Frontal E/I Balance Mediation of tACS Effects on Behavioral Flexibility
Phase: NA
Conditions: Behavioral Flexibility, Healthy Volunteers, High Risk Alcohol Use
Sponsor: University of North Carolina, Chapel Hill

Protocol and Statistical Analysis Plan 
 
 
Frontal E/I balance mediation of tACS effects on behavioral flexibility 
 
 
 
NCT number NCT06634771 
Document Date 10/02/2024 
 

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Master Protocol Document 
 
Title 
Frontal E/I balance mediation of tACS effects on behavioral 
flexibility 
Sub-Title 
FLAME Study 
Principal Investigator 
Charlotte A. Boettiger, Ph.D., Department of Psychology & 
Neuroscience 
(919) 907-0585 
Co-Investigators 
 
Jillian Battista, Department of Psychology & Neuroscience 
Grace M. Elliott, Department of Psychology & Neuroscience 
Statistical  Co-Investigators 
 
 
University of North Carolina, Chapel Hill 
Version Number  2.0 
2 October 2024 
 
 
 
 
I have read, understood, and approved this version of the protocol.   [electronic signatures 
accepted]  
 
Principal Investigator:      
 
 
 
 
________ 
 
Date:  
 
 
 
 
 
Statement of Confidentiality and Nondisclosure     This document is a confidential communication.  Acceptance of 
this document constitutes agreement by the recipient that no unpublished information contained herein will be published 
or disclosed without prior written approval of the Principal Investigator or other participating study leadership, except that 
this document may be disclosed in any medium to appropriate investigators, Institutional Review Boards, Scientific 
Review Committees, and others who are directly involved in the study specified herein under the condition that they keep 
the information confidential. 
 
 
10/02/2024 

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Table of Contents 
Statement of Compliance ............................................................................................................................... 4 
Table of Abbreviations .................................................................................................................................... 5 
1. 
Protocol Synopsis ............................................................................................................................... 7 
2. 
Introduction ......................................................................................................................................... 10 
2.1. 
Background Information .................................................................................................................. 10 
2.2. 
Scientific Rationale .......................................................................................................................... 11 
3. 
Specific Aims ...................................................................................................................................... 12 
3.1. 
Aim 1 .................................................................................................................................................. 12 
3.2. 
Aim 2 .................................................................................................................................................. 12 
3.3. 
Aim 3 .................................................................................................................................................. 12 
4. 
Investigational Plan ........................................................................................................................... 13 
5. 
Study Participants ............................................................................................................................. 15 
5.1. 
Numbers of Participants ................................................................................................................. 15 
5.2. 
Eligibility Criteria .............................................................................................................................. 15 
5.3. 
Randomization  and  Blinding ........................................................................................................ 17 
5.4. 
Recruitment and Retention ............................................................................................................ 18 
6. 
Schedule of Activities and Procedures ....................................................................................... 18 
6.1. 
Table of Events ................................................................................................................................ 18 
6.2. 
Screening .......................................................................................................................................... 19 
6.3. 
Enrollment ......................................................................................................................................... 19 
6.4. 
Study Visit Procedures .................................................................................................................... 19 
6.5. 
Final Visit........................................................................................................................................... 21 
6.6. 
Phone Contacts ............................................................................................................................... 21 
6.7. 
Enrollees May Drop Out ................................................................................................................. 21 
7. 
Statistical Analysis Plans ................................................................................................................ 21 
7.1. 
Strategies that Apply to all the Aims ............................................................................................. 21 
7.2. 
Statistical Design ............................................................................................................................. 22 
8. 
Sample Size Rationale ...................................................................................................................... 23 
9. 
Data Collection and Management ................................................................................................. 24 
10. 
Collection and Management of Tissue Specimens .................................................................. 24 
10.1. Use in Current and Future Studies ............................................................................................... 24 
10.2. Sample Preparation ......................................................................................................................... 25 
10.3. Record Keeping and Monitoring .................................................................................................... 25 
10.4. Storage and Security ....................................................................................................................... 25 
11. 
Safety Monitoring and Management ............................................................................................. 26 
11.1. Risk / Benefit Assessment .............................................................................................................. 26 
11.2. Safety Monitoring ............................................................................................................................. 27 
11.3. Adverse Events and Serious Adverse Events Reporting Procedure ....................................... 28 

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12. 
Regulatory, Ethical, and Study Oversight Specifications ....................................................... 29 
12.1. Informed Consent Process ............................................................................................................. 29 
12.2. Confidentiality and Privacy ............................................................................................................. 30 
12.3. Future Use of Stored Data ............................................................................................................. 31 
12.4. Key Roles and Study Governance ................................................................................................ 31 
12.5. Publication and Data Sharing Plan ............................................................................................... 31 
13. 
References .......................................................................................................................................... 33 
 
 
 
 
 

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Statement of Compliance 
This study will be conducted as specified in the protocol and in accordance with the International 
Conference on Harmonisation Guidelines for Good Clinical Practice (ICH E6) and the Code of Federal 
Regulations on the Protection of Human Subjects (45 CFR Part 46).  
The protocol, informed consent form(s), recruitment materials, and all participant 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 participant 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 participants who provided consent, using a previously 
approved consent form. 
If required by the IRB, the master protocol document, informed consent form(s), recruitment materials, 
and all participant materials will be submitted to the Scientific Review Committee (SRC) prior to IRB 
review (research.unc.edu/clinical-trials/src). 
The statistical analysis plans will be consistent with guidance in CONSORT Statement [1] or STROBE 
Statement [2], ICMJE recommendations [3], the 2016 and 2019 statements of the American Statistical 
Association [4,5], and recommendations in Nature [6,7].*  
All personnel involved in the conduct of this study have completed human subjects protection training. 
 
 
 
 
 
 
 
 
 
 
*  [1]   www.consort-statement.org  
   [2]  www.strobe-statement.org  
   [3]  www.icmje.org  
   [4]  Wasserstein RL, et al. (2016), The ASA's Statement on p-Values, The American Statistician, 70:2, 129-133  
   [5]  Wasserstein RL, et al. (2019), Moving to a World Beyond p < 0.05, The American Statistician, 73:sup1, 1-19  
   [6]  Amrhein, et al. (2019) Scientists rise up against statistical significance, Nature 567, 305-307  
   [7]  Editorial (2019) It’s time to talk about ditching statistical significance: Looking beyond a much used and abused measure 
          would make science harder, but better. Nature 567, 283-283. 

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Table of Abbreviations 
AE / SAE 
adverse event / serious adverse event 
aIC 
anterior insular cortex 
AIE 
adolescent binge drinking 
ANCOVA 
analysis of covariance 
AUD 
alcohol use disorder 
CFR 
U.S. Code of Federal Regulations (www.eCFR.gov) 
CI 
confidence interval 
CITI 
Collaborative Institutional Training Initiative 
CON 
control group 
CONN 
functional connectivity toolbox (web.conn-toolbox.org) 
CONSORT 
Consolidated Standards of Reporting Trials (www.consort-statement.org) 
CZ 
central midline in the 10/20 EEG system 
DOI 
digital object identifier 
dLPFC 
dorsolateral prefrontal cortex 
DSA 
Data Submission Agreement 
DSMB 
data and safety monitoring board 
EEG 
electroencephalogram 
E/I 
excitatory/inhibitory 
F3 
left dorsolateral prefrontal cortex in the 10/20 EEG system 
F4 
right dorsolateral prefrontal cortex in the 10/20 EEG system 
FC 
functional connectivity 
FDA 
U.S. Food and Drug Administration   (www.fda.gov) 
FDR 
false discovery rate 
fMRI 
functional magnetic resonance imaging 
GABA 
Gamma-amino-butyric acid 
Glx 
glutamate + glutamine 
GUID 
Global Unique Identifier 
HABIT 
Hidden Association Between Images Task 
ICMJE 
International Committee of Medical Journal Editors 
IL-6 
interleukin-6 
IRB 
institutional review board 
MPD 
master protocol document 
MR 
magnetic resonance 
MRI 
magnetic resonance imaging 
MRS 
magnetic resonance spectroscopy 
N 
number of enrolled participants 
NADIA 
Neurobiology of Adolescent Drinking in Adulthood 
NDA 
NIMH Data Archive 
NIAAA 
National Institute on Alcohol Abuse and Alcoholism 
NIAAADA 
NIAAA Data Archive 
NIH 
National Institutes of Health 
NIMH 
National Institute of Mental Health 
NoA 
Notice of Award 
PHI 
protected health information 
PI 
principal investigator 
QA 
quality assurance 
QC 
quality control 
REDCap 
Research Electronic Data Capture system 
ROI 
region of interest 
RPPR 
Research Performance Progress Report 
SRC 
UNC Scientific Review Committee   (research.unc.edu/clinical-trials/src) 
STROBE 
STrengthening the Reporting of OBservational studies in Epidemiology. 
SUD 
substance use disorder 
tACS 
transcranial alternating current stimulation 
TNF-alpha 
tumor necrosis factor alpha 

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UNC 
The University of North Carolina 
UNCCH 
UNC Chapel Hill 
WHO 
World Health Organization 
 
 

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1. Protocol Synopsis 
 
Title 
Frontal E/I balance mediation of tACS effects on behavioral 
flexibility 
Study Description 
This proposal is designed to probe the role of excitatory and 
inhibitory (E/I) signaling in key nodes of control circuitry in 
mediating the relationship between alcohol misuse and inflexible 
behavior. In addition, we aim to determine whether adolescent 
binge alcohol exposure amplifies the effects of binge exposure in 
adulthood. We will accomplish this goal via a single multi-session 
study. Participants (n=66) will comprise three groups: adults self-
reporting high risk drinking (WHO risk levels 2, 3, or 4), with (n=22) 
or without (n=22) a history of adolescent alcohol misuse (AIE), and 
lifetime low risk drinking adults (WHO risk levels 0 or 1; n=22). 
Design: a 3-session study that includes an initial screening session 
and behavioral training (Session 1), behavioral testing and an MRI 
scan session (Session 2), bifrontal 10Hz-tACS (true or sham) 
during behavioral testing with pre- and post-EEG recording in a 
resting-state, followed by a second MRI scan session (Session 3). 
We predict that adolescent and adult binge history will have 
interacting effects on E/I balance indices derived from EEG and 
MR-spectroscopy and on behavioral flexibility measured in the 
HABIT Test and that E/I balance indices will mediate the 
relationship between alcohol misuse and behavioral flexibility. We 
also propose to test a causal relationship between E/I balance and 
behavioral flexibility by testing whether 10Hz-tACS to bilateral 
dlPFC alters habitual action selection in the HABIT Test in 
proportion to its effects on the dlPFC 1/f EEG slope and/or the 
MRS-derived GABA/Glx ratio. We predict that changes in indices 
of E/I balance induced by tACS will inversely associate with 
changes in habitual response selection. We will collect a small 
amount of blood from a finger prick in Sessions 1 and 3 and will 
use the collected dried blood spots to measure inflammatory 
markers. 
Specific Aims 
(objectives) 
Aim 1.  Compare groups in terms of Session 2 (baseline) 
perseverative errorsin the HABIT Test and MRS-derived 
GABA:Glx, and Session 3 baseline 1/f EEG slope. 
Aim 2.  Change behavioral flexibility via (transcranial alternating 
current stimulation (tACS), and test whether changes in E/I 
measures mediate this change. 
Aim 3.  Test whether MRS-derived GABA:Glx, or 1/f EEG slope in 
fronto-limbic nodes correlate with functional MRI 
connectivity of these nodes. 
Target Population 
Inclusion Criteria 
• 
22-50 years old 
• 
Medically healthy 
• 
Fluent in English 
• 
High school educated 
• 
Group 1 (CON): no AIE and current (past month) WHO risk 
drinking levels of either 0 or 1 (abstinent or low-risk) 
• 
Group 2 (RISK): no AIE and current WHO level 2, 3 or 4 
(medium risk, high risk, or very high risk) 
• 
Group 3 (RISK+AIE): history of adolescent binge drinking 
(4 or more binge episodes before age 18; per NIAAA, a 

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binge episode = 4+ drinks in a 2-hr period for females and 
5+ drinks in a 2-hr period for males) and current WHO risk 
level 2, 3, or 4. 
 
Exclusion Criteria  
• 
Lifetime history of an SUD (including nicotine) but 
participants will not be excluded for an AUD.  
• 
Neurological disease such as dementia, seizures or head 
trauma 
• 
History of psychosis or psychotic episodes 
• 
Diagnosis of ADHD 
• 
Systemic disease such as cancer, cardiovascular or 
inflammatory disease which could influence cognitive 
functioning 
• 
Motor or visual disturbance (including color blindness) 
which precludes them from performing the tasks 
• 
Current (past month) use of psychoactive drugs (aside from 
caffeine or alcohol)  
• 
Current (past month) use of psychotropic medication 
including antidepressants, mood stabilizers, antipsychotics, 
anxiolytics, stimulants, or hypnotics with the exception of 
stable doses of antidepressants for one month or more.  
• 
Past month use of therapeutic brain stimulation (e.g. TMS 
or ECT) 
• 
Any MRI contraindication based on BRIC’s MRI safety 
checklist 
• 
History of brain surgery  
• 
History of migraine headaches 
• 
Pregnancy 
• 
Brain implants/devices 
• 
First degree relative with primary epilepsy 
• 
Claustrophobia 
• 
Breath alcohol above 0.0% at time of session 
• 
Positive urine drug screen at time of session 
 
Numbers of Enrollees 
A total of N = 66 eligible individuals will be enrolled. We anticipate 
that at least n = 64 of the enrollees will complete all aspects of the 
protocol and have complete data. 
                                                  
Interventions 
In this double-blinded randomized design, participants will receive 
a single session of 10 Hz bi-frontal tACS or active Sham tACS 
during the second half of the HABIT Test in Session 3. 
 
10 Hz bi-frontal tACS: Alternating current stimulation is delivered 
by an XCSITE 100 device (Pulvinar Neuro, Chapel Hill, NC), 
through three conductive carbon-rubber electrodes. Electrodes are 
placed over the apex of the head (Cz) and the prefrontal cortex 
bilaterally (F3 and F4). Stimulation parameters: 2mA peak-to-peak 
10Hz sine-wave flanked by 10 second linear envelope ramps in 
and out for a total duration of 30 min and 20 seconds. 
 
Sham tACS:  The procedure for sham stimulation will be identical, 
but stimulation will last for 2 minutes within the “stimulation” period 
of  30 minutes.  

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Outcome Measures 
For Aim 1.    
1) Habitual action selection, calculated as the proportion of 
perseverative errors after Stimulus-Response contingency change 
for highly trained (Familiar) stimuli in the HABIT. 
2) Prefrontal GABA:Glx ratio measured via single voxel MRS.  
3) Power spectral slope (1/f) of the EEG signal localized to the 
dlPFC. 
4) Concentrations of circulating inflammatory markers, including C-
reactive protein, IL-6, and TNF-α measured from dried blood 
spots. (Secondary outcome) 
For Aim 2.    
1) Change in habitual action selection after 10Hz- or sham-tACS. 
2) Change in prefrontal GABA:Glx ratio after 10Hz- or sham-tACS. 
3) Change in 1/f slope of the EEG signal localized to the dlPFC 
after 10Hz- or sham-tACS. 
4) Change in concentrations of circulating inflammatory markers, 
including C-reactive protein, IL-6, and TNF-α after 10Hz- or sham-
tACS. 
For Aim 3. Change Z-transformed Pearson correlation between 
bilateral dlPFC and striatum after 10Hz- or sham-tACS. 
Statistical Analysis 
Plans  
for Each Aim  
Aim 1 Plans. To test the prediction that E/I balance indices will 
differ from low-risk drinkers more among high-risk drinkers with 
adolescent (<18 years old) binge history, we will analyze the 
baseline (i.e. pre-tACS) data collected in Sessions 2 and 3. We will 
conduct Analyses of Covariance (ANCOVA) to compare groups in 
terms of Session 2 (baseline) perseverative errors in the HABIT 
Test and MRS-derived GABA:Glx, and Session 3 baseline 1/f EEG 
slope, controlling for age, sex, education, psychiatric diagnoses, 
and medication status.  
 
We will use similar experimental designs to those described to 
evaluate a number of secondary outcomes. These include C-
reactive protein measured from blood spots and other immune 
activation markers (TNF-α and IL-1β) derived from salivary 
microvesicles, as well as exploratory/alternative group comparison 
analyses. The latter include effects of tACS on GABA:Glx ratios in 
the dlPFC and aIC, and on EEG 1/f slope. 
 
Aim 2 Plans. Effects of tACS on habitual action selection in the 
HABIT task (primary outcome, Aim 2) will be assessed based on 
the proportion of perseverative errors (relative to all errors) when 
the participant must replace a highly trained response to a set of 
familiar stimuli. We will use a general linear model that includes 
group and stimulation condition as between-subjects factors; we 
will also include baseline perseverative error, age, sex, 
handedness, and anxiety or mood disorder diagnoses as 
covariates. 
 
As for Aim 1, we will use similar experimental designs to those 
described to evaluate a number of secondary outcome responses 
to tACS. These include C-reactive protein measured from blood 
spots and other immune activation markers (TNF-α and IL-1β) 
derived from salivary microvesicles, as well as 
exploratory/alternative outcome analyses. The latter include effects 

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of tACS on GABA:Glx ratios in the dlPFC and aIC, and on EEG 1/f 
slope. 
 
Aim 3 Plans. We will test whether the indirect effect of tACS on 
HABIT perseverative error is mediated by resting-state functional 
connectivity. For our mediation analyses, functional connectivity 
(FC) values derived from a priori ROI-to-ROI (e.g., dlPFC, aIC, and 
limbic striatum) analyses will be used as the mediating variable; 
change in HABIT perseverative errors will serve as the dependent 
variable and will be adjusted for sex, age, handedness, and 
anxiety or mood disorder diagnoses as covariates; tACS condition 
(stim/sham) will serve as the independent variable; and AIE and 
current alcohol misuse scores will be included as moderators. 
 
We will also conduct exploratory analyses testing for associations 
between indices of E/I balance in the dlPFC and dlPFC FC at 
baseline, and whether changes in E/I balance indices associate 
with changes in dlPFC FC. 
Study Duration 
We anticipate the study duration to last 4 years. 
Participation Duration 
We anticipate the participation duration to include 3 sessions over 
the course of 3 days (minimum) or within 2 weeks. 
 
Enrollment Duration 
Slightly less than 4 years. 
 
2. Introduction 
2.1.   Background Information 
Problematic alcohol use is a major health crisis in the United States, with approximately 13% of 
Americans meeting the diagnostic criteria for an AUD (Grant et al., 2017; Grant et al., 2015). AUD 
diagnoses are associated with significant decreases in quality of life, as well as increased risk of early 
mortality (Hasin et al., 2007; Rehm et al., 2016). Even below the clinical threshold, binge drinking 
contributes significantly to risk of death, head injuries, hospitalizations and other severe consequences – 
particularly in adolescents (CDC, 2012; Siqueira & Smith, 2015). The development of tolerable and 
efficacious interventions for risky drinking would benefit a large portion of the American population, 
especially given the current gaps in treatment utilization for individuals with AUDs (Cohen et al., 2007). 
 
A major risk factor for later development of an AUD Is binge drinking during adolescence (Siqueira & 
Smith, 2015; Viner & Taylor, 2007). Preclinical and human literature both indicate that alcohol use during 
adolescence can result in lasting changes to the brain (Crews et al., 2016; Ewing et al., 2014; Guerri & 
Pacual, 2010). Individuals who engage in binge drinking during adolescence are likely to continue these 
patterns into early adulthood (Degenhardt et al., 2013). Adolescent alcohol use may even increase 
vulnerability to future assaults, particularly those resulting from heavy drinking in adulthood.  
 
One behavioral feature associated with heavy alcohol or substance use is overreliance on habitual 
action selection, as opposed to goal-directed decision-making (Ersche et al., 2016; McKim et al., 2016). 
The regions involved in top-down modulation of behavior are commonly referred to as the cognitive 
control circuitry (Friedman & Robbins, 2022; Miller & Cohen, 2001). The dlPFC is one element of this 
fronto-striatal circuitry involved in habit formation and decision-making (Boettiger, 2005; Brosnan & 
Wiegand, 2017; Smittenaar et al., 2013). The involvement of the dlPFC in adaptive decision-making has 
been repeatedly demonstrated in the human literature, as has involvement of the analogous rodent 

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prelimbic cortex in stimulus-response tasks (Smittenaar et al., 2013; Wood & Rünger, 2016). The dlPFC 
is a major hub of the cognitive control circuitry involved in adaptive decision-making, and it represents a 
good target for non-invasive neuromodulation due to its proximity to the scalp (Gbadeyan et al., 2016; 
Vanderhasselt et al., 2013).  
 
Chronic alcohol exposure modifies GABAergic activity in the CNS long-term, causing plastic changes 
that suppress inhibition mediated by GABA receptors (Davies, 2003; Olsen & Liang, 2017). Unpublished 
MRS data from our lab indicate that binge drinking in adulthood is associated with decreased levels of 
GABA in the dlPFC. Binge drinking was also associated with higher levels of glutamate in the region, 
resulting in a disrupted balance between excitatory and inhibitory firing. These data indicate that heavy 
drinking may shift E/I balance toward excitation. Behaviorally, frontal glutamate to GABA ratio was 
positively correlated with habitual action selection. This is consistent with the literature, in which higher 
E/I balance has been associated with impulsivity in decision making (Lam et al., 2022). We hypothesize 
that E/I balance in control circuitry hubs may be shifted toward excitation by repeated binges during a 
critical developmental period, and that this shift results in overreliance on habitual action selection. This 
shift in E/I balance may make one more susceptible to problematic drinking in adulthood through 
disruptions in adaptive and goal-based decision making. This process may be a positive feedback cycle 
in which shifted E/I balance leads to more drinking, and more drinking further shifts E/I balance and thus 
habitual tendencies. 
 
Previous research has demonstrated that non-invasive neurostimulation of the dlPFC can modulate 
decision-making (Ostlund, 2005). However, the literature as a whole is equivocal on the efficacy of non-
invasive neurostimulation to modulate behavior (Brunye et al., 2020; Sellers et al., 2015). The 
mechanisms of neurostimulation remain poorly understood, but a reasonable assumption is that efficacy 
and directionality of effects would be dependent on a number of factors, including scalp-to-cortex 
distance, priming, and individual differences in circuitry (Brunye et al., 2020; Kearney-Ramos et al., 
2018). Data from our lab showed that controls and individuals with a history of SUD were differentially 
affected by 10Hz tACS bilaterally to the dlPFC, such that controls showed an increase in habitual 
behavior, whereas individuals with a history of SUD trended toward improved adaptive behavior (McKim 
et al., 2021). We hypothesize that the directionality of behavioral modulation by dlPFC tACS may be 
dependent on E/I balance at baseline.  
 
The main purpose of this study is to examine the role of frontal excitatory/inhibitory (E/I) balance in 
manifesting the adult behavioral phenotypes associated with adolescent binge drinking, as well as 
determining the potential efficacy of tACS to modulate E/I balance and associated behaviors. We 
hypothesize that risky drinking in adulthood will shift resting dlPFC E/I balance toward excitation, and that 
adolescent ( before age 18) alcohol use will exacerbate this shift. We predict that this shift will mediate 
behavior such that higher E/I ratios will be associated with an increase in perseverative errors in an 
adaptive learning task, and that tACS-induced changes in E/I balance will mediate behavioral changes 
after stimulation. 
2.2.   Scientific Rationale 
We have selected 10Hz bilateral tACS to the dlPFC as an intervention to target disrupted E/I balance 
resulting from adolescent binge drinking and contributing to an excessively habitual behavioral profile 
and risky drinking in adulthood. Transcranial electrical stimulation is a non-invasive, temporary 
intervention that is generally well-tolerated and has a very low risk of adverse side effects (Matsumoto & 
Ugawa, 2017). As described in Background, our lab previously performed 10Hz bilateral tACS to the 
dlPFC in a sample of individuals with a history of SUD and saw a trend toward improvement in 
performance on the HABIT task (McKim et al., 2021). In this study we aim to examine the role of 
stimulation-induced shifts in frontal E/I balance in these behavioral changes, specifically by recording 
resting-state EEG immediately prior to and immediately after stimulation. We will also be examining the 
lasting effects of tACS (on the scale of hours) using baseline and post-stimulation fMRI scanning. The 
literature indicates that tACS allows for entrainment of brain activity to a specific frequency, although 

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circuit effects are poorly understood (Herrmann et al., 2013; Zaehle et al., 2010). Additionally, this study 
will provide the first test of modulating E/I balance – determined through resting-state EEG 1/f slope and 
MRS – using 10Hz tACS. Our lab has performed hundreds of sessions of tACS using the Pulvinar Neuro 
XSCITE100 system with no adverse events reported.  
3. Specific Aims 
3.1. Aim 1 
Compare groups in terms of Session 2 (baseline) perseverative errors in the HABIT Test 
and MRS-derived GABA:Glx, and Session 3 baseline 1/f EEG slope. 
 
1a: Compare perseverative error rates in the HABIT test at session 2 (pre-tACS) across all 3 
groups 
 
1b: Compare MRS-derived GABA:Glx at session 2 (pre-tACS) across all 3 groups 
 
1c: Compare Session 3 baseline 1/f EEG slope across all 3 groups 
 
Hypothesis 1: Perseverative error rates will be higher in the Hazardous drinking groups, with the 
highest rates in the adolescent binge alcohol exposure group, and GABA:Glx ratio and 1/f EEG 
slope will be lower in the hazardous drinking groups, with the lowest ratios and slopes in the 
group with adolescent binge alcohol exposure, tracking with the theory that hazardous alcohol 
use, especially in the adolescent period, drives reduced inhibitory signaling in executive control 
circuitry, shifting E/I balance and reducing behavioral flexibility. 
 
3.2.  Aim 2 
Change behavioral flexibility via (transcranial alternating current stimulation (tACS), and 
test whether changes in E/I measures mediate this change.  
 
2a: Compare 10Hz and sham tACS effects on perseverative error in the HABIT test (session 3) 
compared to baseline (session 2). 
 
2b: Compare 10Hz and sham tACS effects on GBA:Glx ratio and 1/f EEG slope (session 3) 
compared to baseline. 
 
2c: Test whether tACS induced changes in GABA:Glx of1/f EEG slope mediate changes in 
behavioral flexibility after tACS. 
 
Hypothesis 2: 10Hz-tACS to bilateral dlPFC will change habitual action selection in the HABIT 
Test in proportion to its effects on the dlPFC 1/f EEG slope and/or the MRS-derived GABA/Glx 
ratio. We predict that changes in reductions in 1/f slope and in MRS-derived GABA:Glx induced 
by 10Hz-tACS will associate with increased habitual response selection. 
 
3.3.  Aim 3 
Change functional MRI connectivity (fcMRI) of fronto-limbic circuitry via tACS. 
3a: Compare 10Hz and sham tACS effects on fcMRI of a priori ROI (dlPFC, aIC, and limbic 
striatum) in session 3 compared to baseline (session 2). 
3b: Conduct mediation analyses with fcMRI changes to identify connections that mediate the 
effects of tACS on habitual responding. 

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3c: Conduct exploratory analyses testing for associations between indices of E/I balance in the 
dlPFC and dlPFC fcMRI at baseline, and whether changes in E/I balance indices associate with 
changes in dlPFC fcMRI. 
Hypothesis 3:  fcMRI of the dlPFC will correlate with indices of E/I balance and habitual action 
selection before and after 10Hz-tACS to bilateral dlPFC  
4. Investigational Plan 
 
Study Design:   This is a randomized, double-blind, sham-controlled tACS study. 
Brief overview of study events: Potential participants will complete eligibility screening for the study. 
Participants meeting eligibility criteria will complete a baseline session including further eligibility 
screening, training on the computerized HABIT task, and collection of urine, saliva, and blood samples. 
Participants will then complete a session involving the full version of the HABIT task and MRI scanning. 
The final session will involve the full HABIT task, resting-state EEG, sham or active tACS, MRI, and 
collection of another blood spot. Participants will also complete a series of online questionnaires outside 
of their scheduled sessions. See Figure 1 for depiction of study procedures. 
Study Duration: 4 years 
Target Number of Participants: 66 adults comprising three groups: high risk drinkers with (n=22) or 
without (n=22) a history of adolescent alcohol misuse, and lifetime low risk drinkers. 
tACS: For Session 3, participants will complete a HABIT Test Session in the Howell Hall 
Neurostimulation Core Lab or EEG Core Lab, which is located one floor below the Boettiger Lab. 
Participants will receive either 10Hz bi-frontal tACS or sham tACS. 
10 Hz bi-frontal tACS: Alternating current stimulation is delivered by an XCSITE 100 device (Pulvinar 
Neuro, Chapel Hill, NC), through three conductive carbon-rubber electrodes. Electrodes are placed over 
the apex of the head (Cz) and the prefrontal cortex bilaterally (F3 and F4). Stimulation is delivered during 
the second half of the HABIT Test session. Stimulation parameters: 2mA peak-to-peak 10Hz sine-wave 
flanked by 10 second linear envelope ramps in and out for a total duration of 30 min and 20 seconds. 
Sham tACS: The procedure for sham stimulation will be identical, but the actual stimulation will last for 2 
minutes instead of 30 minutes. Participants generally report that stimulation is felt most strongly at the 
beginning of active stimulation, before they adjust to the sensation. Sham stimulation is meant to mimic 
this progression in terms of tactile salience. Stimulation is delivered for 2 minutes at the beginning of the 
HABIT reversal task, flanked by 10 second linear envelope ramps. The stimulating electrodes are left on 
the head until completion of the HABIT task, as is the case in active stimulation. There is no visual or 
auditory indication to the participant or researcher when the 2-minute sham stimulation period has 
ended, allowing the sham stimulation condition to feel similar to active stimulation. The XSCITE 100 
device allows for the programming of stimulation codes corresponding to sham or active stimulation, both 
of which are set to a total duration of 30 minutes, allowing for reliable double-blinding.  
HABIT Task: The HABIT task is a computerized measure of behavioral flexibility; it has been used by our 
lab in multiple previous studies. The task is composed of two parts: the adaptive learning task and the 
reversal task. The HABIT is also preceded by a necessary training module. The task is composed of a 
series of visual patterns defined by consistent color blocking. Patterns come in sets and participants 
learn to pair each pattern within a set of two with a specific button-press response by pressing buttons 
and receiving auditory and visual feedback. During training, participants are presented with one set of 
two patterns and are allowed to learn the button-press responses associated with each pattern. This is 
the familiar set and will reappear throughout the task. During the adaptive learning portion of the task, 

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participants are presented with two or three new sets of patterns and are allowed to learn the button-
press responses corresponding to the patterns within each set. These are the novel sets – they will only 
be presented during this iteration of the adaptive learning and reversal tasks. During the reversal task 
(part 2), the novel and familiar sets are presented again, but the button-press responses have changed 
for one familiar set and one novel set. The adaptive learning portion of the task is intended to measure 
one’s ability to learn stimulus-response associations. The reversal portion of the task, performance on 
which is our primary behavioral interest in this study, is intended to measure behavioral flexibility, or 
one’s ability to update stimulus-response associations that have changed by adapting to feedback. 
Timing of Visits: Eligibility screening will occur up to two weeks before session one. Session 1 will occur 
on day 0, and session 2 will occur at least one night’s sleep after session one to allow for memory 
consolidation of HABIT training sets (max 2 weeks). Session 3 can occur at any time after the completion 
of session 2, ideally within 2 weeks, and the online questionnaires can be completed any time after 
session 1 and prior to session 3. We anticipate most participants will choose to complete the 
questionnaires between sessions at the time and location of their choosing; if not they will be given the 
option to complete them onsite during session one, two, or three. If the online questionnaires are not 
completed prior to session 3, they will be completed by the participant onsite at the end of session 3 prior 
to payment. 
 
 

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Figure 1.  Randomized Protocol Schematic.  
 
  
Recruitment 
and Screening 
 
 
 
 
 
Verification of Eligibility 
and  Enrollment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit 1 
 
 
 
 
Visit 2 
  
 
 
 
Visit 3 
 
 
 
 
 
5. Study Participants 
5.1.  Numbers of Participants 
We plan to enroll 66 individuals total (22 in each group). With possible attrition or loss of data, we expect 
64 individuals to complete the study. 
5.2.  Eligibility Criteria 
We chose the criteria described here in order to ensure participant safety, minimize confounds, and 
optimize recruitment opportunities. The following exclusion criteria are meant to exclude individuals who 
may be at an elevated risk for adverse events during tACS: history of brain surgery, history of migraine 
Obtain informed consent.   
Obtain screening data and baseline data. 
Apply inclusion and exclusion criteria.  
Train participants on task.  
Collect baseline  HABIT and MRI measures 
10Hz tACS 
1/2 N = 33 
Allocations by 1:1 Randomization 
Sham tACS 
1/2 N = 33 
Enrollment of Eligible 
Participants 
Collect baseline EEG, HABIT during tACS, then post-tACS EEG and MRI measures. 
Debriefing and study termination. Participation completed.  

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headaches, family history of epilepsy, or pregnancy. The safety of tACS for these populations has not yet 
been determined, and our lab has administered hundreds of sessions of tACS with no adverse events 
using these exclusion criteria. The following exclusion criteria are meant to exclude individuals who 
should not enter the MRI scanner: presence of any brain implants or devices, or claustrophobia.  
In order to reduce the number of confounding variables, we have chosen to exclude individuals whose 
task performance, brain activity, or inflammatory markers may be affected by external factors unrelated 
to drinking history. Diagnosis of any neurological disease or ADHD, symptoms of psychosis, diagnosis of 
systemic or inflammatory disease, presence of motor or visual disturbances, or use of psychotropic 
medications or psychoactive drugs will all be exclusionary for this reason. A high school diploma or 
equivalent and fluency in English will also be required to control for confounds in task performance. 
Although use of psychotropic medications will generally be exclusionary, individuals who have been on a 
stable dose of an antidepressant for 1 month or more will still be eligible. We believe this will allow for the 
recruitment of a sample that is more representative of Americans who drink alcohol. 
In order to ensure that individuals are fully lucid and able to consent to participate in this study, a breath 
alcohol level above 0.0% or a positive urine drug screen at the beginning of any visit will be exclusionary. 
If this occurs, the participant will be given the option to reschedule their visit. 
There is currently no established threshold for what constitutes meaningful adolescent alcohol exposure 
in the human literature. For this reason, we have chosen to define our AIE group as those who report 4+ 
binge episodes before the age of 18 based on previous data from our lab (Elton et al, 2021). We are 
using the established WHO risk drinking level criteria to define levels of adult drinking (low risk vs. high 
risk). 
 
5.2.1. Inclusion Criteria  
 
All of the below criteria will be self-reported. 
 
In order to be eligible to participate in this ssitudy, an individual must meet all of the following 
criteria: 
• 
 22-50 years old 
• 
Have a high school diploma or equivalent 
• 
Medically healthy 
• 
Fluent in English 
 
For RISK group only: 
• 
High risk alcohol use in the past month (WHO risk level 2-4) 
• 
No history of adolescent binge drinking 
 
For RISK+AIE group only: 
• 
High risk alcohol use in the past month (WHO risk level 2-4) 
• 
High levels of adolescent alcohol use (4 or more binge drinking episodes before age 18) 
 
For CON group only: 
• 
Low risk alcohol use throughout the lifetime (WHO risk level 0-1) 
• 
No history of adolescent binge drinking 
• 
No lifetime history of AUD 
 
5.2.2. Exclusion Criteria  
 

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Any individual who meets one or more of the following criteria will be excluded from participation 
(excluding BAC, urine drug screen, psychiatric diagnoses, and color blindness, all will be self-
reported): 
• 
Lifetime history of an SUD (including nicotine) but participants will not be excluded for an 
AUD.  
• 
Neurological disease such as dementia, seizures or head trauma 
• 
History of psychosis or psychotic episodes 
• 
Diagnosis of ADHD 
• 
Any systemic or inflammatory disease that could affect cognitive functioning (e.g., cancer, 
cardiovascular disease) 
• 
Any motor or visual disturbances that could hinder task performance (e.g., color blindness) 
• 
Use of psychoactive recreational drugs in the past month (excluding caffeine and alcohol) 
• 
Use of psychotropic medications in the past month including antidepressants, mood 
stabilizers, antipsychotics, anxiolytics, stimulants, or hypnotics (excluding antidepressant use 
when dosage has been stable for 1 month or longer) 
• 
Use of therapeutic brain stimulation (e.g. TMS or ECT) in the past month 
• 
Any history of brain surgery 
• 
History of migraine headaches 
• 
Pregnancy 
• 
Any brain implants or devices 
• 
First degree relative with primary epilepsy 
• 
Claustrophobia 
• 
Any MRI contraindication based on BRIC’s MRI safety checklist 
• 
Breath alcohol above 0.0% at time of session 
• 
Positive urine drug screen at time of session 
 
If a participant should have an exclusion criterion arise in the course of their participation (e.g. pregnancy 
or psychotic episode), their participation in the study will end unless the circumstances are transitory in 
nature (e.g. positive breath alcohol or urine drug screen). 
 
5.3.   Randomization  and  Blinding 
Our study design consists of three groups of participants:  
1. (CON): no adolescent binge drinking (AIE) and current (past month) World Health Organization 
(WHO) risk drinking levels of either 0 or 1 (abstinent or low-risk) 
2. (RISK): no AIE and current WHO level 2, 3, or 4 (medium risk, high risk, or very high risk) 
3. (RISK+AIE): history of adolescent binge drinking and current WHO risk level 2, 3, or 4.  
Within each group (n=22), participants will be randomly assigned to either the 10Hz tACS (stim) group or 
the sham tACS (sham) group. 
 
Our study design requires randomly assigning individuals to a stimulation group after completing Session 
1. Prior to study onset, Dr. Boettiger will create a randomization table in which stimulation type (true or 
sham) is pseudo randomly ordered within groups, stratified by sex. Dr. Boettiger will provide assignment 
to the study team based on this predefined randomization table in order to ensure balanced sex and 
group for each stimulation type. The study team will be provided a numeric code to enter into the 
XCSITE 100 tACS system, which will conceal the stimulation parameters from the study team, ensuring 
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5.4. Recruitment and Retention 
The research team has extensive experience in successfully recruiting participants into multi-session 
research studies. Dr. Boettiger has conducted many such studies, including those with challenging 
populations (e.g. alcohol or substance use disorders), or with challenging interventions (e.g. lengthy 
pharmacological challenges or brain stimulation studies). She has been the PI on 8 different challenge 
protocols, has been continuously NIH-funded for 14 years, and is currently the Co-PI of an NIAAA-
funded (P60AA011605) 4-5 session study investigating the neural circuit bases of behavioral flexibility in 
adults (22-40). That study entails an EEG and behavioral training session, three transcranial brain 
stimulation sessions, and an MRI scanning session, with a similar scan protocol to that proposed here. 
Moreover, the Boettiger laboratory has extensive expertise in working with individuals with a history of 
hazardous alcohol use. Dr. Boettiger’s team is trained in all technical aspects of the proposed study, 
including screening, cognitive testing, EEG, fMRI and MRS, and tACS. Thus, the team is highly qualified 
and prepared to recruit and retain sufficient sample sizes for the proposed study.  
 
Recruitment  
Participants will be recruited through IRB-approved flyers and advertisements distributed in the local 
community, which includes the UNC campus and the larger “Research Triangle” area, including Durham, 
Orange, and Chatham counties. No Protected Health Information (PHI) will be accessed prior to 
contacting participants. Potential subjects who respond to these advertisements will be contacted initially 
via telephone or encrypted email. If contacted via email, subjects will be scheduled for an initial 
telephone screening to determine eligibility status. Those who meet inclusion criteria based on the phone 
interview will be scheduled for all study sessions. We have completed data collection for a 4-5 session 
study( n=66) that includes multiple brain stimulation sessions with a drop out rate of only 4%. Moreover, 
that study included three groups similar to those proposed here but with additional exclusion criteria. 
Thus, we do not anticipate difficulties with either participant recruitment or retention. Participants are free 
to discontinue the experiment at any time. If a participant does not complete the entire experiment, we 
will recruit a replacement participant.  
 
Target Communities for Recruitment: UNC Chapel Hill, North Carolina Central University (an HBCU), 
Duke University, Durham, Carrboro, Hillsborough, Pittsboro, Apex, Morrisville, and Cary, NC.  
 
Retention  
We will promote retention by building rapport with study participants during study sessions, by 
maximizing ease of participation through flexible session scheduling and by fairly compensating 
participants for their time. Approaches used by our team in other studies will be used to maximize 
retention including bonuses for exceeding task performance targets and a study completion bonus. We 
will also obtain several types of contact information, including phone, email, and social media during the 
initial screening and at subsequent study sessions. Participants will be paid a bonus of $70 for 
completing all study sessions. Participants will also receive an email reminder from the study team 24 
hours prior to each scheduled session. Given that this study involves only three visits and that the 
protocols are minimally invasive and generally well-tolerated, we anticipate a high retention rate. 
6. Schedule of Activities and Procedures 
6.1. Table of Events 
Table 3. Schedule of activities and procedures 
Procedure 
Screening by 
phone 
Visit 
1 
Visit 
2 
Visit 
3 
Early 
Stop 
Informed consent 
X 
X 
X 
X 
 

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Recruit a Sample of 
Participants 
Eligibility assessments 
X 
X 
 
 
 
Enrollment 
 
X 
 
 
 
Methods 
Electroencephalography (EEG) 
 
 
 
X 
 
Transcranial alternating current stimulation 
(tACS) – real or sham 
 
 
 
X 
 
Magnetic resonance imaging (MRI) – MRS and 
resting state fMRI 
 
 
X 
X 
 
Safety 
Monitoring 
Review contraindications to procedures 
X 
X 
X 
X 
 
Medical history 
X 
X 
 
 
X 
Assessment of AEs 
 
X 
X 
X 
X 
Screening 
Labs 
Pregnancy test 
 
X 
X 
X 
X 
Urine drug test 
 
X 
X 
X 
X 
Breathalyzer 
 
X 
X 
X 
X 
Research 
Labs 
Dried Blood Spot collection 
 
X 
 
X 
 
Saliva collection 
 
X 
 
 
 
Urine collection 
 
X 
 
X 
 
Computerized Tasks  
HABIT training  
 
X 
 
 
  
HABIT testing 
 
 
X 
X 
  
REDCap Questionnaires 
 
X 
X 
 
 
6.2.   Screening 
Individuals who are interested in participating can contact the research team via email or phone. These 
individuals will complete an optional online prescreening (collected with REDCap) followed by a required 
phone screening. Individuals may also choose to complete only the phone screening. The online prescreening 
includes a short e-consent form, followed by questions concerning limited demographic information, 
pregnancy, medical history, history of alcohol use, and current alcohol and substance use. Individuals who 
appear to meet the eligibility criteria will be contacted via phone to complete the full phone screening, up to 14 
days prior to enrollment. The phone screening includes a brief description of study purpose and requirements, 
followed by collection of verbal consent. The phone screening includes questions pertaining to demographic 
information, pregnancy, medical history, medication use, contraindications to MRI or tACS, history of alcohol 
use, psychiatric symptoms, and current use of alcohol and other psychoactive substances. The phone 
screening will also involve scheduling for the first in-person session.  
The purpose of screening is to identify individuals who are members of the target population/s. 
6.3. Enrollment  
Eligibility will be determined using the screening process described in 6.2. A number of additional 
eligibility checks will also occur during session one. These include a color blindness test, a survey 
assessing health history, and administration of the MINI to rule out symptoms of psychosis. At the 
beginning of every session the participant will be asked to undergo an alcohol breath test and urine drug 
screen, and will be asked by the research team if there have been any changes to their health since their 
last session or phone screen. 
“Baseline” metrics will be collected throughout the study, as the tACS intervention will not occur until 
session three. A blood spot for baseline inflammatory markers will be collected during session one, a 
baseline fMRI scan will be collected during session two, and baseline resting-state EEG data will be 
collected immediately prior to stimulation during session three. 
6.4.   Study Visit Procedures 
 Visit 0 (Day –14 to Day –1): Eligibility screening 
• 
Explanation of study requirements 

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• 
Verbal consent to continue with screening 
• 
Eligibility questions 
• 
Scheduling the first session 
 
Visit 1 (Day 0) 
• 
Informed consent 
• 
Alcohol breath test + urine drug screening 
o Aliquoting of urine sample for future analysis 
• 
Ishihara color blindness test 
• 
MINI 
• 
Subject information sheet 
• 
Collection of saliva samples 
• 
Collection of blood spot 
• 
HABIT task training 
• 
Participant departure 
• 
Participant completes questionnaires at home prior to final session 
 
Visit 2 (Day 1 at earliest, Day 7 at latest) 
• 
Informed consent 
• 
Alcohol breath test + urine drug screening 
• 
HABIT practice 
• 
HABIT task 
• 
HABIT reversal task 
• 
Screening for metal prior to entering the magnet 
• 
MRI scan session 
• 
Participant departure 
 
Visit 3 (Day 2 at earliest, Day 14 at latest) 
• 
Informed consent 
• 
Alcohol breath test + urine screening 
• 
HABIT practice 
• 
HABIT task 
• 
Head measurements for tACS electrode placement 
• 
Placement of tACS electrodes 
• 
Placement of EEG net 
• 
Resting-state EEG  
• 
tACS stimulation during HABIT reversal task 
• 
Resting-state EEG 
• 
Removal of tACS electrodes + EEG net 
• 
Collection of blood spot 
• 
Screening for metal prior to entering the magnet 
• 
MRI scan session 
• 
Participant payment 
• 
Participant departure 
   

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6.5.  Final Visit 
At the conclusion of the final testing session, the purposes and predictions of the research will be clearly 
explained to the participants and any questions they have will be answered.       
6.6.   Phone Contacts  
Considering the complexity of activities, the study team will frequently communicate with participants to 
remind them about study visits. Study visit reminders will be sent once a day starting 2 days before the 
study visit via text messaging. 
6.7. Enrollees May Drop Out 
Participants may voluntarily withdraw from participation at any time, for any reason, with no penalty or 
loss of rights. The reasons for drop-out and missing data will be documented in the database.  
7. Statistical Analysis Plans 
7.1. Strategies that Apply to all the Aims 
All statistical procedures will be performed in SPSS, and in-house programs written in MATLAB, R, and 
Python. The PI, Dr. Charlotte Boettiger, currently serves at the biostatistics consultant for the NIAAA-
funded NADIA Consortium led by the Bowles Center for Alcohol Studies at the University of North 
Carolina at Chapel Hill. As such, she will oversee all data analyses. When needed, she will consult with 
statistical collaborators, including Dr. Hongtu Zhu, or Dr. Daniel Bauer. 
 
• 
To help ensure replicability of the research, the analysis plans have been finalized prior to collection 
of data (a priori). For each specific aim, the analysis plans specify detailed steps for obtaining 
estimates of the population parameters of interest (e.g., treatment effects) and for making inferences.   
• 
For each aim, sensitivity analyses will be performed to assess the robustness/fragility of the main 
results as indicated by their sensitivity to reasonable perturbations of the choices of the methods and 
assumptions used. Any question about the optimal choice of methods and assumptions are best 
handled by relegating competing approaches to roles in the domain of sensitivity analyses. Results of 
the sensitivity analyses will be used to guide our level of trust in the main results.  
• 
Human studies are prone to drop-out, missing data, and interval-censored values. The reasons for 
drop-outs, missing / censored data values, and protocol departures will be documented in the 
database.  Best practices for dealing with incomplete data will depend on the documented causes of 
those occurrences.  In the analysis plans established a priori, the strategies for coping with 
incomplete data will be based on anticipated causes. Alternative methods for dealing with incomplete 
data will play important roles in the sensitivity analyses.  
• 
The analyses for each aim will focus on the magnitude and direction of point- and interval-estimates 
of the population parameters of interest. To indicate precision, all statistical estimates of population 
parameters will be tabulated along with corresponding confidence intervals (CI). The CI will be 
interpreted as the set of potential values of the population parameter that are most compatible with 
the observed data. 
• 
All hypothesis tests yielding large p-values will be reported as being inconclusive. For all sample 
sizes, all hypothesis test procedures are (by design) incapable of establishing that the null hypothesis 
is true.  
• 
If p-values are computed they will be reported to several decimal places without categorizing or 
dichotomizing the p-value; that is, the words “significant” and “non-significant” should be avoided.  
The p-value should be reported and interpreted as a continuous measure indicating the availability of 
information against the (null) hypothesis being tested.  

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• 
The proposed statistical analysis strategy acknowledges that no p-value can reveal the plausibility, 
presence, truth, or importance of an association or effect –which is consistent with the statements of 
the American Statistical Association [4,5], the recommendations in Nature [6,7], and guidance, such 
as the CONSORT Statement [1], STROBE Statement [2], and ICMJE guidance [3].†] 
• 
The analysis plans will include outcome-dependent exploratory analyses to generate new 
hypotheses. 
• 
Graphical methods such as forest plots will be used to visualize the analysis results.   
• 
Possible study limitations are that this is a relatively small sample, and the experiments include 
numerous variables. As such, our analyses may prove underpowered to detect significant effects, 
and null findings should not be interpreted as definitive. 
7.2.   Statistical Design 
Our study design consists of three groups of participants: 1 (CON): no adolescent binge drinking (AIE) 
and current (past month) World Health Organization (WHO) risk drinking levels of either 0 or 1 (abstinent 
or low-risk); 2 (RISK): no AIE and current WHO level 2, 3, or 4 (medium risk, high risk, or very high risk); 
and 3 (RISK+AIE): history of adolescent binge drinking and current WHO risk level 2, 3, or 4. Within 
each group, participants will be randomly assigned to either the 10Hz tACS (stim) group or the sham 
tACS (sham) group. Effects of tACS on habitual action selection in the HABIT task (primary outcome, 
Aim 2) will be assessed based on the proportion of perseverative errors (relative to all errors) when the 
participant must replace a highly trained response to a set of familiar stimuli. We will use a general linear 
model that includes group and stimulation condition as between-subjects factors; we will also include 
baseline perseverative error, age, sex, handedness, and anxiety or mood disorder diagnoses as 
covariates.  
 
We will enroll 66 participants, divided into three groups of 22, who will be randomly assigned within group 
to condition (stim vs. sham, n= 11 each), each of which will be divided into cohorts, based on best 
practices.  
 
Our study design requires randomly assigning individuals to a stimulation group after completing Session 
1. Dr. Boettiger will provide assignment to the study team based on a predefined randomization table in 
order to ensure balanced sex and group in each stimulation arm.  
 
To test the prediction that E/I balance indices will differ from low-risk drinkers more among high-risk 
drinkers with adolescent binge history, we will analyze the baseline (i.e. pre-tACS) data collected in 
Sessions 2 and 3. We will conduct Analyses of Covariance (ANCOVA) to compare groups in terms of 
Session 2 (baseline) perseverative errors in the HABIT Test and MRS-derived GABA:Glx, and Session 3 
baseline 1/f EEG slope, controlling for age, sex, education, psychiatric diagnoses, and medication status. 
We may also consider using data from multiple timepoints within the HABIT Test session, for which we 
would either employ a mixed model ANCOVA or multilevel modeling, as we have used in our prior 
studies.  
 
For Aim 2, we will test for effects of tACS (true vs. sham) on habitual action selection in the HABIT task 
(primary outcome, Aim 2) based on the proportion of perseverative errors (relative to all errors) when the 
participant must replace a highly trained response to a set of familiar stimuli. We will use a general linear 
model that includes group and stimulation condition as between-subjects factors; we will also include 
baseline perseverative error, age, sex, handedness, and anxiety or mood disorder diagnoses as 
 
†[1] www.consort-statement.org   ,  [2] www.strobe-statement.org,   [3] www.icmje.org  
  [4]  Wasserstein RL, et al. (2016), The ASA's Statement on p-Values, The American Statistician, 70:2, 129-133.  
  [5] Wasserstein RL, et al. (2019), Moving to a World Beyond p < 0.05, The American Statistician, 73:sup1, 1-19.  
  [6] Amrhein,  et al. (2019),  Scientists rise up against statistical significance, Nature 567, 305-307.  
  [7] Editorial (2019) It’s time to talk about ditching statistical significance: … Nature 567, 283-283. 
 

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covariates. For our mediation analyses, change in E/I balance measures (GABA:Glx and 1/f EEG slope) 
analyses will be used as the mediating variables; change in HABIT perseverative errors will serve as the 
dependent variable and will be adjusted for sex, age, handedness, and anxiety or mood disorder 
diagnoses as covariates; tACS condition (stim/sham) will serve as the independent variable; and AIE and 
current alcohol misuse scores will be included as moderators.   
 
For Aim 3 we will test whether the indirect effect of tACS on HABIT perseverative error is mediated by 
resting-state functional connectivity. For our mediation analyses, functional connectivity (FC) values 
derived from a priori ROI-to-ROI (e.g., dlPFC, aIC, and limbic striatum) analyses will be used as the 
mediating variable; change in HABIT perseverative errors will serve as the dependent variable and will 
be adjusted for sex, age, handedness, and anxiety or mood disorder diagnoses as covariates; tACS 
condition (stim/sham) will serve as the independent variable; and AIE and current alcohol misuse scores 
will be included as moderators. We will use CONN for initial FC analysis of fMRI data. We will also 
conduct exploratory analyses testing for associations between indices of E/I balance in the dlPFC and 
dlPFC FC at baseline, and whether changes in E/I balance indices associate with changes in dlPFC FC. 
Significance testing will be based on a bootstrapping procedure and 95% CI, with FDR-correction 
(q=0.05) for multiple ROI-ROI connections. Additional ROI-to-ROI and seed-to-voxel exploratory 
analyses will also be conducted with these data in collaboration with other components.  
 
Secondary outcomes: We will use similar experimental designs to those described to evaluate a 
number of secondary outcomes. These include C-reactive protein measured from blood spots and other 
immune activation markers (TNF-α and IL-1β) derived from salivary microvesicles, as well as 
exploratory/alternative outcome analyses. The latter include effects of tACS on GABA:Glx ratios in the 
dlPFC and aIC, and on EEG 1/f slope. 
8. Sample Size Rationale 
We will enroll 66 participants, divided into three groups of 22, who will be randomly assigned within group 
to condition (stim vs. sham, n= 11 each), each of which will be divided into cohorts, based on best 
practices. Based on Cohen’s method for calculating power of a linear model, as implemented in G*Power 
3.1, in order to detect a large effect size (f = 0.4), n=64 will give us 80.3% power to detect an interaction 
between tACS and group on habitual responding (primary outcome for Aim 2) at an alpha value of .05. 
Our prior study found a moderately large effect size (Cohen’s f = 0.21) in a repeated measure design 
with a large nuisance effect of session. By changing to a between subjects design and eliminating 
session effects, we expect a large effect size in this study. Thus, the needed total sample to recruit is 64 
participants. We propose collecting n=66 to account for possible attrition or data loss, and so that each 
participant subgroup contains the same number of stim/sham participants (11/11).  
 
To test the prediction that E/I balance indices will differ from low-risk drinkers more among high-risk 
drinkers with adolescent binge history, we will analyze the baseline (i.e. pre-tACS) data collected in 
Sessions 2 and 3. We will conduct Analyses of Covariance (ANCOVA) to compare groups in terms of 
Session 2 (baseline) perseverative errors in the HABIT Test and MRS-derived GABA:Glx, and Session 3 
baseline 1/f EEG slope, controlling for age, sex, education, psychiatric diagnoses, and medication status. 
A power analysis in G*Power 3.1 indicates that a sample size of 64 will allow 80.1% power to detect 
moderately large group differences (Cohen’s f=0.4; alpha=0.05). Thus, our proposed sample size of 66 
participants will also be adequate for testing the hypotheses of Aim 1. We may also consider using data 
from multiple timepoints within the HABIT Test session, for which we would either employ a mixed model 
ANCOVA or multilevel modeling, as we have used in our prior studies.  
 
For Aim 3 we will test whether the indirect effect of tACS on HABIT perseverative error is mediated by 
resting-state functional connectivity. Our previous study using a similar statistical design to evaluate brain 
functional connectivity measures that mediate the effect of dopamine depletion on a measure of 
behavioral flexibility detected statistically significant effects with a smaller sample size (n=34).  

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9. Data Collection and Management 
 
Questionnaire Data Collection: The study data will be entered into a REDCap database developed by 
the study personnel.  REDCap is a 21 CFR Part 11-compliant data capture system provided by the NC 
TraCS Institute at UNC. The data system includes password protection and internal quality checks, such 
as automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate.  Study data 
will be entered directly by participants or from the source documents by study personnel.  
 
Behavioral Task Data Collection: Behavioral task data will be collected electronically and will be stored 
on a password-protected lab server only accessible by IRB-approved study personnel.  
 
MRI Data Collection: MRI data will exist in digital form on computer disk. These data will be collected at 
the UNC Biomedical Research Imaging Center (BRIC) and be stored securely on UNC’s longleaf secure 
server.  
 
EEG Data Collection: EEG Data will be collected digitally using Net Station software installed on a 
computer in Howell Hall. This data will be stored on a password-protected lab server only accessible by 
members of the IRB study team.  
 
Data Quality and Monitoring: All data are collected by trained bachelor’s-level or Master’s level study 
staff. Data quality and safety will be monitored by the PI and research team members. Any issues will be 
discussed with the study PI. The study PI assumes responsibility for ensuring study staff is complying with 
the investigational plan and IRB regulations, as well as ensuring any changes to the protocol have received 
IRB approval and have been reported to the sponsor, and that accurate, complete, and timely reports are 
made to the IRB. A weekly study team meeting will be conducted to discuss research procedures.  
 
• 
All neuroimaging (MRI or EEG) data will be recorded in the database using alphanumeric identifiers 
that are unrelated to any personal identifying information about the participant. Neuroimaging data 
are coded by subject number only on password-protected secure servers located at the BRIC or 
UNC’s longleaf secure server. 
   
• 
All sources of electronic data (i.e., data collected via UNC REDCap) are saved on a password-
protected file on a secure UNC server, of which only the research team members will have access.   
 
• 
All sources of non-electronic research data will be coded by a unique subject number and stored in 
a dedicated locked file cabinet. Materials identifying participants by name (e.g., signed consent 
forms) will be stored in a separate locked file cabinet. The computer file matching participant names 
with code numbers will be saved in a double password-protected file on a secure UNC server 
directory only accessible by the PI and key research team. All data will be entered into the dataset 
using the subject identification number only. Participants are not identified by name in any analysis 
of these data, or any presentation or publication resulting from the analysis of these data. 
 
10. Collection and Management of Tissue Specimens 
10.1. Use in Current and Future Studies 
Three types of biological samples will be collected for use in this study: saliva samples for DNA/RNA 
analysis, blood spots, and urine samples.  

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Saliva samples: During session one, two samples of approximately 2mL saliva will be self-collected by 
participants with researcher instruction. These samples will be marked with the participant’s study ID 
number (not identifiable) and stored at –80 degrees Celsius in a secure research freezer in Howell Hall.  
Urine samples: At the beginning of each session participants will self-administer a urine drug screen with 
researcher instruction. After session one only, a member of the research team will extract a small volume 
of urine from this sample to be kept for later analysis. Each urine sample will be marked with the 
participant’s study ID number and stored at –80 degrees Celsius in a secure research freezer in Howell 
Hall. The urine samples will be shipped to a collaborator at the UNC Nutrition Research Institute to be 
analyzed for the presence of certain metabolites. 
Blood spots: Blood spots will be collected by a trained researcher during sessions one and three. The 
researcher will sanitize the participant’s chosen finger, apply a finger prick, and extract a small amount of 
blood. The blood spots will be marked with the participant’s study ID number and session number and 
stored at –80 degrees Celsius in a secure research freezer in Howell Hall. These samples will be 
analyzed in-house by our research team to quantify inflammatory markers in the blood. 
The samples collected during this study will be analyzed for this study and will be stored for possible use 
in future research studies. The possibility of future use by our study team or others is included in our 
consent form, and will be explained to participants during the informed consent process during session 
one. 
10.2. Sample Preparation 
Saliva samples: The saliva samples can be kept stable for multiple years when stored at subzero 
temperatures in order to ensure sample integrity. 
Urine samples: These samples will be stored at subzero temperatures in order to maximize sample 
integrity over time. A researcher will transport these samples to the lab freezer within 2 hours of 
collection. These samples will then be driven by a member of the study team or shipped to our 
collaborator in Kannapolis, NC. 
Blood spots: These samples will be stored at subzero temperatures in order to maximize sample integrity 
over time. After collection the samples will be left to dry at room temperature, and then a researcher will 
transport these samples to the lab freezer promptly.  
10.3. Record Keeping and Monitoring 
A record of samples collected will be kept and continuously updated. A spreadsheet listing samples 
collected for each participant will be kept according to study ID number. This spreadsheet will be kept on  
a protected server only accessible to the research team and will contain no identifying information. 
Researchers will update this spreadsheet after each session to ensure fidelity in record keeping. 
10.4. Storage and Security 
As described in 10.1, all samples will be kept in a dedicated research freezer located in Howell Hall. The 
freezer itself is designed to constantly monitor internal temperature and door openings. In the event of a 
drop in temperature, an alarm sounds and the PI is notified. All samples taken during this study will be 
de-identified from the time of collection. 

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11. Safety Monitoring and Management 
11.1. Risk / Benefit Assessment 
Potential Risks:  
• 
Cognitive Testing There are no known risks for injury associated with the 
neuropsychological/behavioral components of these cognitive studies. Other possible risks 
include frustration, boredom, or fatigue. Moreover, answering detailed questionnaires and 
participating in computerized cognitive tasks may make subjects anxious or uncomfortable. 
Based on our previous experience, it is unlikely that participants will find these potential 
psychological risks extreme enough to end their participation.  
• 
Questionnaire Data Collection Answering questionnaires may make subjects uncomfortable and 
could cause emotional distress.  
• 
Blood sample collection via finger stick: The risks of drawing blood via a lancet finger stick include 
temporary discomfort from the stick, bruising, and rarely, infection.  
• 
Functional MRI Studies Potential minor risks and discomforts are associated with MRI acquisition. 
No known health risks are associated with these types of MRI studies, although the MRI makes 
loud noises as it acquires data, which could affect a participant’s hearing. The only significant risk 
associated with MRI is the presence of ferromagnetic materials. This is a noninvasive technique 
involving no catheterizations or injection of exogenous tracers. A great many participants have 
now undergone magnetic resonance studies without apparent harmful consequences. 
Radiofrequency power levels and gradient switching times used in these studies are within the 
FDA approved ranges. Subjects must lie still in the MRI scanner for approximately 90 minutes, 
which subjects may find uncomfortable. Some people may become claustrophobic while inside 
the magnet, and will be pulled out of the scanner to end testing If this should occur. A relative 
contraindication to MRI studies is pregnancy, as the risk of MRI to fetuses is unknown.  
• 
Electroencephalography (EEG) Risks of EEG methods are considered minimal. Potential risks 
include discomfort during the placement of the cap or boredom during the EEG recording.  
• 
Transcranial Alternating Current Stimulation (tACS) tACS is a non-invasive brain stimulation 
technique in which a weak electrical current is applied to the scalp. Our tACS protocol involves 
the application of three surface electrodes, one serving as the cathode and the other two as 
anodes. The current flows from the anodes to the cathode, with some current passing along the 
scalp and some passing through the brain. The current passing through the brain produces small 
changes in the excitability of the brain regions falling within the current flow. Minimal risks are 
associated with tACS. tACS may cause the onset of a mild headache of short duration. The 
device could cause discomfort or pain to the head. The use of conductive gel to affix the 
electrodes to the skin may cause skin irritation and dryness. Stimulation devices may cause 
muscle twitching in hands or face that can be uncomfortable.  
• 
Confidentiality Information collected for the purpose of this research study will be kept confidential 
as required by law. All copies of testing records and results will be kept in locked filing cabinets, in 
locked offices, or in password-protected computer files. All information will be accessible only to 
authorized personnel. Alphanumeric codes will be used on all data sheets and data files in place 
of names. No subjects will be individually identified in any report or publication about this study. 
No personal identifiers will be associated with any study data. Screening information from 
participants will be kept in a locked filing cabinet along with their consent forms. The information 
collected during the initial telephone screening is not retained.  
• 
Privacy Telephone interviews will be conducted only in the Boettiger laboratory or office space. 
All laboratory staff have undergone the CITI ethics training for conduct of human subjects 
research. All neuropsychological and behavioral testing will occur in private testing rooms. All 
virtual training will require a meeting password and subjects will remain in waiting room until entry 

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is accepted by teacher. No mailed or emailed materials or messages will include subject specific 
data.  
 
Potential Benefits: Subjects will not experience any direct benefit from participating in these 
studies. We anticipate that the results of these studies will improve our understanding of behavioral 
inflexibility in people who engage in high risk drinking. Eventually this knowledge could lead to 
differentiated treatment options for high risk drinkers with a history of adolescent binge drinking. 
Participants may benefit psychologically from knowing that they are participating in clinically 
motivated research.  
 
11.2. Safety Monitoring 
We propose testing the efficacy of a single session of 10 Hz transcranial alternating current stimulation 
(tACS) on behavioral flexibility, brain mediators of behavioral effects, and inflammatory markers. 
Participants will be healthy adults ages 22-50 years in one of three groups: low risk drinkers (WHO level 
0 or 1) with no adolescent binge history, and adults who currently engage in hazardous alcohol use 
(WHO level 2, 3, or 4) with or without a history of adolescent binge drinking. Our participants are not 
considered to belong to a vulnerable population. Completion of computerized behavioral tasks, tACS, 
and MRI scanning is minimal risk. Therefore, the data and safety monitoring plan (DSMP) does not 
include a Data Safety Monitoring Board (DSMB), consistent with NIAAA policy: “Phase III clinical trials 
must have an independent DSMB. Phase I and II studies that either involve multiple clinical sites, 
use high risk interventions, or involve vulnerable research participants may require a DSMB at 
the discretion of NIAAA.” (from https://www.niaaa.nih.gov/research/guidelines-and-resources/data-and-
safety-monitoring-guidelines, accessed 6/4/2021).  
 
The primary risks anticipated for the proposed study are psychological effects and loss of confidentiality 
or privacy. As detailed in the Confidentiality and Privacy section, comprehensive measures will be 
implemented to protect participants’ data and anonymity.  
 
The proposed research poses minimal risk. In the unlikely event of a Serious Unanticipated Adverse 
Event (AE) during the behavioral testing, tACS, or brain imaging session, study personnel will contact 
campus and local emergency resources and the PI will report the event to the University of North 
Carolina, Chapel Hill IRB. In addition, for all MRI scans, at least two trained individuals will always be 
present in the MRI control room, one of whom will be a trained MRI technician, and a defibrillator/monitor 
is available on-site in case of emergency.  
 
Reporting Procedure for Serious Unanticipated Adverse Events  
In the event of an unanticipated serious adverse event, such as hospitalization of a subject due to study 
enrollment, the PI will ensure that these events are reported to the UNC-CH IRB within 24 hours by 
phone, fax, and/or email, and we will submit a written report no more than two days later. The project 
staff will also use the following reporting procedures:  
 
1. When the study staff and/or PI become aware of a serious adverse event, reporting requirements must 
be implemented in a timely manner.  
2. Dr. Boettiger will complete an “Adverse Event Reporting Form” and submit the form to the UNC-CH 
IRB.  
3. The UNC-CH IRB, with the input of the PI, will review the study protocol and determine what further 
action, if any, to take based on the best interests of the participants.  
 
Suicidality 
In addition to possible AEs from study procedures, the use of the MINI screening interview could lead to 
disclosure of participants’ suicidal ideation which would require further action. Unfortunately, no 
measures are available to determine in a reliable and valid way whether someone is likely to engage in 

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life-threatening behavior within 48 hours. Thus, for the past 15 years, we have followed a protocol that 
allows us to identify and intervene in a cautious and safe manner. This protocol is based on that 
successfully used by Dr. Mitchell Prinstein, within our department. Dr. Prinstein is a licensed Clinical 
Psychologist specializing in the area of depression and self-injury in youth. This protocol is described 
below. 
 
Step 1: Screening with MINI: Any participant endorsing a current state of suicidality is flagged and 
brought to the attention of the PI, Dr. Charlotte Boettiger.   
 
Step 2: Contact participants: We intervene with cases determined to be at potential imminent risk 
by contacting participants.  We do not have a therapeutic relationship with the participants, and our 
measures are research, NOT clinical instruments and cannot be used to detect psychological processes 
with absolute certainty. We therefore cannot be sure that there is reason for major concern, but we would 
rather be safe and alert participants about our concerns. We will explain that the responses made on 
MINI are quite rare, and express our concern that this might indicate a risk of suicide. Dr. Boettiger will 
then help them determine how to get a psychological evaluation, and encourage them to do so.  These 
options include:   
 
1. The UNC Department of Psychology Community Clinic, which offers sliding scale treatment to 
anyone in the community (919) 962-6906.  
2. For University of North Carolina students: The UNC Counseling and Psychological Services, 
CAPS, Monday-Friday, from 8am-5pm. Their phone number is 919-966-3658. After hours call the 
same number to speak to ProtoCall service. For medical services for UNC students, HealthLink 
can be accessed 24/7 at 919-966-2218.  
3. The national suicide hotline at 988, available 24/7, or the suicide text line by texting HOME to 
741741. 
 
4. Escort to the local emergency room. 
 
Oversight  
The PI is responsible for the general oversight for all study activities and will inform the Program Officer 
about changes and requirements for the DSMP. Yearly review of the DSMP will be conducting during the 
regular continuing review process, outlining the following points:  
 
• Reassessment of the risks and benefits to study participants  
• Participant recruitment, accrual, and retention  
• Data quality and confidentiality  
• Consideration of external scientific or therapeutic developments with impact on the safety of 
participants or the ethics of the study  
• Review any adverse events 
 
 The PI will update the general DSMP procedures as needed. 
 
11.3. Adverse Events and Serious Adverse Events Reporting Procedure 
 
See 11.2 for reporting procedure. 
 
Adverse events of any type seem highly unlikely for this study given the safety profile of the intervention 
being used. We will define adverse events as any medical issues arising after study enrollment. We will 

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define serious adverse events as any medical issues arising after study enrollment that require 
hospitalization. Participants will be asked by the research team at the beginning of each session whether 
they have had any notable changes to their medical record. If a participant reports an adverse event, it 
will be recorded by the research team and reported to the study PI within 24 hours. The study PI will 
determine whether they believe the adverse event was related to study activities and data collection will 
proceed accordingly. If a serious adverse event occurs, data collection for all participants will be 
suspended temporarily until the study PI, the study medical advisor, and the UNC-CH IRB determine 
resuming sessions is safe for participants. 
12. Regulatory, Ethical, and Study Oversight 
Specifications 
12.1. Informed Consent Process 
 
Individuals interested in participating will be asked to sign an online consent form before filling out the 
optional RedCap pre-screening form. Potential participants, whether or not they have previously 
completed the optional online pre-screening form, will be asked to provide verbal consent to participate in 
a phone screening to determine eligibility. Acquisition of verbal consent will only occur after a member of 
the research team has described the study and given an overview of the screening questions to follow. 
Enrolled participants will be asked to sign an informed consent form at the beginning of every session. 
During the first session a member of the research team will review the terms of the consent form with the 
participant and will confirm the participant’s full understanding before obtaining consent.  
 
 
All subjects will participate in the informed consent process. Participants will be familiarized with the 
protocol by the Dr. Boettiger or qualified study personnel, including its risks and benefits, and informed 
consent will be documented according to the regulations governing human subject research at the 
University of North Carolina, Chapel Hill, which meet the standards of the NIH. A copy of the informed 
consent document will be given to the participants for their records. The informed consent process will be 
conducted, and the form signed, before the participant undergoes any study-specific procedures. 
 
Recruiting Participants will be recruited through IRB-approved flyers and advertisements distributed in 
the local community, as well as through emails sent to surrounding college campus communities. No 
Protected Health Information (PHI) will be accessed prior to contacting participants. Potential subjects 
who respond to these advertisements will be contacted initially via telephone or email. If contacted via 
email, subjects will be called for an initial telephone screening to determine whether the subject meets 
our inclusion criteria. Those meeting criteria based on the phone interview will be scheduled to come to 
the lab to participate in the study.  
 
Inclusion of Women and Minorities Every attempt will be made to recruit women and minorities, including 
posting advertisements at the Sonja Haynes Stone Center for Black Culture and History; the Lesbian 
Gay Bisexual, Transgender Office; the Student Union; the Carolina Women’s Center; and the Medical 
Campus buildings.  
 
Initial Telephone Screening Potential participants are first screened via telephone for eligibility. This 
process includes a complete explanation of the protocol procedures. At that time, a potential participant 
can decline to participate.  
 
Obtaining Informed Consent Upon arrival to participate in Session 1, participants will read and have 
explained to them the informed consent document. Informed consent is obtained in a comfortable, 
private area in the Boettiger lab. Participants’ questions about the protocol will be thoroughly answered 
prior to obtaining consent, with no time limit to this procedure. Upon consent, the subject is free to 

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withdraw that consent at any time, which will be emphasized to the subject. Consent for behavioral 
testing and imaging will be obtained by approved study staff.  
 
Waiver of written consent for telephone pre-screening We will obtain a waiver of written consent for the 
initial telephone screening. This request derives from our need to conduct a health and medical history 
telephone screen in order to ensure that individuals who respond to our IRB approved recruitment efforts 
are eligible to participate in our research project. The health screen is preparatory to our research and its 
sole purpose is to ensure that participants meet our inclusion/exclusion criteria.  
 
A screening procedure that is efficient for both volunteers and staff members is necessary. These studies 
have strict inclusion/exclusion criteria and the telephone screen efficiently provides the study staff with 
information needed to determine eligibility. To require potential study participants to come to our lab for a 
qualifying screen that can be done over the telephone would place an unnecessary burden on potential 
participants, as well as on study staff. In addition to the health screen, we have created a phone script 
that includes elements of informed consent and explains to potential participants how their privacy will be 
protected. The potential subject is informed that they may stop the phone conversation at any time. The 
health and medical information documented during the telephone screen is not disclosed to other 
investigators. Screens from participants who are excluded are destroyed. Screens from participants who 
are included are kept in a locked filing cabinet along with their consent form and other screening 
information. 
 
12.2. Confidentiality and Privacy 
Confidentiality  
Information collected for the purpose of this research study will be kept confidential as required by law. 
All copies of testing records and results will be kept in locked filing cabinets, in locked offices, or in 
password-protected computer files. All information will be accessible only to authorized personnel. 
Alphanumeric codes will be used on all data sheets and data files in place of names. No subjects will be 
individually identified in any report or publication about this study. No personal identifiers will be 
associated with any study data. Screening information from participants will be kept in a locked filing 
cabinet along with their consent forms. The information collected during the initial telephone screening is 
not retained. 
 
Privacy  
Telephone interviews will be conducted only in the Boettiger laboratory or office space. All laboratory 
staff have undergone the CITI ethics training for conduct of human subjects research. All 
neuropsychological and behavioral testing will occur in private testing rooms. All virtual training will 
require a meeting password and subjects will remain in waiting room until entry is accepted by teacher. 
No mailed or emailed materials or messages will include subject specific data.  
 
 All participant data will be de-identified at the time it is stored on computing resources, such that only 
unique participant ID codes will associate the names of individuals to corresponding data. Access to the 
file linking these two sources of information will be maintained on a secure server that is separate from 
the one housing the actual MRI, biochemical, and behavioral performance data. All behavioral 
performance data will be stored on computers in locked rooms, password protected, and will be backed 
up to a secondary, secured location, all behind the firewall of the University of North Carolina, Chapel 
Hill. All MRI data will be uploaded to secure data servers, access to which will be restricted to personnel 
involved in the administration of the proposed studies.  
 
Shortly after data upload, at least one member of the study team will perform an initial quality check to 
ensure that all files are intact and that there were no errors in transfer. All analyses will be conducted 
when the targeted sample size (n=66) is obtained. The conduct of research according to the documented 
study protocol will be monitored by at least two individuals from the study team, including one senior/key 

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personnel (Dr. Boettiger). All data analyses will also be quality checked by at least two individuals from 
the study team. 
 
12.2.1. 
Certificate of Confidentiality 
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
12.3. Future Use of Stored Data 
Data collected for this study will be analyzed and stored at UNC Chapel Hill (UNCCH). As required by 
NIAAA, the de-identified, archived data will be transmitted to and stored at the NIAAADA (see below) for 
use by other researchers including those outside of the study. Permission to transmit data to the NDA 
should be included in the informed consent.  
     When the study is completed, access to study data will be provided through the NIAAADA. 
 
12.4. Key Roles and Study Governance 
Principal Investigator 
 
Medical Advisor 
Charlotte A. Boettiger, PhD, Professor, 
Psychology & Neuroscience 
 
Jeffrey W. Cooney, MD, Assistant Professor, 
Neurology 
UNC Chapel Hill  
 
Duke University School of Medicine 
919-907-0585 
 
919-668-2498 
cab@unc.edu 
 
jeffrey.cooney@duke.edu 
 
Co-Lead Investigator Graduate Student 
 
Co-Lead Investigator Graduate Student 
Jillian Battista, PhD Student, Behavioral & 
Integrative Neuroscience 
 
Grace Elliott, PhD Student, Behavioral & 
Integrative Neuroscience 
UNC Chapel Hill 
 
UNC Chapel Hill 
919-843-9193 
 
919-843-9193 
jillian.battista@unc.edu 
 
gelliott@unc.edu 
 
Statistical Co-Investigator 
Charlotte A. Boettiger, PhD, Professor, Psychology & Neuroscience 
UNC Chapel Hill  
919-907-0585 
cab@unc.edu 
 
Dr. Boettiger currently serves as the Statistical Consultant for the U24 Neurobiology of Adolescent 
Drinking in Adulthood (NADIA) Consortium. 
12.5. Publication and Data Sharing Plan  
DISSEMINATION PLAN 

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We will comply with the NIH policy on dissemination of NIH-funded clinical trial information by registering 
the details of this study on ClinicalTrials.gov no later than 21 days after the start of data collection. We 
will adhere to all reporting requirements, including posting a summary of results and outcomes within a 
year after the completion of data collection. Plans for disseminating results and the timeline for doing so 
will be included in the informed consent documents for this study. The UNC Office of Sponsored 
Research will ensure compliance for registering and reporting as part of their post-award monitoring. 
We will also present our findings at relevant scientific conferences, including that of the Research Society 
on Alcoholism and will submit the results of our planned analyses for publication in peer-reviewed 
journals.  
NIAAA Data Archive (NIAAADA) Data Sharing Plan (DSP) 
Per NOT-AA-22-011, this study will submit and share data with NIAAA Data Archive (NIAAADA), a data 
repository housed within the NIMH Data Archive (NDA). To this end, this study will fulfill the following 
summary of tasks and expectations: 
Common to all studies: 
• 
Obtain Informed Consent that allows for broad sharing of the research subjects’  de-
identified data and/or approval of the Institutional Review Board, consistent with the Data 
Submission Agreement (DSA) 
• 
Collect Personally Identifiable Information (PII) from research subjects that will allow for 
the creation of the NDA GUID (a de-identified subject ID). 
• 
Create an NDA Account with access to the GUID Tool. 
• 
Complete and submit a Data Submission Agreement (DSA) within 6 months of the 
Notice of Award (NoA) issue date. 
• 
Create a list of data items to be collected in the project in the Data Expected tab of the 
NDA Collection within 6 months of NoA issue date. 
• 
Create a GUID for each research subject using the NDA GUID tool. 
• 
Format and submit data according to the NDA  Data Dictionary and/or work with 
NDA staff to define an appropriate data structure, if one does not already exist. 
• 
Submit data on or before the NDA submission due dates (April 1 and October 1 each 
year) in accordance with the applicable Data Sharing Terms and Conditions of award. 
• 
Perform QA/QC checks on data within 4 months after the submission due dates and address 
any issues identified by the NDA. 
• 
Share data in accordance with the default NIAAADA data sharing schedule in accordance 
with the applicable Data Sharing Terms and Conditions of award. 
• 
Upload supporting documents that allow future analysts to effectively use the data, 
including: study protocols, assessment schedules, operating procedures, original 
assessment instruments (if not proprietary), and analytic/statistical algorithms used to 
derive variables in publications. 
• 
Create an NDA Study, linking relevant publications to submitted data at the time of 
publication, and use the appropriate Digital Object Identifier (DOI) and Acknowledgement 
template in these publications. 
• 
Submit a statement of progress on data sharing in non-competing renewals and progress 
reports. Further instructions are available in the RPPR Instruction guide under section 
C.5.b. Resource Sharing. Refer to specific Tutorials about data submission and sharing via 
the NDA or refer to the data sharing planning section where the data will be shared. 
 
Study-specific details: 
Brief summary of the assessment schedule for data that will eventually be shared with the NIAAADA: 
 
In this 3-visit study, we will first determine eligibility at the screening and randomization visit; dried 
blood spot measures of inflammatory markers will also be collected. In visit 2, we will measure 

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baseline habitual action selection in the HABIT, and collect structural MRI, baseline resting-state 
fMRI, and single-voxel MRS measuring GABA and glutatmate/glutamine. At visit 3, we will collect 
resting-state EEG before and after 10Hz- or sham-tACS, as well as habitual action selection in the 
HABIT during 10Hz- or sham-tACS, as well as repeat structural and functional MRI and MRS, and 
dried blood spot collection following 10Hz- or sham-tACS. Demographic, psychometric, and 
substance-related information will be collected via REDCap at the participant’s convenience. 
 
Listing of proposed data collection instruments (assessments) that do not currently exist in the NDA: 
1. Alcohol Related Blackout Questionnaire  
2. Alcohol-Induced Blackout Measure-2 
3. Munich Parasomnia Screening 
4. Alcohol Use Questionnaire [Q10-12] 
5. Carolina Alcohol Use Pattern Questionnaire (version 2) 
6. Value-Driven Attention Questionnaire 
7. Hidden Association Between Images Task (HABIT) 
 
Proposed schedule for running the data validation tool once data collection begins:  
The PI (Boettiger) has established an NIMH Data Archive (NDA) account. Data will be uploaded to 
the NDA by the deadlines (June 1; Dec 1) and will be subject to quality control checks by the 
investigators within 4 months (Oct 1; Apr 1) of submission to the NDA. Upon notice of award, Data 
Expected will be submitted to the NDA, and will include (i) a data dictionary, (ii) targeted enrollment, 
and (iii) anticipated data submission and sharing dates. For each manuscript based on findings from 
this project, investigators will submit to the NIAAADA: data, list of variables, analytic plan, and results. 
We will determine who will have access to which aspects of the project data based on NIH’s data 
access principles. We will provide access to person-level data for research purposes only, based on 
Data Use Certifications. Data on UNC servers will be made available to investigators with appropriate 
IRB approval and Data Use Agreements. 
13. References 
 
Boettiger, C. A. (2005). Frontal Networks for Learning and Executing Arbitrary Stimulus-Response 
Associations. Journal of Neuroscience, 25(10), 2723-2732. 
https://doi.org/10.1523/jneurosci.3697-04.2005  
Brosnan, M. B., & Wiegand, I. (2017). The Dorsolateral Prefrontal Cortex, a Dynamic Cortical Area to 
Enhance Top-Down Attentional Control. The Journal of Neuroscience, 37(13), 3445-3446. 
https://doi.org/10.1523/jneurosci.0136-17.2017  
Brunye, T., Elliott, G., & Loverro, K. (2020). Examining state-dependent effects of transcranial direct 
current stimulation on visual search and executive function tasks. NeuroReport, 32(1).  
CDC. (2012). Vital signs: binge drinking prevalence, frequency, and intensity among adults - United 
States, 2010. Morbidity and mortality weekly report.  
Cohen, E., Feinn, R., Arias, A., & Kranzler, H. R. (2007). Alcohol treatment utilization: findings from the 
National Epidemiologic Survey on Alcohol and Related Conditions. Drug and Alcohol 
Dependence, 86, 214-221.  
Crews, F. T., Vetreno, R. P., Broadwater, M. A., & Robinson, D. L. (2016). Adolescent Alcohol Exposure 
Persistently Impacts Adult Neurobiology and Behavior. Pharmacological Reviews, 68(4), 1074-
1109. https://doi.org/10.1124/pr.115.012138  
Davies, M. (2003). The role of GABAA receptors in mediating the effects of alcohol in the central nervous 
system. J Psychiatry Neurosci, 28(4), 263-274.  

<FLAME Study> 
  
 
Protocol Number <23-1702> 
 
<02 October 2024> 
Interventional or Observational Protocol                                                                                                                                                                                 Page   34 
Template Version  1.0   2021-09-09 
Degenhardt, L., O'Loughlin, C., Swift, W., Romaniuk, H., Carlin, J., Coffey, C., Hall, W., & Patton, G. 
(2013). The persistence of adolescent binge drinking into adulthood: findings from a 15-year 
prospective cohort study. BMJ Open, 3(8), e003015. https://doi.org/10.1136/bmjopen-2013-
003015  
Ersche, K. D., Gillan, C. M., Jones, P. S., Williams, G. B., Ward, L. H. E., Luijten, M., De Wit, S., 
Sahakian, B. J., Bullmore, E. T., & Robbins, T. W. (2016). Carrots and sticks fail to change 
behavior in cocaine addiction. Science, 352(6292), 1468-1471. 
https://doi.org/10.1126/science.aaf3700  
Ewing, S. W. F., Sakhardande, A., & Blakemore, S.-J. (2014). The effect of alcohol consumption on the 
adolescent brain: A systematic review of MRI and fMRI studies of alcohol-using youth. 
Neuroimage Clin. https://doi.org/10.1016/j.nicl.2014.06.011  
Friedman, N. P., & Robbins, T. W. (2022). The role of prefrontal cortex in cognitive control and executive 
function. Neuropsychopharmacology, 47(1), 72-89. https://doi.org/10.1038/s41386-021-01132-0  
Gbadeyan, O., McMahon, K., Steinhauser, M., & Meinzer, M. (2016). Stimulation of Dorsolateral 
Prefrontal Cortex Enhances Adaptive Cognitive Control: A High-Definition Transcranial Direct 
Current Stimulation Study. The Journal of Neuroscience, 36(50), 12530-12536. 
https://doi.org/10.1523/jneurosci.2450-16.2016  
Grant, B. F., Chou, S. P., Saha, T. D., Pickering, R. P., Kerridge, B. T., Ruan, W. J., Huang, B., Jung, J., 
Zhang, H., Fan, A., & Hasin, D. S. (2017). Prevalence of 12-Month Alcohol Use, High-Risk 
Drinking, and <i>DSM-IV</i> Alcohol Use Disorder in the United States, 2001-2002 to 2012-2013. 
JAMA Psychiatry, 74(9), 911. https://doi.org/10.1001/jamapsychiatry.2017.2161  
Grant, B. F., Goldstein, R. B., Saha, T. D., Chou, S. P., Jung, J., Zhang, H., Pickering, R. P., Ruan, W. 
J., Smith, S. M., Huang, B., & Hasin, D. S. (2015). Epidemiology of DSM-5 Alcohol Use Disorder. 
JAMA Psychiatry, 72(8), 757. https://doi.org/10.1001/jamapsychiatry.2015.0584  
Guerri, C., & Pacual, M. (2010). Mechanisms involved in the neurotoxic, cognitive, and neurobehavioral 
effects of alcohol consumption during adolescence. Alcohol  
Hasin, D. S., Stinson, F. S., Ogburn, E., & Grant, B. F. (2007). Prevalence, Correlates, Disability, and 
Comorbidity of DSM-IV Alcohol Abuse and Dependence in the United States. Archives of General 
Psychiatry, 64(7), 830. https://doi.org/10.1001/archpsyc.64.7.830  
Herrmann, C. S., Rach, S., Neuling, T., & Struber, D. (2013). Transcranial alternating current stimulation: 
a review of the underlying mechanisms and modulation of cognitive processes. Frontiers in 
Human Neuroscience.  
Kearney-Ramos, T. E., Dowdle, L. T., Lench, D. H., Mithoefer, O. J., Devries, W. H., George, M. S., 
Anton, R. F., & Hanlon, C. A. (2018). Transdiagnostic Effects of Ventromedial Prefrontal Cortex 
Transcranial Magnetic Stimulation on Cue Reactivity. Biological Psychiatry: Cognitive 
Neuroscience and Neuroimaging, 3(7), 599-609. https://doi.org/10.1016/j.bpsc.2018.03.016  
Lam, N. H., Borduqui, T., Hallak, J., Roque, A., Anticevic, A., Krystal, J. H., Wang, X.-J., & Murray, J. D. 
(2022). Effects of Altered Excitation-Inhibition Balance on Decision Making in a Cortical Circuit 
Model. The Journal of Neuroscience, 42(6), 1035-1053. https://doi.org/10.1523/jneurosci.1371-
20.2021  
Matsumoto, H., & Ugawa, Y. (2017). Adverse events of tDCS and tACS: A review. Clinical 
Neurophysiology Practice, 2, 19-25.  
McKim, T. H., Bauer, D. J., & Boettiger, C. A. (2016). Addiction History Associates with the Propensity to 
Form Habits. Journal of Cognitive Neuroscience, 28(7), 1024-1038. 
https://doi.org/10.1162/jocn_a_00953  
McKim, T.H., Dove S.J., Robinson D.L., Fröhlich F., Boettiger C.A.. (2021) Addiction history moderates 
the effect of prefrontal 10-Hz transcranial alternating current stimulation on habitual action 
selection. J Neurophysiol. 125(3):768-780. https://doi: 10.1152/jn.00180.2020.  
Miller, E. K., & Cohen, J. D. (2001). AN INTEGRATIVE THEORY OF PREFRONTAL CORTEX 
FUNCTION. Annual Review of Neuroscience, 24, 167-202.  
Olsen, R. W., & Liang, J. (2017). Role of GABAA receptors in alcohol use disorders suggested by 
chronic intermittent ethanol (CIE) rodent model. Molecular Brain, 10(1). 
https://doi.org/10.1186/s13041-017-0325-8  

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Ostlund, S. B. (2005). Lesions of Medial Prefrontal Cortex Disrupt the Acquisition But Not the Expression 
of Goal-Directed Learning. Journal of Neuroscience, 25(34), 7763-7770. 
https://doi.org/10.1523/jneurosci.1921-05.2005  
Rehm, J., Shield, K. D., Roerecke, M., & Gmel, G. (2016). Modelling the impact of alcohol consumption 
on cardiovascular disease mortality for comparative risk assessments: an overview. BMC Public 
Health, 16(1). https://doi.org/10.1186/s12889-016-3026-9  
Sellers, K. K., Mellin, J. M., Lustenberger, C. M., Boyle, M. R., Lee, W. H., Peterchev, A. V., & Fröhlich, 
F. (2015). Transcranial direct current stimulation (tDCS) of frontal cortex decreases performance 
on the WAIS-IV intelligence test. Behavioural Brain Research, 290, 32-44. 
https://doi.org/10.1016/j.bbr.2015.04.031  
Siqueira, L., & Smith, V. C. (2015). Binge Drinking. American Academy of Pediatrics, 136(3). 
https://doi.org/10.1542/peds.2015-2337  
Smittenaar, P., Thomas, Romei, V., Nicholas, & Raymond. (2013). Disruption of Dorsolateral Prefrontal 
Cortex Decreases Model-Based in Favor of Model-free Control in Humans. Neuron, 80(4), 914-
919. https://doi.org/10.1016/j.neuron.2013.08.009  
Vanderhasselt, M.-A., De Raedt, R., Brunoni, A. R., Campanhã, C., Baeken, C., Remue, J., & Boggio, P. 
S. (2013). tDCS over the Left Prefrontal Cortex Enhances Cognitive Control for Positive Affective 
Stimuli. PLoS ONE, 8(5), e62219. https://doi.org/10.1371/journal.pone.0062219  
Viner, R. M., & Taylor, B. (2007). Adult outcomes of binge drinking in adolescence: findings from a UK 
national birth cohort. Journal of Epidemiology &amp; Community Health, 61(10), 902-907. 
https://doi.org/10.1136/jech.2005.038117  
Wood, W., & Rünger, D. (2016). Psychology of Habit. Annual Review of Psychology, 67(1), 289-314. 
https://doi.org/10.1146/annurev-psych-122414-033417  
Zaehle, T., Rach, S., & Herrmann, C. S. (2010). Transcranial Alternating Current Stimulation Enhances 
Individual Alpha Activity in Human EEG. PLoS ONE, 5(11), e13766. 
https://doi.org/10.1371/journal.pone.0013766  
Appendices 
 
Appendix 1:  XCSITE 100 User Guide Version 1.1 (May 2017)