACS for the Treatment of MDD  
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1.2 SCHEMA 
 
 
 
Pre-screening Session (remote) 
Screening Session (remote/in person) 
Medication 
Handedness 
 
MINI 
C-SSRS 
HDRS-17 
WHODAS 
 
Day 1 
Vitals 
C-SSRS 
HDRS-17 
BDI II 
IDAS 
YMRS 
Rumination 
PSQI 
SHAPS 
Q-LES-Q-SF 
BAS/BIS 
STAI 
Preg Test 
Medication 
RSEEG 
Stimulation 
Stim AE  
Structured AE  
Days 2-4 
Vitals 
Medication 
RSEEG 
Stimulation 
Stim AE Q 
Structured AE  
Daily suicide 
survey 
2-week FU (in person) 
Vitals 
C-SSRS 
HDRS-17 
BDI II 
IDAS 
YMRS 
Rumination 
PSQI 
SHAPS 
Q-LES-Q-SF 
BAS/BIS 
STAI 
Medication 
RSEEG 
Structured AE  
Daily suicidality survey emailed 
Maudsley 
 
Day 5 
Vitals 
C-SSRS 
HDRS-17 
BDI II 
IDAS 
YMRS 
Rumination 
PSQI 
SHAPS 
Q-LES-Q-SF 
BAS/BIS 
STAI 
Medication 
RSEEG 
Stimulation 
Stim AE  
Structured AE  
Randomization 
ACTIVE 
Arm 1 
n =10 
SHAM 
Arm 2 
n =10 
Stimulation Week (in person) 
Electronic consent 
Study information 
Exclusion criteria 

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1.3 SCHEDULE OF ACTIVITIES (SOA) 
 
Procedures 
Pre-Screening (remote) 
Screening  
(in person / remote) 
Day 1 of Stimulation (D1) 
Day 2 of Stimulation (D2) 
Day 3 of Stimulation (D3) 
Day 4 of Stimulation (D4) 
Day 5 of Stimulation (D5) 
Interim  
Day 6-Day 21 +/-3 days 
2-week Follow up (FU) 
Day 22 +/-3 day s 
Electronic Informed consent (S) 
X 
 
 
 
 
 
 
 
 
HIPPA authorization 
 
 
X 
 
 
 
 
 
 
Medication & Psychotherapy 
X 
X 
X 
X 
X 
X 
X 
 
X 
Determine Eligibility 
X 
X 
 
 
 
 
 
 
 
Handedness (S) 
 
X 
 
 
 
 
 
 
 
Demographics + Medical Hx (S) 
 
X 
 
 
 
 
 
 
 
M.I.N.I. 
 
X 
 
 
 
 
 
 
 
C-SSRS 
 
X 
X 
 
 
 
X 
 
X 
HDRS-17 
 
X 
X 
 
 
 
X 
 
X 
WHODAS 2.0 (S) 
 
X 
 
 
 
 
 
 
 
Maudsley Staging Method 
 
X 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
Urine Pregnancy Test 
 
 
X 
 
 
 
 
 
 
Beliefs about Treatment (S) 
 
 
X 
 
 
 
 
 
 
IDAS (S) 
 
 
X 
 
 
 
X 
 
X 
Rumination (S) 
 
 
X 
 
 
 
X 
 
X 
SHAPS (S) 
 
 
X 
 
 
 
X 
 
X 
PSQI (S) 
 
 
X 
 
 
 
X 
 
X 
BDI II (S) 
 
 
X 
 
 
 
X 
 
X 
Q-LES-Q-SF (S) 
 
 
X 
 
 
 
X 
 
X 
BAS/BIS (S) 
 
 
X 
 
 
 
X 
 
X 
STAI Y1 / Y2 (S) 
 
 
X 
 
 
 
X 
 
X 
YMRS 
 
 
X 
 
 
 
X 
 
X 
Vitals 
 
 
X 
X 
X 
X 
X 
 
X 
EEG 
 
 
X 
X 
X 
X 
X 
 
X 
tACS Stimulation (alpha/sham) 
 
 
X 
X 
X 
X 
X 
 
 
Daily Stimulation Q (S) 
 
 
X 
X 
X 
X 
X 
 
 
AE Structured Interview + Review 
 
 
X 
X 
X 
X 
X 
 
X 
Blinding Q (subject) (S) 
 
 
 
 
 
 
X 
 
 
Blinding Q (operator) (S) 
 
 
 
 
 
 
X 
 
 
Symptom Improvement Q. (S) 
 
 
 
 
 
 
X 
 
 
Daily Suicide Survey (S) 
 
 
X-------------------------------------------------------------X    
X-------------- X 
(S): implemented as survey in RedCap, Q is used as abbreviation for questionnaire.  
Daily Suicide Surveys will be conducted in RedCap throughout the study 
 
 

Mechanism of Action of tACS for the Treatment of MDD  
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2. INTRODUCTION 
 
2.1 STUDY RATIONALE  
 
Major depressive disorder (MDD) is a common, severe psychiatric illness that affects 6.7% of adults every 
year in the United States [1]. MDD is characterized by depressed mood, loss of interest or pleasure in 
activities that used to be enjoyable (anhedonia), sleep disturbances, cognitive impairment, and other 
symptoms that can severely affect quality of life. Current recommended pharmacological treatments are 
non-targeted, resulting in undesirable side effects. Furthermore, the STAR*D trial demonstrated that a 
large percentage of patients are treatment-resistant, and that remission is less likely following multiple 
drug trials[2]. More effective and safer therapies for the treatment of MDD are desperately needed.    
   
2.2 BACKGROUND  
  
MDD has been associated with hypoactivity in the left dorsolateral prefrontal cortex [3], characterized by 
elevated alpha oscillations [4]. Therefore, targeting and reducing alpha oscillations in the left dorsolateral 
prefrontal cortex could prove beneficial to patients with MDD. Targeted stimulation modalities, such as 
transcranial alternating current stimulation (tACS), can directly engage and modulate oscillatory activity. 
TACS utilizes sine-wave stimulation waveforms that can enhance specific oscillatory activity [5]. The 
proposed study is a follow-up to a recently concluded trial run by the Carolina Center for Neurostimulation 
under the direction of Drs. Frohlich and Rubinow [6]. 
 
This recently published trial compared the efficacy of 10Hz-tACS (therapeutic frequency, n = 10), 40Hz-
tACS (control frequency, n = 10), and sham/placebo stimulation (n = 10). The intervention consisted of 5 
consecutive days of 40-minutes of stimulation. Results demonstrated that 2-weeks after completion of 
the intervention, the 10Hz-tACS group had more subjects responding to treatment (i.e., at least a 50% 
decrease in depressive symptoms) as measured by the clinician-administered assessments (Montgomery-
Asberg Depression Rating Scale, p = 0.026, and Hamilton Depression Rating Scale, p = 0.026). Concurrently, 
we found that 10Hz-tACS successfully reduced alpha oscillations in the left frontal regions after 
completion of the stimulation protocol (p < 0.05). While these data demonstrate preliminary efficacy, it is 
unclear how tACS caused this change, indicating a need to elucidate neurophysiological mechanism of 
action. Previous work from other groups demonstrate an increase in alpha oscillations following tACS (e.g., 
[[7]–[9]]), whereas our stimulation paradigm resulted in a decrease in alpha oscillations. Importantly, what 
is missing is the temporal course of these physiological changes and how they correspond to clinical 
outcomes. By including high-definition EEG before and after each stimulation, we will be able to delineate 
the neurophysiological reorganization of alpha oscillations in response to repeated application of tACS. 
 
2.3 RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
 
Risk of Confidentiality Breach: In the unlikely event of a breach of confidentiality, people might discover 
that an individual was involved in this research study. This is especially sensitive because the clinical 
population recruited for this study may be subjected to negative consequences caused by the stigma of 
mental disorders. Furthermore, some might not agree with the principle of participating in research or of 

Mechanism of Action of tACS for the Treatment of MDD  
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changing natural brain activity. To avoid breaches in confidentiality, study documents that contain 
personal information, including the informed consent document, and the document that links study ID 
numbers to personal identifying information are kept in locked filing cabinets in locked rooms, separate 
from any source documents containing subject dummy identifiers. All data is stored in locked cabinets 
inside locked offices; electronic data will be stored only on password-protected computers, and data 
encryption methods will be used during communication between investigators. Interviews will be 
conducted over the HIPAA approved Zoom account. Only study personnel will have access to the data. All 
study staff participate in annual human subject training that includes education about responsibilities to 
the minimize risk of confidentiality breach. 
 
Risk of Embarrassment: Self-report assessments contain questions regarding sensitive personal 
information. This risk is necessary in order to assess mood symptoms and associated psychopathology. 
Subjects will be assured upon intake that only study personnel will see any clinical ratings and that self-
identifying information will not be collected alongside HIPAA protected information.  
 
Risk of Injury and Discomfort: The side effects of tACS are mild and transient; in fact, low intensity 
transcranial current stimulation, such as tACS, has been used for several years without any report of 
serious side effects [10]. Furthermore, this stimulation mode has nothing to do with electroconvulsive 
therapy that applies many orders of magnitude higher stimulation current. Rather, transcranial current 
stimulation is so weak that it does not cause super-threshold activation of neurons [11]. However, tACS 
does have some mild side effects, such as transient mild tingling, burning, or itching under the electrode 
sites. In our previous trial, subjects from all three groups of stimulation reported either absent or mild 
side effects, and there was no difference between the groups with the exception of “flickering lights” (or 
phosphenes, p = 0.014)[6]. To monitor these mild side effects, we will be administering a stimulation 
questionnaire after each stimulation session to determine whether these effects were experienced and 
at what intensity. Research personnel is present during the full experimental. If subject is experiencing 
severe discomfort (as determined by the questionnaire or by self-report), the stimulation will be stopped 
immediately.  
 
Patients with MDD have an about 20 times higher rate of suicide than average. We have no evidence that 
our treatment paradigms will in any way increase this likelihood. In the previous iteration of this trial, 4 
subjects in the sham/placebo stimulation group experienced an increase in suicidal thoughts and only one 
of those 4 subjects reported suicidal intent [6]. No subjects who received tACS reported an increase in 
suicidal ideation from baseline. Regardless, subjects with high suicide risk will not be included in this study. 
If an enrolled subject shows signs of suicide risks that were not apparent during enrollment, a referral to 
UNC Psychiatry will be made. Dr. Schiller, Co-I, will facilitate this process. 
 
We will be using the Suicide Item included in the HDRS-17 [12] to assess suicide risk, as well as the 
Columbia Suicide Severity Rating Scale (C-SSRS) [13] to determine intent for suicide during the screening 
interview. Inclusion criteria state that the subject must be low suicide risk and that potential subjects with 
an above “low risk” designation will not be eligible for the study. After the subject enrolls in the study, we 
will evaluate suicidal thoughts daily through an online questionnaire administered through REDCap. 
Additionally, the C-SSRS will be administered at D1, D5, and at the 2-week follow-up visit. In the event 
that suicide risk increases during participation in the study, the subject will be asked to stop the study. 
The subject will be provided with a referral to UNC Department of Psychiatry, and their mental health 
care or family medical doctor will be contacted. 
 

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2.3.2 KNOWN POTENTIAL BENEFITS  
 
This study has not been designed to benefit the individual subjects. However, subjects in this study may 
experience some degree of relief from mood symptoms because of the tACS treatment. Our last trial 
revealed preliminary efficacy, as participants who received 10 Hz tACS stimulation showed a 50% decrease 
in symptoms of depression two weeks later [6].  
 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
 
The risks and benefits presented above are no more serious than for other clinical trials in this population. 
Based on the need for complementary and alternative treatments for MDD, the potential risks are worth 
the potential benefits. Justification as to why the risks of participation in the study outweigh the value of 
the information to be gained. 
 
3. OBJECTIVE AND OUTCOME MEASURES  
 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR ENDPOINTS 
Primary 
 
 
To determine the effect of 10 Hz 
tACS on brain physiology as 
measured with 
electroencephalogram (EEG). 
 
 
Resting state EEG 
recordings taken 
before/after every 
stimulation session, as 
well as at a 2-week 
follow up visit. 
The focus is to determine the 
mechanism of action for five 
consecutive days of forty-minute tACS 
stimulation sessions. These endpoints 
will allow the examination of both 
immediate and long-term effects of 10 
Hz tACS on physiology. 
Secondary 
 
 
To elucidate the relationship 
between change in physiology and 
change in clinical symptoms  
Correlations between 
Hamilton Depression 
Rating Scale (HDRS), 
Beck Depression 
Inventory (BDI) and 
alpha oscillation 
power (as measured 
by resting state EEG 
recordings).  
Previous work indicates that 10 Hz 
tACS modulates mood and physiology, 
but there is no clear indication that 
these changes are related. 
 
 
4. STUDY DESIGN  
 
4.1 OVERALL DESIGN 
 
The design for this study is a pilot, randomized, double-blind, sham-controlled, clinical trial which will be 
used to investigate the mechanism of action of 10 Hz tACS in subjects with MDD, as well as determine the 
relationship between physiological changes and clinical changes. We are recruiting from a clinical 
population. Participants will be 20 males and non-pregnant females ages 18-70 with unipolar, non-

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psychotic MDD, free of benzodiazepine and anticonvulsant medication, who have a HDRS-17 > 8 and are 
at a low risk for suicide according to the Hamilton Depression Rating Scale as well as the Columbia Suicide 
Severity Rating Scale (C-SSRS).  All women of child-bearing potential will be asked to take a pregnancy test 
during the initial session to determine eligibility for the study; nursing or pregnant subjects will be 
excluded from participation, and all women of child-bearing potential will be required to use an 
appropriate form of birth control throughout their participation. These individuals will be outpatients and 
may or may not seek mental health care from a family practitioner, therapist, or psychiatrist. 
 
This is a single-site, pilot clinical trial with 2 arms. Due to the Covid pandemic recruitment was slower than 
expected. We anticipate to complete study enrollment until 12/2022. Subjects will be randomly assigned 
to one of two arms: active sham stimulation or 10 Hz (alpha) tACS. We will use the XCSITE100 stimulator 
designed by PulvinarNeuro for investigational purposes to deliver stimulation over five consecutive days. 
Subjects will stay in a relaxed yet experimentally controlled state by watching a nature movie such as 
“Reefscape” during stimulation. The initial session (D1) and D5 will take approximately 4 hours. D2-D4 will 
take 90 minutes. The 2-week follow-up will take approximately 3 hours. We estimate that total 
participation to be approximately 17 hours. 
 
 
4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
  
This study is a double-blind, randomized, sham-controlled interventional study. The choice of double blind 
and randomization is important for the integrity of our data, especially for the clinical assessments. All 
individuals involved with data collection (as well as all randomized subjects) will be blind to the subject’s 
arm assignment until all data has been collected. This will reduce implicit and explicit bias in the data 
collection process. In this study, subjects will be randomized in to one of two arms: active sham (i.e., 
placebo) stimulation or 10 Hz tACS. This is a follow-up to a previous study that compared 40 Hz tACS 
(control frequency), 10 Hz tACS and sham/placebo stimulation. The greatest effect was found when 
comparing 10 Hz tACS and sham/placebo stimulation; no effect was found with 40 Hz tACS. Furthermore, 
for 40 Hz tACS the study blind was compromised by stimulation-induced phosphenes, with 80% of subjects 
correctly guessing that they had received stimulation. As a result, we choose to further explore 10 Hz tACS 
in comparison to sham/placebo stimulation, omitting 40 Hz tACS as our control frequency. While having 
only a single control (placebo/sham) may be problematic, we have determined that our smaller, second 
part of this study does not require the same 3 arms as previously used, as the primary outcome is target 
engagement measured with the EEG and to further understand the mechanism of action of tACS in a 
multi-day study. In this design, our sham/placebo stimulation is active, including 80 seconds of stimulation 
(20 seconds ramp-in to 40 seconds of 10 Hz tACS at 1 mA with 20 seconds of ramp-out). This choice of 
active sham/placebo is important to mimic the skin sensations and other transient side effects associated 
with stimulation. Typically, stimulation side effects only last approximately a minute into stimulation; 
indicating that theoretically sham/placebo stimulation should be difficult to differentiate from 10 Hz tACS. 
 
4.3 JUSTIFICATION FOR DOSE 
 
As a continuation study, we are utilizing the exact same dosage as given before: 5 consecutive days of 40-
minutes of stimulation [6]. This dosage was chosen based on a previous study, which examined 
transcranial direct current stimulation (tDCS) for the treatment of MDD [14].   
 

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4.4 END OF STUDY DEFINITION 
 
The end of this study is defined as when the last subject completes the final study visit (i.e., the 20th 
subject completes their 2-week follow-up visit). 
 
5. STUDY POPULATION 
 
5.1 INCLUSION CRITERIA 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
• 
Ages 18-70 years 
• 
DSM-IV diagnosis of MDD; unipolar, non-psychotic  
• 
Hamilton Rating Depression Rating Scale (HRDS-17) score >8 
• 
Low suicide risk as determined by a score of <3 on the Suicide Item on the HDRS-17 and based 
on additional information from the C-SSRS (no intent) 
• 
Capacity to understand all relevant risks and potential benefits of the study (informed consent) 
5.2 EXCLUSION CRITERIA 
 
An individual who meets any of the following criteria will be excluded from participation in this study: 
• 
DSM-V diagnosis of moderate or severe alcohol use disorder (AUD) within the last 12 months. 
• 
DSM-V diagnosis of moderate to severe substance use disorder (excluding tobacco) within the 
last 12 months.  
• 
Current axis I mood, or psychotic disorder other than major depressive disorder 
• 
Lifetime comorbid psychiatric bipolar or psychotic disorder 
• 
Eating disorder (current or within the past 6 months) 
• 
Obsessive-compulsive disorder (lifetime) 
• 
Post-traumatic stress disorder (PTSD, current or within the last 6 months) 
• 
Attention deficit hyperactivity disorder (ADHD, currently under treatment) 
• 
Current use of benzodiazepines or anti-epileptic drugs 
• 
Antidepressant drugs taken for less than 4 weeks (i.e., recently initiated) 
• 
Neurological disorders, including but not limited to history of seizures (except childhood febrile 
seizures and ECT-induced seizures), dementia, history of stroke, Parkinson's disease, multiple 
sclerosis, cerebral aneurysm. 
• 
Medical or neurological illness (unstable cardiac disease, AIDS, malignancy, liver or renal 
impairment) or treatment for a medical disorder that could interfere with study participation; 
comorbid neurological condition (i.e. seizure disorder, brain tumor) 
• 
History of traumatic brain injury that required subsequent cognitive rehabilitation, or cause 
cognitive sequelae. 
• 
Prior brain surgery and/or any brain devices/implants, including cochlear implants and 
aneurysm clips 
• 
Current pregnancy or lactation. If the ability to become pregnant exists, unwillingness to use 
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• 
Anything that, in the opinion of the investigator, would place the participant at increased risk or 
preclude the participant’s full compliance with or completion of the study 
• 
Non-English speakers 
5.3 LIFESTYLE CONSIDERATIONS 
  
Benzodiazepine use will be prohibited during this study, unless used as needed. If subjects are prescribed 
benzodiazepines as needed (PRN), they will be requested not to use benzodiazepines within 48 hours of 
any study session. Current medication intake is queried every day during the stimulation period. 
 
5.4 SCREEN FAILURES 
 
In the design of this study, initial phone or zoom screening procedures should identify the majority of 
subjects who could potentially become screen drop outs in the clinical trial. However, the phone/zoom 
screening process does not necessarily account for all exclusion criteria. 
In the case that a subject enrolls to participate in the trial and the first interview reveals that they do not 
meet study criteria, the study personnel completing the interviewing process will clearly explain why the 
subject does not meet criteria. However, in the case that a subject does not qualify based on suicide risk, 
procedures will be followed to ensure subject safety. 
 
5.5 STRATEGIES FOR RECRUITMENT AND RETENTION 
This clinical trial will utilize multiple recruitment strategies to communicate this opportunity to as many 
potential participants as possible.  Our first means is through a referral process.  Participants can be 
referred to the study through their primary mental health care provider or family doctor during routine 
visits. This type of recruitment will take place in doctors’ offices throughout Chapel Hill, Carrboro, Durham 
and Raleigh areas. We estimate that approximately 15 participants will be enrolled from the Chapel 
Hill/Carrboro area and 5 between Durham and Raleigh. Clinicians will be informed of inclusion criteria 
through email and listserv announcements and be asked to mention this clinical trial to appropriate 
patients and offer them a flyer/ brochure with contact information. We will also be using the UNC i2b2 to 
send request forms to the Carolina Data Warehouse to recruit participants who have been seen at UNC 
Hospitals that meet the inclusion criteria. 1 
In addition to referrals through primary care providers, we will advertise the study directly to the public 
on websites such as ClinicalTrials.gov, studypages.com, Research For Me, frohlichlab.org and 
Carolinaneurostimulation.org. We will have contact information and a summary of the clinical trial posted 
on the Frohlich Lab Facebook and Twitter pages. Will we also be launching a Facebook or Instagram 
advertisement to identify potential patients. This advertisement will include a link to a brief screening 
survey on RedCap to help identify participants. We will also be using the UNC Mass email and department 
listserv to send out an email that has the link to the survey. All patient identifiers will be stored in REDCap 
 
 
 

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until recruitment is over. When recruitment is over, all patients who do not consent or are not eligible for 
participation in the study will have their responses permanently deleted in REDCap. 
We will send unencrypted emails to facilitate the initial contact to potential participants. Medical 
information is never requested per email. All medical information is recorded through HIPAA conform 
Zoom meetings and RedCap surveys.  
Our retention strategy includes a payment schedule of three times per subject. The subject will receive 
payment in form of a Visa Card at D1 ($75). This card will be charged again on D5 ($140), and the final 
follow-up session ($40). Thus, completion of the study will result in a financial compensation of $255. 
  
Research personnel will be easily available for the subjects to contact via email or phone. The inclusion 
criteria state that each subject must be able to understand all risks and benefits associated with this study. 
We will be asking each subject to answer questions about the consent form to determine that the study 
process and the duration of participation are completely understood by all subjects. We will aim to have 
a specific research team member assigned to complete all sessions with the same subject. However, we 
will not require the same researcher to be present during stimulation sessions 2 through 4.  The study 
team will work hard at forming rapport with the subject so they feel comfortable and willing to discuss 
what may be sensitive information. Retention will be quantified by the fraction of subjects coming to each 
scheduled session (the data from each session will be scored and documented the day of the session).     
 
As this study requires several days of participation in a row, we will allow subjects to miss one stimulation 
session, if it is made up at the end of the stimulation week. If two consecutive stimulation sessions are 
missed, the subject will be withdrawn from participation. This schedule would permit the subject to still 
receive the full intervention (5 days of stimulation) without being withdrawn from the study due to 
unforeseen circumstances (e.g., inclement weather, car trouble). In addition, the 2-week follow-up 
session will be scheduled on the first day of enrollment to allow for planning but within ± 3 days of the 
final day of stimulation to account for possible scheduling problems. 
 
6. STUDY INTERVENTION 
 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
 
We will be using the XCSITE100 stimulator designed by PulvinarNeuro in the Frohlich Lab for 
investigational purposes. The device is not implanted and has not been designed for or used to support 
or sustain human life. This device does not have a potential for serious risk to the health, safety, or welfare 
of the subject. There has never been an instance of serious side-effect reported due to the use 
of transcranial brain stimulation.  
 
Previous studies in the Frohlich lab that used the XCSITE 100 and comparable devices (i.e., the commercial, 
CE-certified Neuroconn Plus stimulator) have been conducted under an IDE and classified as “non-
significant risk” by the full UNC IRB. The Neuroconn Plus stimulator and the XCSITE100 stimulator are 
electrically equivalent and provide the same stimulation. While the Neuroconn device is commercially 

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available, it cannot be preprogrammed for 10Hz-tACS, and is not designed for tACS clinical trials. This 
makes the use of the Neuroconn device not appropriate for this study. 
 
The XSCITE100 stimulator may apply tACS for up to 40-minutes (2400 seconds) with appropriate current 
ramp-up at the beginning of stimulation and ramp-down at the end of stimulation. tACS may be applied 
for currents between 100 μA and 2 mA (peak-to-peak for tACS). For the purposes of this study, this 
device will be set to deliver either sham stimulation or 10Hz-tACS. The XCSITE100 device runs on a single 
9V rechargeable battery. The device itself is run through a device on a tablet. For more instructions, please 
see the submitted instruction manual. 
 
The stimulator has two main components: 
1. Android tablet with user interface application (i.e., App) 
2. Stimulator with: 
a. Microprocessor 
b. Function generator chip 
c. Voltage controlled current source 
d.  Safety circuitry 
Current Sensor Circuit. A 33.2 Ω sense resistor is placed in series with the stimulation electrodes on the 
high side. Since high-side current sensing is used, any short circuit of the electrode terminals to ground 
will be detected. The stimulation current flows through this resistor and creates a voltage. The voltage 
across this resistor is sensed and amplified by the AD628 difference amplifier. The gain of the difference 
amplifier is set to 9.9039. The current sensor voltage is then shifted before it is read by the microprocessor 
and the hardware current safety feature.  
 
Voltage Sensor Circuit. The differential voltage across the electrodes is measured so that the impedance 
can be calculated. The voltage is measured by buffering the positive electrode and negative electrode 
each with a unity gain op-amp circuit. The voltage output is then shifted before it is read by the 
microprocessor using the same level shifting circuit described in the current sensor section.  
 
The device is equipped with 4 different stages of safety precautions, all of which protect the participant 
from high currents. The stages are as follows: 
1. Automatic software current cutoff. The output of the current sensor described above is read by a 
microprocessor, which compares the reading to a value of +3 mA peak. If the current exceeds 
these limits, stimulation is turned off. The user is then given the option to investigate the issue, 
and cancel or resume the test. Since high-side current sensing is used (described above), any short 
circuit of the electrode terminals to ground will be detected.  
2. Automatic hardware current cutoff. The output of the current sensor is fed into a pair of 
comparators which detect if the current exceeds + 4.5 mA. If so, the fault is latched such that the 
relay in series with the electrodes is opened. Additionally, the microprocessor is notified of this 
instance through an interrupt. Upon this interrupt, the microprocessor immediately stops 
stimulation  
3. Permanent hardware current cutoff. A 5 mA fast-acting fuse is in series with the electrode 
connector. If the above two over-current detection methods fail, the fuse will blow, and the 
stimulator will no longer be electrically connected to the device.  
4. Power supply fuse. Finally, if for any other reason the entire device draws too much current, the 
main power supply fuse is blown. This fuse is sized with a cutoff of 200% of steady-state operating 
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6.1.2 DOSING AND ADMINISTRATION 
 
The research team will first measure each subject's head using the 10-20 system to determine the 
electrode locations. Subjects will then be fitted with the 3 electrodes for stimulation: two 5x5cm 
electrodes placed over F3 and F4, and one 5x7cm electrode placed over Cz. Electrodes will be carbon 
rubber, with Ten20 conductive paste applied. During stimulation, the subject will be in a relaxed and 
experimentally controlled state by watching a nature movie (such as Reefscapes). One session of 
stimulation will be performed per day, for 40-minutes. Before and after each stimulation session, an 
eyes open and eyes closed resting state EEG will be performed. 
 
The stimulation waveforms used in this study are sham/placebo stimulation and 10 Hz tACS at 1 mA 
zero-to-peak amplitude. Sham/placebo stimulation includes 20 seconds of ramp-in to 40 seconds of 10 
Hz tACS at 1 mA to 20 seconds of ramp-out, for a total of 80 seconds of stimulation. This is intended to 
mimic the skin sensations (e.g., itching, burning, tingling) that are experienced at the onset of 
stimulation, assisting with blinding the subject's assignment. 10 Hz tACS also has a ramp-in and ramp-out 
of 20 seconds, with 40-minutes of stimulation at an amplitude of 1 mA, for a total of 2440 seconds of 
stimulation. 
 
Stimulation devices will be pre-programmed, and codes will be randomized to one of the two 
experimental arms. Researchers will enter the subject-specific code into the tablet that controls the 
XCSITE100 device and will monitor subjects during the 40-minutes of the stimulation. Research 
personnel will be thoroughly trained and have trainings documented on the transcranial stimulation 
device and will be present during all stimulation sessions. To monitor side effects of stimulation 
a stimulation effects questionnaire will be administered after each stimulation session. 
 
6.3 MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 
Randomization 
Dr. Mengsen Zhang, a Frohlich Lab member, will randomize 20 6-digit codes, which will be used by the 
study coordinator and research assistants. These codes are directly linked to which treatment subjects 
receive (sham/placebo or 10 Hz tACS at 1 mA) and will be entered into the XCSITE tablet. The 
assignment of each subject cannot be determined by looking at the codes (e.g., codes are not 
sequential, code assignment is not based on “odd” or “even” numbers). Dr. Mengsen Zhang has no other 
responsibility in the study other than providing these randomized codes. If Dr. Mengsen Zhang leaves the 
Frohlich Lab, another equivalent researcher who does not work with human subjects will perform this 
task. 
 
Blinding 
The study is double-blind. This means that the subject and the researchers are unaware of each subject's 
assignment until the completion of all data collection. This is accomplished using the randomization codes 
described above. Furthermore, this study utilizes an active sham stimulation. This means that the 
sham/placebo condition includes some stimulation, mimicking the skin sensations associated with tACS. 
Blinding success is tested on D5 with two separate questionnaires for operator and participant. In our 

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previously concluded trial, subjects in the sham and 10 Hz tACS groups responded similarly to the blinding 
questionnaire, indicating that our active sham stimulation successfully blinded the subjects. 
 
6.4 STUDY INTERVENTION COMPLIANCE 
 
Full compliance with the intervention is defined as completing all 5 daily stimulation sessions for 40 
minutes each day. As the intervention is applied and monitored by research personnel, compliance can 
be directly observed. 
 
6.5 CONCOMITANT THERAPY 
  
Eligible subjects will be permitted to be receiving concomitant therapy, such as psychotherapy, 
antidepressants, or other medications. The only medications not permitted during this trial are 
anticonvulsants and recent benzodiazepines. The use of antidepressants or other therapies may affect 
our outcome, as subjects may experience relief of their depressive symptoms from these treatments. 
However, eligibility for this trial includes stable medication for the past 4 weeks. An adequate dose of 
antidepressants includes at least 4 weeks of use, so we anticipate that this requirement will reduce the 
potential bias for permitting the use of antidepressants and other medications. We also aim to ensure a 
stable dosing of antidepressant medication during the study. 
 
To ensure that concomitant therapies are logged appropriately, subjects will be requested to report any 
changes to the researchers. Furthermore, concomitant therapies (pharmacotherapy, psychotherapy) will 
be logged at Screening, D1, D5 and follow-up visit. Subjects will be requested to include the dosing for 
these therapies (i.e., how often per day, how much in each pill, how many pills) as well as when they were 
first prescribed the medication. 
 
6.5.1 RESCUE MEDICINE 
 
N/A 
  
7. STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
  
7.1 DISCONTINUATION OF STUDY INTERVENTION 
 
Discontinuation from the week of stimulation does not mean discontinuation from the study, and 
remaining study procedures should be completed as indicated by the study protocol. If a clinically 
significant finding is identified (including, but not limited to changes from baseline) after enrollment, the 
investigator or qualified designee will determine if any change in subject management is needed. 
 
The study intervention (i.e., the 5 consecutive days of stimulation) will be discontinued for the following 
reasons: 
• 
A subject develops significantly increased suicidal risk, as determined by an acute assessment by 
Dr. Schiller or Dr. Rubinow. 
• 
A subject has a YMRS score greater than or equal to 12. 

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• 
The subject misses a single day of stimulation and is unable to make it up at the end of the 
stimulation week. 
• 
The subject fails to complete the online suicidality questions within 72 hours of receiving the 
survey email and a personal phone contact does not explain the omission.   
• 
Any clinical adverse event (AE), laboratory abnormality, intercurrent illness or other medical 
condition, or situation occurs such that continued participation in the study would not be in the 
best interest of the subject. 
• 
The subject meets any exclusion criteria (either newly developed or not previously recognized). 
 
If the subject withdraws from the intervention, the subject will be contacted and invited for the 2-week 
follow-up assessment including all respective questionnaires. This assessment is not intended as a clinical 
follow-up with the participants but serves research purpose only.  
 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
 
Participants are free to withdraw from participation in the study at any time and without having to give a 
reason. But if the subject is willing to give a reason for discontinuation or withdrawal from the study it will 
be recorded with the subject files. Subjects who sign the informed consent form and are not randomized 
will be replaced. Subjects who sign the informed consent form, are randomized, and receive the full study 
intervention (5 consecutive days of 40-minutes of stimulation), and subsequently withdraw or are 
withdrawn or discontinue from the study will not be replaced. However, subjects who sign the informed 
consent form, and are randomized and receive only part of the study intervention, and subsequently 
withdraw or are withdrawn or discontinue from the study will be replaced.  
 
7.3 LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if he or she fails to return for the scheduled follow-up 
visit and is unable to be contacted by the study site staff. All efforts will be made to ensure subjects are 
not lost to follow-up, including developing rapport and ensuring enrolled subjects are reminded of their 
session dates. To ensure that subjects attend the follow-up session, research personnel will be flexible in 
timing, including offering sessions later in the day as well as some weekends. 
 
Every effort will be made to contact subjects who are lost to follow-up, including contacting via email 
and phone. However, if a subject is lost to follow-up, the missed sessions will be labeled as missing data 
and our pre-determined analysis plan takes into consideration missing data. 
 
 
8. STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 EFFICACY ASSESSMENTS  
Inclusion and exclusion criteria will be determined at the initial two session (pre-screening and 
screening), including concomitant therapies, medical history, and diagnoses, to ensure that subjects are 
diagnosed with MDD, with low suicide risk, and free of benzodiazepines and anticonvulsant medications. 
 

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8.1.1 PHYSIOLOGICAL CHANGES 
  
1. Resting state EEG (RSEEG) recordings will be completed several times during the study. On every 
day of the intervention week, subjects will complete an eyes open resting-state EEG (RSEEG) 
immediately before and after the 40-minutes of stimulation. This measure is used to determine 
the immediate after-effects of tACS on brain activity, specifically on alpha oscillation power, as 
well as how consecutive stimulation sessions may affect these after-effects. RSEEG will also be 
recorded at the follow-up visit to determine the lasting effects of stimulation. An eyes-closed 
RSEEG will also be collected on Day 1 and at the follow-up visit. 
 
8.1.2 CLINICAL EVALUATIONS 
  
1. The M.I.N.I is a diagnostic tool that will be used during the screening session to confirm diagnosis 
of unipolar, non-psychotic MDD. 
2. The Hamilton Depression Rating Scale 17-item (HDRS17) [12] will be administered during the initial 
session, day 1 of stimulation, day 5 of stimulation and during the 2-week follow-up visit. This scale 
is used to determine eligibility and to monitor the severity of the subject’s depressive symptoms, 
as well as determine suicide risk. 
3. The C-SSRS is a standardized method to access lifetime suicidality and current suicidal ideations 
and intends.  
4. The Maudsley Staging Method is a method of measuring treatment resistance in subjects with 
depression on a scale of 3 to 15 (mild = 3-6; moderate = 7-10; severe=11-15)[16]. 
5. The Young Mania Rating scale (YMRS) to detect symptoms of mania and hypomania during the 
study.  
 
8.1.3 SELF-REPORT ASSESSMENTS 
  
1. The Beck Depression Inventory (BDI) [17], [18]will be administered at day 1, day 5 of stimulation 
and at the follow-up visit. The BDI is a 21-item, self-report rating inventory that measures 
characteristic attitudes and symptoms of depression and will be used to monitor the severity of 
the patient’s depression throughout the study. 
2. The inventory of Depression and Anxiety Symptoms (IDAS) [19] will be completed day 1 of 
stimulation, day 5 of stimulation and during the 2-week follow-up visit. This inventory is used to 
monitor the perceived severity of the subject’s symptoms of depression. 
3. The Quality of Life Enjoyment and Satisfaction Questionnaire, short form (Q-LES-Q-SF)[20], will be 
administered at day 1 of stimulation, day 5 of stimulation, and the 2-week follow-up visit. This 
self-report questionnaire assesses quality of life. 
4. The Pittsburg Sleep Quality Index (PSQI) [21], [22] will be administered at day 1 of stimulation and 
the 2-week follow up visit. This self-report questionnaire assesses sleep quality over the course of 
the past month. It will be used to determine changes in sleep quality. 

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5. The WHO Disability Assessment Schedule 2.0 (WHODAS 2.0) [23], [24] will be administered at 
baseline to assess any comorbid disability that could potentially affect quality of life and/or 
response to stimulation.  
6. The Behavioral Inhibition and Behavioral Activation Self Report Scales (BAS/BIS) [25] will be 
completed during day 1 of stimulation, day 5 of stimulation and during the 2-week follow-up visit. 
These scales are used to monitor the perceived sensitivity to reward and punishment.  
7. The State-Trait Anxiety Inventory (STAI) [26] will be completed during day 1 of stimulation, day 5 
of stimulation and during the 2-week follow-up visit. These 40 items are used to monitor 
frequencies of feelings of anxiety.  
8. The Snaith-Hamilton Pleasure Scale (SHAPS) [27] will be completed at day 1 of stimulation, day 5 
of stimulation, and the 2-week follow-up visit. This scale is used to assess anhedonia. 
9. The Beliefs about treatment questionnaires comprises four questions regarding participants 
expectations.  
10. The Rumination scale assess recurring thought process on a 1-4 Likert Scale.  
11. Blinding questionnaires for participant and operator are applied at D5. 
12. Adverse Event questionnaires are administered after every stimulation session and ask about 
somatic symptoms/experiences during the tACS application.  
 
 
8.2 SAFETY AND OTHER ASSESSMENTS 
 
 
8.2.1 SUICIDAL IDEATIONS AND SUICIDAL BEHAVIOR 
 
Only subjects with a HDRS-17 suicidality item score of <3 and low suicide risk based on the C-SSRS are 
enrolled in this study. Furthermore, suicidal thoughts and behaviors will be assessed using a brief daily 
suicide survey (IRB# 14-0600) consisting of three questions. Participants will be asked if they had any 
thoughts of hurting themselves in the last 24 hours (suicidal ideation, SI) and whether they have hurt 
themselves in the past 24 hours (suicidal behavior, SB). They will also be asked to rate the severity of their 
depression on a scale of 1 to 10. Participants receive a link to answer the daily suicide survey on RedCap. 
To maximize participant’s safety the daily suicide survey is send during the follow-up period as well. 
Responses will be monitored by study personnel daily and the last survey will be administered at the day 
of follow-up visit.  In addition, the C-SSRS [13] will be administered by trained research personnel at 
screening, D1, D5 and the follow-up session to thoroughly assess suicide risk. 
 
If a participant report experiencing either SI or SB on either tool, research personnel will collect more 
information from the participant to deliver to either Dr. Schiller or Dr. Rubinow. Clinical personnel will 
decide if an acute assessment is required. Acute assessment may include facilitating contact of the subject 
with their psychiatrist or primary care physician to establish a plan for safety, continued care, and follow-
up. If the participant does not have an established provider, Dr. Schiller or Dr. Rubinow will assist in 
establishing a care plan. If at any point during the assessment, the participant is deemed an imminent risk 
of harm to self or others, study personnel will enlist the aid of campus security to ensure that the 
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Rubinow will decide if participation should be stopped after the acute assessment. If the participant is 
hospitalized, their participation will end. 
 
8.2.2 DEVELOPMENT OF MANIA 
 
The Young Mania Rating Scale (YMRS) [28] was designed to assess the severity of manic symptoms at 
enrolment, with the ability to track changes over the duration of participation. When undergoing 
treatment for depression, a possible side effect (as inferred from clinical trials with antidepressant 
medications) is to alter levels of serotonin, potentially associated with mania [29], [30]. Although we do 
not expect such an event to occur since we are not using a medication that targets serotonin levels, we 
will be conducting this assessment at Day 1, Day 5 and during the 2-week follow-up as a precautionary 
measure. If a participant develops any sign of mania (YMRS > 12), Dr. Schiller will conduct an acute 
assessment to determine if they are experiencing mania. If this occurs during the week of stimulation, 
participation in the study will be stopped and their primary mental health care provider or family physician 
will be contacted. In the case that they do not see anyone for their depression, Dr. Schiller will assist in 
creating a medical care plan 
 
8.2.3 ADVERSE EVENT ASSESMENT 
 
A structured interview probing for the experience of adverse events will be completed after each day of 
stimulation and at the follow up visit. Dr. Schiller or Dr. Rubinow will use the completed interview to assess 
the severity of the adverse event and its relationship to the study intervention as well determine if 
participation should be terminated.  
 
8.2.4 STIMULATION RELATED SIDE EFFECT ASSESSMENT 
 
A stimulation questionnaire will be administered at the end of each stimulation session. This tool will be 
used as a safety measure and to collect data on the participant’s experience. A similar questionnaire was 
used in a previous study (IRB# 13-2995) to determine ability to successfully blind the participants using 
sham transcranial current stimulation.  
   
8.3 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
Adverse event means any untoward medical occurrence associated with the use of an intervention in 
humans, whether considered intervention-related (21 CFR 312.32 (a)).  
 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of the 
investigator, it results in any of the following outcomes: death, a life-threatening adverse event, inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity or 
substantial disruption of the ability to conduct normal life functions, or a congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require hospitalization may 

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be considered serious when, based upon appropriate medical judgment, they may jeopardize the subject 
and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 
Examples of such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 SEVERITY OF EVENT 
 All adverse events (AEs) will be assessed by the principal investigator and/or co-investigator(s) using the 
following guidelines:  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
 
All adverse events (AEs) must have their relationship to study intervention assessed by the principal 
investigator and co-investigator(s) who examines and evaluates the participant based on temporal 
relationship and their clinical judgment. The degree of certainty about causality will be graded using the 
categories below. In a clinical trial, the study product must always be suspect.  
 
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to study intervention administration and cannot be 
explained by concurrent disease or other drugs or chemicals. The response to withdrawal of the 
study intervention (dechallenge) should be clinically plausible. The event must be 
pharmacologically or phenomenologically definitive, with use of a satisfactory rechallenge 
procedure if necessary. 
• 
Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within a 
reasonable time after administration of the study intervention, is unlikely to be attributed to 
concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this definition. 
• 
Potentially Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, other 
factors may have contributed to the event (e.g., the participant’s clinical condition, other 
concomitant events). Although an AE may rate only as “possibly related” soon after discovery, it 
can be flagged as requiring more information and later be upgraded to “probably related” or 
“definitely related”, as appropriate. 

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• 
Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study intervention administration makes a causal relationship 
improbable (e.g., the event did not occur within a reasonable time after administration of the 
study intervention) and in which other drugs or chemicals or underlying disease provides plausible 
explanations (e.g., the participant’s clinical condition, other concomitant treatments). 
• 
Not Related – The AE is completely independent of study intervention administration, and/or 
evidence exists that the event is definitely related to another etiology. There must be an 
alternative, definitive etiology documented by the clinician. 
 
 
8.3.3.3 EXPECTEDNESS  
 
The principal investigator (Dr. Rubinow), with input from the co-investigator (Dr. Schiller) when necessary, 
will determine whether an adverse event (AE) is expected or unexpected in this population. The co-
investigator (Dr. Flavio Frohlich) is an expert in non-invasive brain stimulation and will provide his expert 
opinion regarding this as well. An AE will be considered unexpected if the nature, severity, or frequency 
of the event is not consistent with the risk information previously described for the study intervention. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention of 
study personnel during study visits, or the study subject may report AE or SAEs outside of a scheduled 
study visit. All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured 
on the appropriate case report form (CRF). Information to be collected includes event description, time 
of onset, clinician’s assessment of severity, relationship to study product (assessed only by those with the 
training and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs 
occurring while on study must be documented appropriately regardless of relationship. All AEs will be 
followed to adequate resolution. Any medical condition that is present at the time that the subject is 
screened will be considered as baseline and not reported as an AE. However, if the study subject’s 
condition deteriorates at any time during the study, it will be recorded as an AE.   Changes in the severity 
of an AE will be documented to allow an assessment of the duration of the event at each level of severity 
to be performed. AEs characterized as intermittent require documentation of onset and duration of each 
episode.   
 
Research personnel will record all reportable events with start dates occurring any time after informed 
consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation. At each study visit, research personnel will inquire about the occurrence of AE/SAEs since 
the last visit. Events will be followed for outcome information until resolution or stabilization. All AEs 
including local and systemic reactions not meeting the criteria for SAEs will be captured on the appropriate 
case report form (CRF). Information to be collected includes event description, time of onset, clinician’s 
assessment of severity, relationship to study product (assessed only by those with the training and 
authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring while 
on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
 
 

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8.3.5 ADVERSE EVENT REPORTING 
 
We will be adopting the following reporting procedures:  
 
What event is reported When 
is 
event 
reported 
By whom is event 
reported 
To whom is event 
reported 
Fatal or life-
threatening 
unexpected, suspected 
serious adverse 
reactions 
Within 24 hours of 
initial receipt of 
information 
Investigator 
• 
Local/internal 
IRB 
Non-fatal, non-life-
threatening 
unexpected, suspected 
serious adverse 
reactions 
Within 48 hours of 
initial receipt of 
information 
Research Personnel 
• 
Local/internal 
IRB 
 
Unanticipated adverse 
device effects 
Within 10 working days 
of investigator first 
learning of effect  
Investigator 
• 
Local/internal 
IRB 
 
Unanticipated Problem 
that is not an SAE 
Within 7 days of the 
investigator becoming 
aware of the problem 
Investigator 
• 
Local/internal 
IRB 
 
  
8.3.6 REPORTING OF PREGNANCY  
 
Pregnancy tests to all women of child-bearing potential at the first laboratory visit, before the stimulation 
session. There is no evidence that suggests tACS would interfere with pregnancy [31]. However, should a 
subject become pregnant during the study their participation will be immediately terminated and they 
will be asked to consult with the Principal Investigator.    
 
8.4 UNANTICIPATED PROBLEMS 
 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 

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• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
8.4.2 UNANTICIPATED PROBLEM REPORTING  
 
If a UE occurs, the IRB will be notified, and the study will be adjusted as needed to protect the health and 
safety of the subjects. Depending on the nature of the UE, the research protocol, inclusion/exclusion 
criteria, and informed consent will be changed to reflect the possibility of this event reoccurring. During 
this time, no new subjects will be recruited and the research procedures for currently enrolled subjects 
will be stopped. Each UE will be recorded and reported throughout the study.   
 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
 
Any new information gained during the study that may affect a participant’s willingness to continue in the 
study will be reported to all currently enrolled subjects. 
 
9. STATISTICAL CONSIDERATIONS  
 
9.1 STATISTICAL HYPOTHESES 
 
• 
Primary Efficacy hypothesis: 
o Null: There is no difference in changes of alpha frequency power between baseline RSEEG 
and RSEEG at completion of stimulation between treatment regimens. 
o Alternate: There is a difference in changes of alpha frequency power between baseline 
RSEEG and RSEEG at completion of stimulation between treatment regimens. 
 
• 
Secondary Efficacy hypothesis: 
o Null: There is no relationship between the changes in alpha frequency power and changes 
in Hamilton Depression Rating Scale, 17 item (HDRS17) scores. 
o Alternate: There is a relationship between the changes in alpha frequency power and 
changes in Hamilton Depression Rating Scale, 17 item (HDRS17) scores. 
 
9.2 SAMPLE SIZE DETERMINATION 
 
In our recently concluded pilot clinical trial we compared the effects of 10 Hz tACS (therapeutic frequency) 
against 40 Hz tACS (control frequency) and sham/placebo stimulation. A total of 32 subjects enrolled, 30 
completed the 5-day intervention, and 26 completed both a 2 and a 4-week follow-up session. With 
approximately 9 subjects in each group, a significant reduction was found in alpha oscillation power on 
the fifth day of stimulation (p < 0.05) [6]. Therefore, with a total of 20 subjects (10 in each group), we 
anticipate we will find a significant difference. Of note, our funding mechanism covers the cost of enrolling 
20 participants.  
 
9.3 POPULATIONS FOR ANALYSES 
 

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Every effort will be made to ensure all enrolled and randomized subjects complete all study sessions as 
described in this protocol. However, a priori, we determined that our population for analysis will be a 
modified intention-to-treat (ITT) analysis dataset. For this study, enrolled eligible subjects will be 
randomized to receive 5 consecutive days of stimulation. If a subject completes the intervention (i.e., 
receives all 5 consecutive days of stimulation), they will be included in all analyses moving forward. 
 
As 
previously 
stated 
in 
Section 
7 
Study 
Intervention 
Discontinuation 
and 
Subject 
Discontinuation/Withdrawal, enrolled subjects who do not complete the full intervention will be 
replaced. Therefore, with this population for analysis plan, we anticipate having data from 20 subjects 
that are eligible for analysis. 
 
9.4 STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
 
All testing described below assumes a significance threshold of p = 0.05. Continuous data will be described 
using means and standard deviations, while categorical data will be described using counts/percentages.  
 
There will be no pre-specified covariates described in this protocol. Data will be assessed for normality 
and, if deemed necessary, corrective procedures will be applied (e.g., log normalization). 
 
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) (EEG) 
 
We will perform spectral analysis of resting state EEG from day 1 of stimulation (baseline), day 5 of 
stimulation, and the 2-week follow-up visit. We will use a repeated measure ANOVA and/or a general 
linear mixed-effects model (GLMM) to assess if there is statistically significant interaction between 
treatment (sham/placebo and 10 Hz tACS) and session (baseline, day 5 of stimulation, 2-week follow-up). 
In the case of missing data (e.g., missing follow-up visits), analysis using a GLMM will be prioritized, as 
GLMM takes into account missing data. Spectral analysis will be performed with multi-tapered estimation 
of the frequency spectrum followed by integration over the classical alpha EEG band (8-12 Hz). 
 
In a separate analysis, we will perform spectral analysis of resting state EEG before and after each separate 
stimulation session. These analyses will be completed on the modified intention-to-treat dataset. Results 
from this section will be described in mean decibel (dB) change and presented using spectral images for 
visualization. 
 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S)(CORRELATION BETWEEN EEG CHANGES 
AND SYMPTOMS OF DEPRESSION) 
 
Using the EEG results described in 9.4.2, mean dB change from baseline will be correlated with normalized 
change in HDRS17 scores.* Correlations will be assessed using Pearson's r and data will be plotted against 
the best fit line to visualize the results. 
 
   *HDRS17 normalization:  
       [[HDRS17 score at Visit #] - [HDRS17 score at baseline]]/[HDRS17 score at baseline] 
 

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9.4.4 SAFETY ANALYSES 
 
As discussed in Section 8.2, Safety and Other Assessments safety will be assessed with the three questions 
from the daily suicide questionnaire, the C-SSRS, an adverse effects questionnaire, and the Young Mania 
Rating Scale (YMRS). 
 
The suicide questionnaire is used as a screening to assess increase in suicidal ideation (SI) or suicidal 
behavior (SB) daily. The C-SSRS will be used to assess suicide risk more thoroughly. If a participant's 
responses on either tool indicate SI or SB, the subject will be contacted, and an acute assessment will be 
completed if deemed necessary by Dr. Schiller or Dr. Rubinow (as described above). Any verified increases 
in suicidal ideation will be described in counts/percentages and compared between groups using chi-
square tests. 
 
The stimulation questionnaire will be administered following every stimulation session, for a total of 5 
administrations per participant. This questionnaire solicits ratings of 14 possible adverse effects 
associated with electrical stimulation, on a scale of 1 (absent) to 4 (severe). Paired t-tests with random 
effect "subject" will be calculated per adverse effect to determine if there are any differences in adverse 
effect severity between groups (sham/placebo, 10 Hz tACS). Severity per adverse effect will be described 
with mean and standard deviation. 
 
The Young Mania Rating Scale (YMRS) will be used to assess any development of mania over the course 
of treatment. A YMRS score of greater than 12 indicates the possible development of a manic episode. 
Any subject who scores greater than 12 on the YMRS will be assessed by Dr. Schiller or Dr. Rubinow. Any 
verified manic episode will be described in counts/percentages and compared between groups using a 
chi-square test. 
 
The Adverse Events Structured Interview will be administered by research personnel following every 
stimulation session and at the follow up visit, for a total of 6 time points per participant. This interview 
solicits open-ended descriptions of 11 possible adverse effects a participant may experience in any clinical 
trial as well as one question of ‘any other experiences’. Responses will be reviewed by the clinician and 
rated as described in Section 8.3. Paired t-tests will be calculated per adverse effect to determine if there 
are any differences in adverse effect severity between groups (sham/placebo, 10 Hz tACS). Severity per 
adverse effect will be described with mean and standard deviation.3.   
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
All baseline descriptive statistics will be analyzed either using paired t-tests or chi-square tests when 
deemed appropriate. Descriptive statistics will be described based on the General Approach described in 
9.4.1. 
 
9.4.6 PLANNED INTERIM ANALYSES  
 
There are no other planned interim analyses. 
 
9.4.7 SUB-GROUP ANALYSES 
 

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Sub-group analyses will not be used in this study, as the sample size is too small to conduct analyses based 
on age, sex, race/ethnicity, or other demographic characteristic(s). 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study 
and continues throughout the individual’s study participation. Extensive discussion of risks and possible 
benefits of tACS will be provided to the participants and their families. Electronic consent and HIPAA 
consent will be obtained in the pre-screening remote session. The informed electronic consent will be 
documented via REDCap. A copy of the electronic consent and the HIPAA consent is handed out to the 
participant at the begin of D1. All consent forms will be IRB-approved and updated with any new 
information as modifications are made throughout the study. 
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
 
At the begin of the remote pre-screening session, the researcher and potential participants will review 
the clinical trial in its entirety. At several intervals during the consent review, the researcher will ask 
questions that will assess the comprehension of the information in the consent. If the participant is unsure 
or does not know, the researcher will return to that section and more carefully explain the information. 
We will especially ensure that stimulation related side effects, the placebo arm of the study and the 
possibility to withdraw from the study at any given moment without prejudice are well understood. If 
needed, the participants will have the opportunity to discuss the study with their surrogates or think about 
it prior to agreeing to participate. 
 
At the beginning of the first visit to the lab (D1), participants will receive a paper copy of the electronic 
consent. The rights and welfare of the participant will be protected by emphasizing to them that the 
quality of their medical care will not be adversely affected if they decline to participate in this study 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly 
inform research staff, study subjects, and the IRB and will provide the reason(s) for the termination or 
suspension.  Study subjects will be contacted, as applicable, and be informed of changes to study visit 
schedule.   
 
Circumstances that may warrant termination or suspension include, but are not limited to: 

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• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Increased suicide risk due to intervention (3 out of 8 participants or, if more than 8 enrolled, > 
25% of participants require additional clinical care for their symptoms of depression)  
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and 
satisfy the IRB. 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, 
and the research team. This confidentiality is extended to cover the clinical information relating to 
participants. Therefore, the study protocol, documentation, data, and all other information generated will 
be held in strict confidence.  In addition, all research activities will be conducted in an as private as possible 
setting. 
 
All data will only be referenced by dummy identifier code. Data will be stored on a password protected 
computer. A key connecting names and identifier code numbers will be kept in a locked cabinet, accessible 
only by research personnel. All data will be stored and analyzed on password protected computers, also 
only accessible by research personnel. Participants will not be identified in any report or publication about 
this study and there is no risk of deductive disclosure. At the end of the study, all records will continue to 
be kept in a secure location for as long a period as dictated by the IRB. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be entered into TraCS Clinical Research Data Management Service (REDCap). The database system 
provides secure web-based data entry with the data stored on servers that are maintained by TrACS. The 
data is encrypted during transmission. The servers are located in a secure campus area with all appropriate 
physical security measures in place. The web and database servers are monitored by the TraCS IT staff, 
patched frequently, and scanned by a third-party vendor to ensure that they are protected against known 
vulnerabilities. The scanning application is the standard service for the entire campus. Access is by 
individual user id and is restricted to the forms and/or functions that the user needs to have.   
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
 
Data collected for this study will be analyzed and stored within the Carolina Center for 
Neurostimulation/Frohlich Lab. After the study is completed, the data will be fully de-identified and 
archived within a locked file cabinet within the Carolina Center for Neurostimulation. 
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
Provide the name and contact information of the Principal Investigator and the Medical Monitor. 
Principal Investigator 
Co-Investigator 
Co-Investigator 
David Rubinow, MD 
 
Flavio Frohlich, PhD 
Crystal Schiller, PhD 

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The University of North Carolina 
at Chapel Hill - Department of 
Psychiatry 
 
919-445-0212 
The University of North Carolina 
at Chapel Hill - Department of 
Psychiatry 
 
919-966-4584 
The University of North Carolina 
at Chapel Hill - Department of 
Psychiatry 
 
919-966-4810  
david_rubinow@med.unc.edu 
Flavio_frohlich@med.unc.edu 
crystal_schiller@med.unc.edu 
  
10.1.6 SAFETY OVERSIGHT 
  
Safety oversight will be under the direction of the Principal Investigator Dr. Rubinow. He will review all 
adverse events timely and serious adverse events and changes in the suicidality and mania ratings 
immediately. Based on his review, continuation of participant’s participation is decided. All SAE or 
unanticipated AE will be reported to the local IRB.  
 
 
10.1.7 CLINICAL MONITORING 
 
The purpose of the monitoring plan is to present the approach of the Carolina Center for Neurostimulation 
to monitoring clinical trials.  The plan facilitates compliance with good clinical practice. 
 
(a) The rights and well-being of human subjects are protected. 
(b) The reported trial data are accurate, complete, and verifiable from source documents. 
(c) The conduct of the trial is in compliance with the currently approved protocol/amendment(s), with 
GCP, and with applicable regulatory requirement(s). 
 
This section identifies key monitoring activities and specifies the data to be reviewed over the course of a 
clinical trial.  This is a single site, investigator initiated, clinical trial so there will be no site monitoring plan 
in place. 
 
 
10.1.7.1 THE CAROLINA CENTER FOR NEUROSTIMULATION MONITORING PLAN 
 
The latest version of the approved IRB application for this clinical trial will be followed at all times. This 
responsibility falls in the hands of the trained research personnel.  If at any time there is a deviation from 
protocol, the deviation form protocol log will be filled out. All team members will be trained on how and 
when to use this log.  
 
Data will be verified for completeness following every study session and all data will be entered into 
REDCap, a secure online database. After a subject has completed their participation (full completion 
through the 2-week follow-up visit or because they withdrew prior to completion), data will be rereviewed 
for completeness and accuracy. After all data has been collected, data will be re-reviewed by another lab 
member who was not involved with the data collection process.  
 
AE and SAE are clearly defined in the Master Protocol. Documents of AE and SAE can be found in the study 
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trained research personnel to report all events to the PI. Reporting of AEs and SAEs is described within 
Section 8.3. 
 
The PI and Co-I will have read-only access to the REDCap database.  This allows the PI and Co-I to view 
reports that provide information on any missing data on an individual subject basis, but does not allow 
them to add, change or input any data. 
  
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
 
The Carolina Center for Neurostimulation will conduct internal quality management of study conduct, 
data collection, documentation, and completion. Following written Standard Operating Procedures 
(SOPs), research personnel will verify that the clinical trial is conducted and data are generated and 
biological specimens are collected, documented (recorded), and reported in compliance with the 
protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable 
regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
 
10.1.9 DATA HANDLING AND RECORD KEEPING  
 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
 
Trained research personnel will be responsible for the informed consent process, review for eligibility, 
questionnaire administration, data entry, device administration, EEG administration, and CRF entries. 
Research personnel will be responsible for AE/SAE documentation and reporting, while the PI will be 
responsible for the AE assessment, review of the AE documentation forms and overview of the research 
staff. 
 
Clinical data (including AEs, concomitant medications, and expected adverse reactions data) will be 
entered into a data capture system provided by TraCS Clinical Research Data Management Service 
(REDCap). The data system includes password protection and internal quality checks, such as automatic 
range checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be 
entered directly from the source documents. Trained research personnel will have complete access to the 
REDCap system, while the PI and Co-I will have read-only ability. This will enable the researchers to enter 
the data and the PI and Co-I to review. 
 
As discussed in Section 10.1.3 , data entered into REDCap is stored on servers that are maintained by 
TrACS. The data is encrypted during transmission. The servers are located in a secure campus area with 
all appropriate physical security measures in place. The web and database servers are monitored by the 
TraCS IT staff, patched frequently, and scanned by a third party vendor to ensure that they are protected 
against known vulnerabilities. The scanning application is the standard service for the entire campus.  
 
10.1.9.2 STUDY RECORDS RETENTION  
  
According to the University of North Carolina at Chapel Hill’s Archives and Record Management Services 
schedule for General Records Retention and Disposition Schedule 6.10, records will be kept for 5 years 
after the completion of the study or grant end date, whichever is later. 
 

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10.1.10 PROTOCOL DEVIATIONS  
  
All deviations from the protocol will be addressed in study participant source documents. The researcher 
will complete a Protocol Deviation Log using the participant code as the identifier. This form will collect 
information such as the date the deviation occurred, details of what the deviation consisted of, any 
corrective and preventative actions that were taken as a result of the deviation, and the date that the PI 
and IRB were notified. The PI will review the information and initial once approved. A completed copy of 
the Protocol Deviation Form will be maintained in the regulatory file, as well as in the participant’s source 
document. The site PI/study staff will be responsible for knowing and adhering to their IRB requirements. 
 
10.1.11 PUBLICATION AND DATA SHARING POLICY 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-reviewed 
journals.   
 
 
10.1.12 CONFLICT OF INTEREST POLICY 
 
The independence of this study from any actual or perceived influence is critical. Any conflict of interest 
for any persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will 
be disclosed and managed by the UNC Conflict of Interest Office. If necessary, for persons who have a 
perceived conflict of interest, management will be provided again by the UNC Conflict of Interest office. 
 
10.2 
ADDITIONAL CONSIDERATIONS 
 N/A 
 
 
 

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10.3 
ABBREVIATIONS 
The list below includes abbreviations utilized in this template.  However, this list should be customized for 
each protocol (i.e., abbreviations not used should be removed and new abbreviations used should be 
added to this list). 
 
AE 
Adverse Event 
BDI 
Beck Depression Inventory 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSM-5 
Diagnostic and Statistical Manual, 5th Edition 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
EEG 
Electroencephalogram 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HDRS 
Hamilton Depression Rating Scale 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS 
Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
PSQI 
Pittsburg Sleep Quality Index 

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Q-LES-Q-SF 
Quality of Life Enjoyment & Satisfaction Questionnaire, Short Form 
QA 
Quality Assurance 
QC 
Quality Control 
RSEEG 
Resting state electroencephalogram 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SCID 
Structured Clinical Interview for the DSM-5 
SHAPS 
Snaith-Hamilton Pleasure Scale 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
YMRS 
Young Mania Rating Scale 
WHODAS 2.0 
World Health Organization Disability Assessment Schedule 2.0 
 
 
 

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10.4 
PROTOCOL AMENDMENT HISTORY 
  
 
Version 
Date 
Description of Change  
Brief Rationale 
2 
05/02/22 
Update of several aspects  
Increase Feasibility 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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11 
REFERENCES   
 
[1] 
I. H. Stanley, T. E. Joiner, and C. J. Bryan, “Mild traumatic brain injury and suicide risk among a 
clinical sample of deployed military personnel: Evidence for a serial mediation model of anger and 
depression,” J. Psychiatr. Res., vol. 84, pp. 161–168, 2017, doi: 10.1016/j.jpsychires.2016.10.004. 
[2] 
A. J. Rush et al., “Acute and longer-term outcomes in depressed outpatients requiring one or 
several treatment steps: a STAR*D report,” Am. J. Psychiatry, vol. 163, no. 11, pp. 1905–1917, Nov. 
2006, doi: 10.1176/ajp.2006.163.11.1905. 
[3] 
A. R. Brunoni et al., “Neuromodulation approaches for the treatment of major depression: 
challenges and recommendations from a working group meeting,” Arq. Neuropsiquiatr., vol. 68, 
no. 3, pp. 433–451, Jun. 2010, doi: 10.1590/s0004-282x2010000300021. 
[4] 
J. B. Henriques and R. J. Davidson, “Regional brain electrical asymmetries discriminate between 
previously depressed and healthy control subjects,” J. Abnorm. Psychol., vol. 99, no. 1, pp. 22–31, 
Feb. 1990, doi: 10.1037//0021-843x.99.1.22. 
[5] 
M. M. Ali, K. K. Sellers, and F. Fröhlich, “Transcranial alternating current stimulation modulates 
large-scale cortical network activity by network resonance,” J. Neurosci. Off. J. Soc. Neurosci., vol. 
33, no. 27, pp. 11262–11275, Jul. 2013, doi: 10.1523/JNEUROSCI.5867-12.2013. 
[6] 
M. L. Alexander et al., “Double-blind, randomized pilot clinical trial targeting alpha oscillations with 
transcranial alternating current stimulation (tACS) for the treatment of major depressive disorder 
(MDD),” Transl. Psychiatry, vol. 9, no. 1, p. 106, 05 2019, doi: 10.1038/s41398-019-0439-0. 
[7] 
T. Neuling, S. Rach, and C. S. Herrmann, “Orchestrating neuronal networks: sustained after-effects 
of transcranial alternating current stimulation depend upon brain states,” Front. Hum. Neurosci., 
vol. 7, p. 161, 2013, doi: 10.3389/fnhum.2013.00161. 
[8] 
A. Vossen, J. Gross, and G. Thut, “Alpha Power Increase After Transcranial Alternating Current 
Stimulation at Alpha Frequency (α-tACS) Reflects Plastic Changes Rather Than Entrainment,” Brain 
Stimulat., vol. 8, no. 3, pp. 499–508, Jun. 2015, doi: 10.1016/j.brs.2014.12.004. 
[9] 
F. H. Kasten, J. Dowsett, and C. S. Herrmann, “Sustained Aftereffect of α-tACS Lasts Up to 70 min 
after Stimulation,” Front. Hum. Neurosci., vol. 10, p. 245, 2016, doi: 10.3389/fnhum.2016.00245. 
[10] A. Antal et al., “Low intensity transcranial electric stimulation: Safety, ethical, legal regulatory and 
application guidelines,” Clin. Neurophysiol. Off. J. Int. Fed. Clin. Neurophysiol., vol. 128, no. 9, pp. 
1774–1809, 2017, doi: 10.1016/j.clinph.2017.06.001. 
[11] F. Fröhlich and D. A. McCormick, “Endogenous electric fields may guide neocortical network 
activity,” Neuron, vol. 67, no. 1, pp. 129–143, Jul. 2010, doi: 10.1016/j.neuron.2010.06.005. 
[12] J. B. Williams, “Standardizing the Hamilton Depression Rating Scale: past, present, and future,” Eur. 
Arch. Psychiatry Clin. Neurosci., vol. 251 Suppl 2, pp. II6-12, 2001, doi: 10.1007/BF03035120. 
[13] K. Posner et al., “The Columbia–Suicide Severity Rating Scale: Initial Validity and Internal 
Consistency Findings From Three Multisite Studies With Adolescents and Adults,” Am. J. 
Psychiatry, vol. 168, no. 12, pp. 1266–1277, Dec. 2011, doi: 10.1176/appi.ajp.2011.10111704. 
[14] A. R. Brunoni et al., “The sertraline vs. electrical current therapy for treating depression clinical 
study: results from a factorial, randomized, controlled trial,” JAMA Psychiatry, vol. 70, no. 4, pp. 
383–391, Apr. 2013, doi: 10.1001/2013.jamapsychiatry.32. 
[15] A. L. Stuart, J. A. Pasco, F. N. Jacka, S. L. Brennan, M. Berk, and L. J. Williams, “Comparison of self-
report and structured clinical interview in the identification of depression,” Compr. Psychiatry, vol. 
55, no. 4, pp. 866–869, May 2014, doi: 10.1016/j.comppsych.2013.12.019. 
[16] A. Fekadu, S. C. Wooderson, K. Markopoulou, and A. J. Cleare, “The Maudsley Staging Method for 
treatment-resistant depression: prediction of longer-term outcome and persistence of symptoms,” 
J. Clin. Psychiatry, vol. 70, no. 7, pp. 952–957, Jul. 2009, doi: 10.4088/JCP.08m04728. 

Mechanism of Action of tACS for the Treatment of MDD  
V.2 
Protocol: 20-1822 
 
05 April 2022 
 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
35 
[17] A. T. Beck, R. A. Steer, and M. G. Carbin, “Psychometric properties of the Beck Depression 
Inventory: Twenty-five years of evaluation,” Clin. Psychol. Rev., vol. 8, no. 1, pp. 77–100, Jan. 1988, 
doi: 10.1016/0272-7358(88)90050-5. 
[18] D. J. A. Dozois, K. S. Dobson, and J. L. Ahnberg, “A psychometric evaluation of the Beck Depression 
Inventory–II,” Psychol. Assess., vol. 10, no. 2, pp. 83–89, 1998, doi: 10.1037/1040-3590.10.2.83. 
[19] D. Watson et al., “Development and validation of the Inventory of Depression and Anxiety 
Symptoms (IDAS),” Psychol. Assess., vol. 19, no. 3, pp. 253–268, Sep. 2007, doi: 10.1037/1040-
3590.19.3.253. 
[20] J. Endicott, J. Nee, W. Harrison, and R. Blumenthal, “Quality of Life Enjoyment and Satisfaction 
Questionnaire: a new measure,” Psychopharmacol. Bull., vol. 29, no. 2, pp. 321–326, 1993. 
[21] D. J. Buysse, C. F. Reynolds, T. H. Monk, S. R. Berman, and D. J. Kupfer, “The Pittsburgh Sleep 
Quality Index: a new instrument for psychiatric practice and research,” Psychiatry Res., vol. 28, no. 
2, pp. 193–213, May 1989, doi: 10.1016/0165-1781(89)90047-4. 
[22] J. S. Carpenter and M. A. Andrykowski, “Psychometric evaluation of the Pittsburgh Sleep Quality 
Index,” J. Psychosom. Res., vol. 45, no. 1, pp. 5–13, Jul. 1998, doi: 10.1016/s0022-3999(97)00298-5. 
[23] G. Andrews, A. Kemp, M. Sunderland, M. Von Korff, and T. B. Ustun, “Normative Data for the 12 
Item WHO Disability Assessment Schedule 2.0,” PLoS ONE, vol. 4, no. 12, Dec. 2009, doi: 
10.1371/journal.pone.0008343. 
[24] J. Rehm et al., “On the development and psychometric testing of the WHO screening instrument to 
assess disablement in the general population,” Int. J. Methods Psychiatr. Res., vol. 8, no. 2, pp. 
110–122, 1999, doi: 10.1002/mpr.61. 
[25] C. S. Carver and T. L. White, “Behavioral inhibition, behavioral activation, and affective responses 
to impending reward and punishment: The BIS/BAS Scales,” J. Pers. Soc. Psychol., vol. 67, no. 2, pp. 
319–333, 1994, doi: 10.1037/0022-3514.67.2.319. 
[26] L. J. JULIAN, “Measures of Anxiety,” Arthritis Care Res., vol. 63, no. 0 11, Nov. 2011, doi: 
10.1002/acr.20561. 
[27] R. P. Snaith, M. Hamilton, S. Morley, A. Humayan, D. Hargreaves, and P. Trigwell, “A scale for the 
assessment of hedonic tone the Snaith-Hamilton Pleasure Scale,” Br. J. Psychiatry J. Ment. Sci., vol. 
167, no. 1, pp. 99–103, Jul. 1995, doi: 10.1192/bjp.167.1.99. 
[28] R. C. Young, J. T. Biggs, V. E. Ziegler, and D. A. Meyer, “A rating scale for mania: reliability, validity 
and sensitivity,” Br. J. Psychiatry J. Ment. Sci., vol. 133, pp. 429–435, Nov. 1978, doi: 
10.1192/bjp.133.5.429. 
[29] E. Y. Kim et al., “Rate of diagnostic conversion to bipolar disorder in adults with unipolar 
depression and psychopharmacological treatment in the republic of Korea: A nationwide register-
based study,” J. Affect. Disord., vol. 273, pp. 240–246, Aug. 2020, doi: 10.1016/j.jad.2020.03.159. 
[30] L. M. Melhuish Beaupre et al., “Antidepressant-Associated Mania in Bipolar Disorder: A Review 
and Meta-analysis of Potential Clinical and Genetic Risk Factors,” J. Clin. Psychopharmacol., vol. 40, 
no. 2, pp. 180–185, Apr. 2020, doi: 10.1097/JCP.0000000000001186. 
[31] G. N. Konstantinou, S. N. Vigod, S. Mehta, Z. J. Daskalakis, and D. M. Blumberger, “A systematic 
review of non-invasive neurostimulation for the treatment of depression during pregnancy,” J. 
Affect. Disord., vol. 272, pp. 259–268, Jul. 2020, doi: 10.1016/j.jad.2020.03.151.