Neural Correlates of the Shift in Social Buffering of Social Evaluative Threat in 11-14 Year Olds
Phase: NA
Conditions: Social Stress, Adolescent Behavior
Sponsor: University of Minnesota

PROTOCOL TITLE:  An MRI Study of Stress and Social Support 
VERSION DATE: v.5.5 2022.09.20 
 
Page 1 of 33 
 
 
Protocol Title 
The Role of Social Partners in Buffering Physiological and Brain 
Indicators of Stress in Children and Adolescents: An MRI Study 
of Stress and Social Support 
Principal 
Investigator/Faculty 
Advisor 
Kathleen M. Thomas, Ph.D. 
Institute of Child Development 
612-625-3389 
thoma114@umn.edu 
Student 
Investigator 
N/A 
 
  
 
 
 
Scientific 
Assessment 
Nationally-based, federal funding organizations 
IND/IDE # (if 
applicable) 
N/A 
IND/IDE Holder 
N/A 
Investigational Drug 
Services # (if 
applicable) 
N/A 
Version 
Number/Date: 
Version 5.5 / 2022.09.20 
 
PROTOCOL COVER PAGE 
 
 
 
 

PROTOCOL TITLE:  An MRI Study of Stress and Social Support 
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Page 2 of 33 
 
 
REVISION HISTORY 
 
Revision # 
Version Date 
Summary of Changes 
Consent Change? 
1 (v2) 
May 29, 2019 
Response to reviewer concerns 
Yes 
2 (v2.01) 
Aug 1, 2019 
Addition of Safety Screen worded 
for Minors. No other change to 
protocol document. 
No 
3 (v2.02) 
Aug 29, 2019 
Clarified that no HIPAA data will be 
given to CMRR; corrected 
misloaded file documenting CMRR 
pre-approval for IRB review 
No 
4 (v3.0) 
Nov 21, 2019 
Added option for online consent 
for parent of “friend” via Redcap – 
changed in protocol and in 
Consent_Friend form; updated 
MRI screening form for minors per 
CMRR requirement; added 
additional self-report 
questionnaire measures for 
participant.  
Yes 
(Consent_Friend 
form is updated to 
include option of 
online 
completion) 
5 (v4.0) 
Jan 28, 2020 
Added option for research 
participants to complete 
questionnaires online using an 
iPad and Redcap. 
No. 
6 (v4.01) 
Nov 11, 2020 
Response to required 
modifications: Addition of clearer 
rationale for deception used in test 
procedures and indication that 
participants may withdraw their 
data and records when they are 
debriefed on this deception.  
No 
7 (v5.0) 
Nov 30, 2020 
Removed urine + oxytocin testing, 
added Certificate of Confidentiality 
language to consent form. 
Changes required for COVID safety 
and budget limits: added online 
consent and assent for all subjects, 
session 1 completely online, 
removed nurse exam, removed 
friend condition 
Yes  
8 (v5.01) 
Jan 4, 2021 
Made required changes: removed 
references to nurse exam from 
No 

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section 17.2 and removed 
references to involvement of a 
friend in section 13.1 
9 (v5.1) 
Jan 15, 2021 
Added video recording of sessions. 
Consent form updated to clarify 
recording. 
Yes 
10 (v5.2) 
May 25, 2021 
Added new NRI questionnaire 
version and contacting past 
participants to complete this 
addition, with payment of $10 via 
online gift card or ClinCard. 
 
11 (v5.3) 
Oct 27, 2021 
Added recruitment method of 
contacting organizations through 
email. 
No 
12 (v5.4) 
Aug 18, 2022 
Added recruitment methods of 
mailing paper flyers and contacting 
people through organization 
listserv. 
No 
13 (v5.5) 
Sep 20, 2022 
Increased child payment to $100 
total and parent payment to $50 
total. 
Yes 
 
 

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Table of Contents 
 
1.0 
Objectives 
6 
2.0 
Background 
6 
3.0 
Study Endpoints/Events/Outcomes 
7 
4.0 
Study Intervention(s)/Investigational Agent(s) 
7 
5.0 
Procedures Involved 
8 
6.0 
Data and Specimen Banking 
13 
7.0 
Sharing of Results with Participants 
13 
8.0 
Study Population 
14 
10.0 
Local Number of Participants 
17 
11.0 
Local Recruitment Methods 
17 
12.0 
Withdrawal of Participants 
18 
13.0 
Risks to Participants 
19 
14.0 
Potential Benefits to Participants 
20 
15.0 
Statistical Considerations 
21 
16.0 
Confidentiality 
26 
17.0 
Provisions to Monitor the Data to Ensure the Safety of Participants 
26 
18.0 
Provisions to Protect the Privacy Interests of Participants 
27 
19.0 
Compensation for Research-Related Injury 
29 
20.0 
Consent Process 
29 
21.0 
Setting 
31 
22.0 
Multi-Site Research:    N/A 
31 
23.0 
Resources Available 
31 
24.0 
References 
32 
 

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ABBREVIATIONS/DEFINITIONS 
● fMRI: Functional Magnetic Resonance Imaging 
● MISTiC: Minnesota Imaging Stress Test in Children 
● TSST: Trier Social Stress Test 
● CORT, AUCi: salivary cortisol, area under the curve from intercept 
● DHEA: dehydroepiandrosterone 
● EKG: Electrocardiogram 
● vmPFC: ventromedial prefrontal cortex 
● ICD: Institute of Child Development 
 
 

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1.0 
Objectives 
1.1 Purpose:  
The goal of this project is to examine the neural systems underlying the 
physiological responses to stress in children and adolescents, and to 
examine the effectiveness of various social partners (parent, stranger) in 
buffering youth from the physiological and brain effects of stress. 
2.0 
Background 
2.1 Significance of Research Question/Purpose & Existing Literature:  
Social buffering, a key concept in the psychobiology of stress, describes a 
phenomenon in which the presence and availability of social partners 
reduces activity of threat- and stress-mediating neurobiological and 
neuroendocrine systems (Gunnar, 2017). It is a key pathway through which 
social support reduces stress (Uchino et al., 2012) and enhances health 
(Kaplan et al., 1977). Impaired social buffering is a primary pathway 
through which adverse childhood experiences get under the skin to affect 
development (Hostinar et al, 2014).  Despite its centrality to stress 
regulation, we know relatively little about the normative development of 
social buffering beyond the infant years. 
 
2.2 Preliminary Data:  
Prior behavioral data from our laboratories demonstrates that parents 
remain effective social buffers beyond infancy and throughout childhood. 
Among 9- and 10-year-olds, preparing for the Trier Social Stress Test (TSST) 
with a parent completely blocked elevations in cortisol to this social 
evaluative stressor (Hostinar et al., 2015). However, our results suggest 
that puberty results in a waning of parental HPA stress buffering 
effectiveness. Preparing for the TSST with the parent vs experimenter had 
no effect on 15- and 16-year-olds or on 11- to 14-year-olds who were at 
more advanced pubertal stages (Doom et al., 2016).  
 
 
2.3 Study Aims:   
The current study examines the neural response, as well as the cortisol 
hormonal response to social evaluative stress across the transition to 
puberty.  We use a modified version of the Trier Social Stress Test (TSST) in 
the MRI scanner (called the Minnesota Imaging Stress Test in Children; 
MISTiC) to elicit social evaluative stress. In this project, we will randomly 
assign youth to complete this testing under one of three social buffering 
conditions: presence of a parent, presence of an experimenter (stranger), 
or no social support present (standard imaging condition). 

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The long-term goal of our research is to enrich understanding of stress 
regulation and its role in human development. The overall objective of this 
project is to understand normative processes in stress regulation and their 
neurobiological correlates as children leave childhood and become more 
autonomous in adolescence. The central hypothesis of this project is that 
early adolescence is a period of time when parental buffering wanes in 
efficacy, leaving many youth experiencing a relative dearth of relationships 
that can provide powerful stress buffers.  
The aim of the current study is to delineate changes in brain activation in 
response to social buffering by parents over the pubertal transition. 
Hypothesis #1: Parental social buffering will result in increased activation in 
brain regions associated with safety (e.g. vmPFC) and lower activation in 
brain regions involved in fear and pain (e.g., insula, amygdala, 
hypothalamus), as well as tighter coupling between the vmPFC and 
amygdala activity, but these relationships will wane with pubertal 
development.  
Hypothesis #2: Neural activity associated with social buffering will mediate 
the effectiveness of physiological buffering by social partners. 
 
3.0 
Study Endpoints/Events/Outcomes 
3.1 Primary Endpoint/Event/Outcome:  
Neural activity in vmPFC, insula, amygdala, and hypothalamus; salivary 
cortisol, heart rate, and respiration  
3.2 Secondary Endpoint(s)/Event(s)/Outcome(s):  
Exploration of possible sex differences in different conditions later in 
puberty but not earlier in puberty. 
4.0 
Study Intervention(s)/Investigational Agent(s) 
4.1 Description:  Note that there are no investigational agent(s) (e.g., drug, 
device) being evaluated. The study qualifies as an intervention due to 
random assignment into social buffering condition, to determine the effect 
of social partner on stress response. 
Child participants (11- to 14-year-olds) will be randomly assigned to one of 
three conditions during an fMRI version of a social evaluative stress 
paradigm: (1) no social buffer, (2) presence of experimenter/stranger, or 
(3) presence of a parent. 
4.2 Drug/Device Handling:   N/A 
4.3 Biosafety:    N/A  
4.4 
Stem Cells:   N/A 
 

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5.0 
Procedures Involved 
5.1 Study Design: The study design is cross-sectional.  200 target children (11- 
to 14-year-olds) will be randomly assigned to one of three conditions: (1) 
no social support (standard MRI scanning condition); (2) presence of a 
stranger (experimenter); or (3) presence of a parent. Outcome measures 
will be compared across the three social buffering conditions. Pubertal 
stage will be included as a continuous variable in analyses of physiological 
and/or brain responses. 
Study Overview:  
Visit 1: Parent and child will connect with a research staff member via 
video conferencing for a session lasting approximately 2 hours. Following a 
detailed overview of study and consent/assent procedures, participants 
will be asked to complete the following measures: 
Parent: 
● 
Questionnaire regarding family income, education level, and family 
composition 
● 
Parent-report questionnaire about child’s general health, behavior, 
and development (MacArthur Health Behavior Questionnaire; HBQ) 
● 
Parent-report MRI safety screening questionnaire about the child to 
rule out any contraindications for MRI scanning 
 
Children/adolescents:  
● 
Questionnaire about general health, behavior, and development 
(MacArthur Health Behavior Questionnaire; HBQ) 
● 
Questionnaire about their relationships with their parents and their 
best friend (Network of Relationships Questionnaire) 
● 
Pubertal development questionnaire (Morris and Udry, 1980) 
● 
Safety screening questionnaire to confirm safety in the MRI scanner 
 
Visit 2: Parent and child will come to the Center for Magnetic Resonance 
Research for a session lasting approximately 2 hours. Following a session 
overview and consent/assent procedures, participants will be asked to 
complete the following measures: 
Parent: 
● 
Required safety screening questionnaire to confirm child’s safety in 
the MRI scanner 
● 
[If the child is randomized to the parent-present buffering condition], 
sit in a separate room while connected via video conference call to 
research staff in the MRI console room for duration of MRI scan 
(approx. 1 hour) 

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Child: 
● 
Required safety screening questionnaire to confirm child’s safety in 
the MRI scanner 
● 
Saliva sampling at 10 time points throughout the testing session to 
assess levels of stress-sensitive compounds, including cortisol, alpha 
amylase, and DHEA. 
● 
Children chew on an absorbent sponge for 1-2 minutes to acquire 
each saliva sample. 
● 
Measure height and weight using a stadiometer. 
● 
Practice in a pretend MRI machine to gain familiarity with the 
scanning environment and assess comfort in the scanner bore (i.e., 
rule out claustrophobia) 
● 
Standard non-invasive structural and functional MRI scanning for 1 
hour at 3-Tesla (clinical standard) field strength. 
● 
Collection of physiological data during the MRI scan (respiration belt, 
and pulse oximeter and/or 3 EKG leads). 
● 
Performance of a social evaluative stress test during MRI scanning.  
(1) Five minutes of silent preparation of a speech about oneself, (2) 
Oral delivery of the speech to two judges in white lab coats, visible by 
video feed via video conference call, and (3) Performance of 
challenging, but age-appropriate math problems under timed limited 
conditions, while observed by the same two judges. To increase the 
social evaluative component of the stress, youth are told that we will 
be video recording their speech and will show it to a panel of kids 
their age who will rate their performance. Although, we do video 
record, we will not show anyone the child’s performance. The social 
evaluative stress created by this test depends upon both the visible 
judges and the belief of a recording that is shown to peers. Without 
one of these aspects, the stress test does not reliably produce a stress 
response in the studied population. The research goals of this study 
cannot be met without a reliable and standard social evaluative stress 
test. This is standard practice of the Trier Social Stress Test. This 
recording will be used for training and data purposes only. 
● 
Completion of a post-scan survey to assess perceived stress at various 
points throughout the session. 
● 
Debriefing of participant to reveal deception (no kids evaluating the 
recording, two judges were also not actually evaluating 
performance). 
 
5.2 Study Procedures: 

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Social Buffering Manipulations: Will include 3 between-subjects conditions 
and both sexes. Participants will be randomly assigned to No Social Partner, 
Stranger/Experimenter, or Parent.  
Stressor Paradigm:  MISTiC paradigm while the child is lying in the MRI 
scanner; includes two judges (one male, one female). After consent, youth 
are separated from their parents and taken for completion of 
questionnaires (approximately 20 minutes). Youth are taken to a private 
changing room to change into hospital scrubs prior to moving to the MRI 
scanner. An MRI-safe respiration belt and pulse oximeter and/or EKG 
electrodes are positioned for autonomic assessment and the child is given 
ear plugs, a button response box for math task, and an emergency call 
button. Youth rest and/or watch a video of their choosing during structural 
MRI sequences (6-10 minutes), then lie quietly with eyes open for a 5-
minute scan with no video or audio material. They are then given an 
overview of the speech task and are told that they have 5 minutes to 
prepare. MRI data are collected during the 5 minutes of speech prep, 
following which the child is introduced to two judges who are visible on the 
child’s video screen and who ask the child to perform their 5 min speech. 
No MRI data are collected during speech performance. At the end of the 
speech, the judges give the child instructions for the math performance 
section, and the child performs multiple-choice math problems that appear 
on the video screen using the button response box. MRI data are collected 
during math performance and judges remain visible on the screen during 
math performance. Following the judged math, judges leave the screen 
and youth rest or watch a video for 10 minutes during diffusion scanning.  
Finally, children perform another set of multiple-choice math problems 
with no judges present (photo of the same judges turned away) while MRI 
data are collected. Children are removed from the scanner and 
immediately complete a stress questionnaire and a debriefing session. 
Saliva samples are collected every 10-20 minutes throughout the session, 
including between scan segments, to assess dynamic changes in stress-
related hormones. In the scanner, this involves handing a long collection 
sponge to the child which hangs out of the mouth to avoid any possibility 
of choking since the child is lying supine.  We have used this method 
successfully in a previous study collecting saliva in the scanner 
environment.  Following debriefing, the child will change back into their 
own clothing and return to the lobby to complete additional questionnaires 
for 30 min while being sampled for cortisol every 10 min.  
Note: As part of the stress challenge, we tell youth that their speech and 
judged math performance will be video recorded and rated by a group of 
kids their age. However, we will not actually share their performance with 
anyone. 

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Pubertal Development: We will create a summary pubertal development 
index from several measures. All youth will complete the Morris and Udry 
(1980) pubertal development questionnaire, which allows placement in the 
gold-standard Tanner stages for hair, testicles, and breast development 
(Tanner, 1962). In all studies target participants will complete the Morris 
and Udry questionnaire within 2 weeks before the experimental sessions 
described below. We will also analyze salivary DHEA to augment our 
measures of puberty (Shirtcliff, Dahl, & Pollak, 2009).  
 
Questionnaires.  
Forms will be completed either on paper or online via Redcap. 
Demographic Questionnaire: Parents will complete information on pre-tax 
family income, education level of parent(s), composition of the household, 
and medications that the child regularly takes. 
Daily Diary: The parent and the youth both will report the following for the 
child that day: the time of wake up, medication usage, illness/fever, 
physical activity, and caffeine consumption.  
Relationship Quality:  Participants will complete the Network of 
Relationships Inventory - Behavioral Systems Version (NRI-BSV, Furman & 
Buhrmester, 2009) for the parent who accompanies them and their close 
friend. This questionnaire is based on an integration of attachment and 
Sullivanian theory and assesses three systems that are expected to be key 
in close relationships: attachment, caregiving and affiliation.  The scales 
are: companionship, seek safe haven, seek secure base, provide safe 
haven, provide secure base, conflict, antagonism and criticism. We will also 
add 3 scales from the NRI-Relationship Qualities Version: Emotional 
Support, Approval and Satisfaction. Each scale consists of 3 items scored on 
a 5-point Likert scale. Psychometric qualities are high. Condition 
differences in quality of relationships will be examined and included as 
covariates if groups differ significantly. Otherwise, in follow-up analyses we 
will examine whether social buffering effects are stronger in relationships 
the child views as more supportive.  
Four additional NRI scales (Seek Safe Haven, Seek Secure Base, Provide 
Safe Haven, Provide Secure Base) will be collected to better measure 
relationship quality. Completed participants will be contacted to 
complete these additional 12 questions and will be compensated for their 
time. 
MacArthur Health and Behavior Questionnaire (HBQ) for 9-18 Year-Olds 
(Parent and Child, 2.1):  This questionnaire was developed by Marilyn 
Essex based on the original HBQ for 4-8 year olds. We have used it many 

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times. The scales we proposed to use here have high reliabilities (Cronbach 
alpha’s> .8). We will use the peer scales (acceptance/rejection, bullied, 
relational victimization, asocial, behavioral inhibition) and broad-band 
symptoms scales (Internalizing, Externalizing). These measures will be 
examined for balance across groups and if differences by condition, 
pubertal stage, or sex are found, will be entered as covariates.   
Self-Report of Stress: As a manipulation check, participants will rate their 
arousal and emotional state at various points during Visit 2. 
Version 3.0 addition: Added Child-self-report RSQ for Peer, Family, & 
Academic Stress, and PALS Student report of adaptation to challenge in 
environment. 
 
Physiological Measures. 
Stress Hormones: We will assay saliva samples for stress sensitive 
hormones: cortisol and alpha-amylase. Saliva will be collected using 
SalivaBio Oral Swabs (SOS) or passive drool and stored frozen at −20°C until 
shipped for assay. All testing will be conducted in the late afternoon to 
control for circadian rhythm. Medications will be recorded from parent 
report and classes of drug codes will be used to group meds by presumed 
mechanisms of action and will be entered as covariates (Granger et al., 
2009). Systemic glucocorticoid medications are an exclusion criterion. For 
each analyte, we will calculate area under the curve from intercept (AUCi) 
to provide a single measure for the main analysis. 
Pubertal Hormones - DHEA, Testosterone, Estradiol: Sufficient saliva will 
be obtained at sampling to assay for these additional pubertal hormones. 
These assays will be performed if we obtain a sex by condition by pubertal 
development (3-way) interaction on key outcome measures. If so, these 
measures may provide insight into the mechanism of these interactions 
(Butler et al., 1989; Mehta, Mor, Yap, & Prasad, 2015). 
 
Physiological Recording:  Participants will wear standard Siemens 
physiological recording equipment for the MRI environment (respiration 
belt, and pulse oximeter or EKG leads). Samples will be collected 
throughout the entire MRI scan. Response measures will be computed by 
regressing the stress condition values on comparison condition values and 
saving the residual as the response measure.  
All questionnaires and surveys are included in the ETHOS portal. Contact 
information and child age will be obtained from an existing registry of 
families who have expressed interest in child development research 
(specifically, parent name(s), address, phone number, email address, child 

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name, child sex, and child birthdate). All other data will be acquired directly 
from the participants, and information obtained from the participant 
registry will be verified verbally with the parent.  
 
5.3 Study Duration: Participants will attend two sessions.  Each session will last 
approximately 2 hours. 
● 
All participants will be enrolled and assessed within a 30-month 
period. 
● 
Primary data analysis endpoints will be available within 1 year of the 
end of data collection (i.e., 42 months from initial enrollment). 
 
5.4 Individually Identifiable Health Information:  This study will collect health 
information directly from participants.  A HIPCO survey has been uploaded 
in the ETHOS portal. 
 
5.5   Use of radiation:    N/A 
5.6 Use of Center for Magnetic Resonance Research: All MRI scanning will 
occur on a 3-Tesla MRI scanner located at the CMRR. Scans will be 
conducted by the PI or her study team. No PHI data will be entered into 
any CMRR computer or equipment; scans will be collected in de-identified 
form. The CMRR will not have access to any PHI from any participant in this 
study. Therefore, HIPAA authorization is not applicable in this case. 
6.0 
Data and Specimen Banking 
6.1 Storage and Access: Saliva samples will be stored in a -20 degree freezer 
until they can be batched with other samples for hormonal assay. Any 
material remaining after the planned assays will be discarded. No tissue or 
biological samples will be stored for future use or shared. 
 
Biospecimen samples will be identified only with a unique study ID, child 
age, child sex, and sample number. No PHI will be associated with any 
biospecimen samples. Samples will not identify the study condition of 
interest (i.e., social buffering condition). 
6.2 Sharing: De-identified data will be shared with qualified researchers when 
requested for scientific purposes including replication of results, use in 
meta-analyses, or appropriate extension of analyses. No biospecimen 
samples will be saved or shared.   
7.0 
Sharing of Results with Participants 
7.1 Results will only be shared with participants in aggregate, with the 
following exceptions:   

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We will take anthropomorphic measures (such as height and weight) as 
well as pubertal staging and these figures will be shared with parents at the 
time of the session. We deliver the figures on a post-card, with links to 
national pediatric norms provided by the CDC.  
Regarding other hormonal or physiological measures in this study, 
abnormal values are commonly due to an interfering substance present in 
the sample (e.g., milk for salivary cortisol) or motion artifact in the case of 
EKG. Without a body of evidence, it seems unethical to alarm families given 
an out-of-range assay value and therefore we do not plan to share 
individual results with participant.  
In the event that the scanner operator or study personnel see something 
unusual in the MRI scan, the images will be submitted for review by a 
radiologist.  Images contain only the study ID number, participant’s age in 
years, participant sex, and test date.  In the event that the radiologist 
determines that the incidental finding should receive clinical follow-up, we 
will share this information with the parent(s) of the minor child participant 
with the recommendation to consult with the child’s primary care 
physician. Incidental findings from research scans are not clinically 
diagnostic and the images may not be added to the child’s medical record. 
8.0 
Study Population 
8.1 Inclusion Criteria: Healthy 11- to 14-year-olds. Additional inclusion criteria 
ensure that youth will be able to follow the study procedures (have 
sufficient vision, hearing, and language skills to provide verbal and written 
assent, see and read stimuli presented on the computer screen, and hear 
verbal instructions provided by the experimenter and/or judges).   
8.2 Exclusion Criteria for child participation: Premature birth (less than 37 
weeks), congenital and/or chromosomal disorders (e.g. cerebral palsy, FAS, 
mental retardation, Turner Syndrome, Down Syndrome, Fragile X), Autism 
Spectrum Disorders, history of serious medical illness (e.g., cancer, organ 
transplant), youth using systemic glucocorticoids or beta-adrenergic 
medications, diagnosed psychiatric illness or psychotropic medication, 
seizure disorder or other neurological disorder, academic delay or 
individualized education plan (IEP), contraindications for MRI (implanted 
medical device; presence of non-removal metal in or on the body, including 
piercings, orthodontic braces or certain permanent retainers), known 
pregnancy, tattoo, or history of significant claustrophobia.  
8.3 Screening: Screening will occur by asking parents questions at the time of 
telephone recruitment. To reduce confounding of age and pubertal stage, 
we will use a stratified recruitment method. During recruitment, parents 
will be asked 3 questions about their child’s physical development, and 
children will be initially categorized into Pre/Early and Mid/Late Puberty. As 

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cells fill, participants will no longer be enrolled into those cells. Also, to 
increase minority representation we will sample across zip codes with 
higher racial and ethnic minority representation.   
9.0 
Vulnerable Populations 
9.1 
Vulnerable Populations:  
 
Population / Group 
Identify whether any of the 
following populations will be 
targeted, included (not necessarily 
targeted) or excluded from 
participation in the study.  
Children 
Targeted Population 
Pregnant women/fetuses/neonates 
Excluded from Participation 
Prisoners 
Excluded from Participation 
Adults lacking capacity to consent 
and/or adults with diminished 
capacity to consent, including, but 
not limited to, those with acute 
medical conditions, psychiatric 
disorders, neurologic disorders, 
developmental disorders, and 
behavioral disorders 
Excluded from Participation 
Non-English speakers 
Excluded from Participation 
Those unable to read (illiterate) 
Excluded from Participation 
Employees of the researcher 
Excluded from Participation 
Students of the researcher 
Excluded from Participation 
Undervalued or disenfranchised 
social group 
Included/Allowed to Participate 
Active members of the military 
(service members), DoD personnel 
(including civilian employees) 
Included/Allowed to Participate 
Individual or group that is 
approached for participation in 
Excluded from Participation 

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research during a stressful situation 
such as emergency room setting, 
childbirth (labor), etc. 
Individual or group that is 
disadvantaged in the distribution of 
social goods and services such as 
income, housing, or healthcare. 
Included/Allowed to Participate 
Individual or group with a serious 
health condition for which there are 
no satisfactory standard treatments. 
Excluded from Participation 
Individual or group with a fear of 
negative consequences for not 
participating in the research (e.g. 
institutionalization, deportation, 
disclosure of stigmatizing behavior). 
Included/Allowed to Participate 
Any other circumstance/dynamic 
that could increase vulnerability to 
coercion or exploitation that might 
influence consent to research or 
decision to continue in research. 
Excluded from Participation 
 
9.2  Additional Safeguards:  
Children are the targeted population for this study.  Others that have been 
checked as “allowed” above are not being intentionally included or 
excluded. For example, we will not inquire about someone’s membership 
in a disenfranchised group, nor whether a target child’s parent is on active 
duty in the military. Our careful consent/assent procedures, designed to 
handle research with children, should be sufficient to cover other 
vulnerable participants who may inadvertently enter our study.  
All of the subjects will be children, ranging 11-14 years old. This study 
focuses on adolescents because our goal is an analysis of the impact of 
social buffering and puberty on stress-mediating systems. Children have 
been the focus of our research for the past 30+ years. Our staff members 
are highly trained in working with children and families. The graduate 
students working on the project will be Child Psychology and/or Child 
Clinical Psychology Ph.D. students. The undergraduate research assistants 
will, for the most part, be majoring in child development.  

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Much of the work will be performed online over video conferencing. Staff 
will guide parents and children through the study procedures and 
participating families will likely be comfortable in their own homes. 
Our laboratory has been scanning both typically developing children and 
adolescents (ages 4-17) and those at risk due to early health or 
environmental risks for over two decades. We have conducted NIH funded 
research with child participants at the CMRR for the past 16 years. We are 
very experienced in putting children at ease in the MRI scanner and 
ensuring that children are aware that they can stop at any time.  We have 
completed a prior MRI study with 11- to 14-year-olds using the MISTiC 
social stress paradigm; therefore, we are confident that we are well 
positioned to properly inform children about the procedures and are not 
stressing children beyond normal everyday levels that they experience in 
school or in extracurricular activities (sports competitions; music, dance, or 
drama performances; etc.). 
Parents provide consent, youth provide assent, and see below where that 
process is fully described to ensure that youth have full understanding and 
are free to decline participation. 
 
10.0 Local Number of Participants 
10.1 Local Number of Participants to be Consented: We plan to analyze data 
from at least 200 target participants. To account for attrition between 
sessions/replacement for incomplete data, up to 300 children may be 
consented/enrolled. 
11.0 Local Recruitment Methods 
11.1 Recruitment Process: Families will be initially contacted by phone, email, or 
paper mail to invite them to participate. Paper mail will include IRB-
approved recruitment flyers. Interested families will be provided a more 
detailed overview of the study procedures in a telephone call. If the family 
is interested in participating, the parent will verbally complete a screening 
interview to assess inclusion and exclusion criteria, and for those who are 
eligible, the first visit will be scheduled. Copies of the consent and assent 
forms will be sent to families prior to the visit, along with directions to the 
University, and a reminder call will be a chance for families to confirm their 
interest in participation after having seen the consent forms. The formal 
consent process will occur at both of the two sessions.  
11.2 Identification of Potential Participants: Potential participants will be 
identified using the ICD registries of families interested in being contacted 
about research. These families have agreed to be contacted, and once ICD 
Registry procedures have been followed, including proof of IRB approval, 

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an encrypted list is provided to study staff. No protected records are 
involved. Study staff will make initial contact. 
11.3 Recruitment Materials: We will recruit via telephone, email, and paper mail 
(as parents have given us these three means of contacting them). We will 
also post flyers and put the flyer information on our website so that 
families can contact us. We will also email organizations what may be able 
to assist with sharing study contact information and spreading interest, 
using the Letter to Organizations template. We will also email 
organizations’ listservs when approved to do so by the listserv 
administrators. Materials are uploaded in ETHOS. 
11.4 Payment: 
● 
Child Participant: $40 debit or gift card for Visit 1; $60 in debit or gift 
card for Visit 2 
● 
Parent of Child Participant: $25 in debit or gift card for Visit 1; $25 in 
debit or gift card for Visit 2 
Cards will be given at the session to parent & child as specified, though we will 
have parents sign receipts for payment. We intend to use Greenphire ClinCard 
prepaid debit cards for compensation and have included appropriate language in 
the consent document. We will provide a Target gift card instead of the ClinCard 
if parents object to the terms and conditions of the ClinCard. 
 
12.0 Withdrawal of Participants 
12.1 Withdrawal Circumstances: If participants are enrolled (consent signed) 
and subsequently determined to meet exclusion criteria, their data will be 
excluded from analyses. Children may be withdrawn from the study 
without their consent if we discover a safety concern for MRI scanning 
after consent/assent. Children may also be withdrawn if the experimenter 
believes that the child is excessively distressed by the stress paradigm or 
MRI scanning procedures. Participants will receive full compensation for 
the session at which they are withdrawn. 
12.2 Withdrawal Procedures: If participants voluntarily withdraw, we will verify 
permission to retain and use any data collected to that point. If participants 
wish to withdraw all permission, we will destroy any data collected from 
the participant but will keep their name in our study records with a note 
that they withdrew. This procedure prevents us from inadvertently 
enrolling participants more than once. If the participant is withdrawn by 
the researcher, we will retain and use any data collected from the 
participant. 
12.3 Termination Procedures: Data will not be used after termination. 

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13.0 Risks to Participants 
Foreseeable Risks: Puberty Staging: There is the risk of embarrassment. 
Both for the youth’s well-being and to prevent study drop out, we are 
motivated to reduce any sense of embarrassment as much as possible in a 
study that requires the measurement of pubertal changes. The Morris and 
Udry questionnaire will be completed in privacy and in the comfort of the 
subject’s home while connected via video conference. 
Questionnaires: Youth or parents may experience concerns when 
completing questionnaires dealing with symptoms of behavior problems. 
Any time one works with youth there is the possibility that they will reveal 
information about abuse and/or indicate that they are thinking of hurting 
themselves or others. We are mandated reporters, a fact that is revealed in 
both consent and assent forms. However, in over a decade of working with 
children and youth drawn from our participant files, we have never 
encountered a reportable incident, so we consider this risk to be very low. 
Minnesota Imaging Stress Test in Children: The MISTiC is a social evaluative 
stressor. Public speaking evokes anxiety in many individuals. 
The MISTiC activates stress-mediating systems because it serves as a threat 
to the social self. Public speaking produces anxiety in most people. 
However, answering questions in class, speaking in front of the class or 
school, and solving problems in front of teachers and classmates is a 
common occurrence in children’s lives. Thus, the MISTiC standardizes a 
common everyday stressor. To reduce risk, youth are told that they can 
stop without prejudice if they choose. Because they are told they are being 
evaluated (and this is a necessary part of the procedure), after the MISTiC 
session, the youth will be debriefed to make it clear that there were no 
judgements made about the speech or math performed. It will be 
explained that performance in a social evaluation task was used in order to 
elicit a physical stress response. The youth will never receive any negative 
evaluations from adults as part of being in this study. As a part of this 
debriefing, participants are told that they may withdraw their data and 
record of participation. 
MRI Scanning: The primary risks associated with magnetic resonance 
scanning involve risk of injury or burn in the event that unsafe metallic 
objects are introduced into the scanner environment. Loose items have the 
potential to become projectiles while stationary metallic surfaces could 
build heat over time and cause a burn. In addition, implanted medical 
devices could become non-functional or experience torque that could 
move metal implants through body tissue. All participants are screened 
multiple times for the presence of metal in or on the body. Final screening 
occurs immediately prior to MRI scanning. 
 

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There is no evidence for any long-term negative effect of MRI procedures 
on the body. The guidelines from the Bureau of Radiological Health (Food 
and Drug Administration) will be followed with regard to specific 
absorption rate (SAR) of energy (rf) and time-variant magnetic fields 
(dB/dt). All safety features of the clinically approved Siemens 3T scanner 
will be used to ensure that levels are well below the peripheral nerve 
stimulation threshold for both children and adults. 
The MRI procedure involves exposure to a high level of noise while in the 
scanner environment. Participants are required to wear sound attenuating 
earplugs to protect their hearing. Beyond the risk to hearing, many 
participants find the scanner sounds annoying and monotonous.  We 
provide additional headphones or foam padding over the top of the 
earplugs to further attenuate the background noise.  This additional 
padding can become uncomfortable after long periods of time. 
To reduce anticipatory stress for the scanner experience and to ensure that 
children feel comfortable with all MRI procedures, youth will complete a 
mock MRI scan in an MRI simulator. This pretend scanner has all of the 
primary features of the actual scanner, including an identifcally sized 
tunnel, the peripheral recording equipment, earplugs, headphones, video 
screen, audio system, button response box, and a speaker to play the 
scanner sounds around the tunnel, as if we were scanning, but with no 
magnetic field and no images produced. 
Saliva Sampling. The saliva is collected on an absorbent swab. Any time 
anything is put in the mouth there is the risk of choking. This is a greater 
concern when participants are lying supine in the MRI environment.  
However, we deliberately use long sponge rolls that that hang out of the 
mouth during saliva collection. The child is instructed to hold on to the end 
sticking out of the mouth while chewing on the other end until it is soggy.  
The age of participants, 11-14 years, also reduces the risk of choking as 
these youth are better able to follow experimenter instructions and to 
realize when they are placing the sponge too far into their mouths to be 
comfortable. 
 
13.1 Reproduction Risks:  
The risks of strong magnetic fields for the developing human fetus are 
unknown.  To protect against these unknown but potential risks, we will 
screen all female youth who have started menstruating for possible 
pregnancy. 
13.2 Risks to Others:   N/A 
14.0 Potential Benefits to Participants 

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14.1 Potential Benefits: This study confers no direct benefits to participants. 
15.0 Statistical Considerations 
15.1 Data Analysis Plan:  
Preliminary Analyses to Determine Covariates Needed for Primary 
Analyses. The purpose of collecting data on potential covariates is to 
identify variables that are correlated with both the independent variable 
(social buffering condition, sex, pubertal stage) and the primary outcome 
measures. Those covariates that are associated with the independent and 
outcome variables will be controlled for in the primary analyses. Those that 
are not will be used to describe the sample, but will not need to be 
included in the analyses to obtain unbiased estimates. For each potential 
covariate, we will conduct correlations or t-tests with pubertal stage, and t-
tests, ANOVAs, or chi-square tests (as appropriate) with sex and condition. 
If data do not confirm to modeling assumptions, we will use nonparametric 
analogs instead. 
 
Preliminary Analyses of Imaging Data to Yield Primary Outcome Measures: 
fMRI data will be analyzed in FSL software (version 5.0.10) to generate 
voxel-wise contrast maps. Mean signal values will be extracted from these 
maps in a priori regions of interest for every participant using the methods 
described below. These preliminary analyses are used to generate the fMRI 
outcome variables of interest. The values of these outcome variables for 
each participant will be used in the general statistical analyses described in 
the next section. In total, these preliminary analyses will yield 6 primary 
task-related outcome variables, and 6 primary connectivity-related 
outcome variables. 
 
Task-based analysis: Math under social evaluative stress In first level 
analyses (single subject, single-run), math performance will be modeled for 
each participant using a voxel-wise multiple regression analysis with a task 
predictor (math trials vs. blank trials), 24 regressors for head motion 
(Satterthwaite et al., 2013) and additional regressors to model censored 
data points (motion spikes). In subsequent analyses, we will explore the 
utility of including other time varying individual differences variables (e.g., 
heart rate, respiration) that are known to contribute to the BOLD signal. 
However, since these measures are expected to vary by stress condition, 
removing the effects of these physiological variables may unintentionally 
remove effects of interest. Particular care will be taken to reduce the 
confound of head motion by censoring (identifying as artifact) any data 
point with absolute motion greater than 2 mm (1 voxel) or a framewise 
displacement greater than 0.5 mm, as well as the immediately preceding 
and following data points. Data points with DVARS values exceeding the 

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individual’s 75th percentile plus two times the interquartile range will also 
be censored. Participants with more than 25% of data points censored for 
artifact will be excluded from further analyses. 
 
Second level analyses (single-subject, multiple runs) will test the within-
subject contrast of math performed with judges (social evaluative stress) 
relative to math performed alone (without social evaluation) to identify 
brain regions differentially activated by social evaluative stress. Based on 
prior literature, we will examine activation in 5 a priori regions of interest, 
including “pain-related” brain regions (dorsal anterior cingulate [dACC], left 
and right anterior insula) and “safety-related” or regulatory brain regions 
(ventromedial prefrontal cortex [vmPFC], and left and right dorsolateral 
prefrontal cortex [dlPFC]). For each subject, the mean percent change in 
MR signal (averaged across voxels) between judged math and math alone 
will be extracted from each of these 6 regions. This analysis will generate 
the 6 task-related brain activation outcome variables (listed in section 4.3) 
for each participant.  
 
Given that the MISTiC paradigm has not been used previously, it is possible 
that the most prominent regions of activity for this task may not match the 
a priori regions identified from the prior stress and social buffering 
literature. Therefore, we will also conduct an exploratory group-level 
analysis to identify primary regions of activation for the same socially 
evaluative math vs. non-evaluative math conditions (judged math vs. math 
alone, cluster corrected p<.005). Because we expect that social buffering 
will alter the activity difference between social evaluative and non-
evaluative math periods, we will use only the control condition (no social 
partner) to identify regions activated by this task. In the resultant group-
level map, we will identify up to 4 primary regions of activation by selecting 
clusters that meet the cluster-corrected p<.005 threshold, and then rank-
ordering those regions by maximal z-statistic. Mean percent change in 
signal will be extracted for each participant (across all four buffering 
conditions) using anatomical masks derived from this analysis conducted in 
the control (no social partner) group. Therefore, exploratory analyses may 
generate up to 4 additional task-related MRI outcome variables. 
 
Connectivity analysis: Speech preparation We are also interested in the 
neural correlates of anticipatory stress during the speech preparation 
period. However, the speech preparation period does not contain 
distinctive event markers needed for task-based fMRI analyses. Thus, to 
address the neural correlates of stress during speech preparation, we will 
use a seed-based connectivity approach to examine strength of functional 
connectivity between a priori seed regions of interest in the limbic system 
(amygdala, insula, hypothalamus) and target regions in the prefrontal 

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cortex (left and right vmPFC and dlPFC). To determine whether connectivity 
varies as a function of stress, we will compare connectivity during the 
speech preparation scan to connectivity during the baseline resting state 
scan. Preprocessing steps will include motion correction, unwarping, and 
eddy-current correction. An initial voxel-wise multiple regression analysis 
will be performed to remove effects of head motion 24 predictors), and 
estimates of CSF and white matter signals (2 predictors) from each 
subject’s data. Initial analyses will not include a predictor for global signal 
given evidence that the inclusion of global signal may falsely introduce 
anticorrelations in the data (Murphy et al., 2009; Saad et al., 2012). 
However, in exploratory analyses, we will examine the degree to which the 
results change when global signal is included in the model. 
 
First-level (single-subject, single run) connectivity analyses on the 
residualized data will include the time course for a seed region and 
predictors for censored data points. Two separate regression models will 
be run, one using the bilateral amygdala as the seed region, and the other 
seeding connectivity in the hypothalamus. 
 
Second-level (single-subject, multiple-runs) analyses will test the within-
subject contrast of connectivity during anticipatory stress (speech 
preparation) relative to baseline resting state for each seed-region. Masks 
of a priori target brain regions of interest will be applied to resultant 
contrast maps to extract stress-related connectivity in the bilateral vmPFC 
and left and right dlPFC for each individual. This analysis will generate the 6 
connectivity-related brain outcome variables (2 seed regions x 3 target 
regions) for each participant.Because difference scores (speech preparation 
minus baseline) can mask the direction of connectivity effects (positive vs. 
negative correlations), follow-up simple-effects analyses will assess 
connectivity separately in the speech preparation and resting state periods, 
using the same a priori seed and target regions. These follow-up simple-
effects analyses will allow us to interpret the directionality of connectivity 
effects. 
15.2 Power Analysis: For the power analysis for the primary analyses, we 
calculated the minimum detectable effect size (MDES) assuming n = 200, a 
= .05, and power = .80 with 12 covariates in our model. Power was 
estimated in R (R Core Team) using the pwr library (Champely, 2018). A 
power analysis was performed to quantify the minimum detectable effect 
size (f2) for a three-way interaction between social buffering condition, sex, 
and pubertal stage within a multiple regression model controlling for the 
12 covariates, all two-way interactions, and the main effects (the most 
complex model considered in our primary analysis, see Equation 1 below). 
Assuming these conditions, we should be able to detect an f2 of 0.056. In 

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other words, adding the three-way interaction to a model containing the 
covariates and all lower order interactions and main effects, the three-way 
interaction would need to explain an additional 5.6% of the variability to be 
detected. This falls between Cohen’s criteria for a small (f2 = .02) and 
medium effect (f2 = .15) (Cohen, 1988). With a sample size of 200, even if 
we lose 40 of the participants to movement artifact for the imaging 
analyses, assuming all the previous conditions but a sample size of 160, we 
will still have an f2 of .070, or still within a small-medium effect size. As the 
number of covariates decreases, the size of the MDES will decrease. 
Therefore, this represents a conservative estimate of the power for our 
primary analyses as we are likely to have fewer covariates in our models 
and will use multiple imputation to correct for missing data. For the two-
way interaction and main effects models, described below, the power will 
be larger than these reported values as the models will be simpler. 
15.3 Statistical Analysis of MRI and non-MRI Outcome Variables:  
The following is a list of the analyses that we will perform on the outcome 
measures. They are classified as to whether they are the primary or 
mediational analyses. All statistical analyses will be performed in R. 
 
The primary analyses will consist of a series of multiple regression models. 
For each dependent variable, variation in neural activity, cortisol response, 
and heart rate response, we will begin by testing the three-way interaction 
between social buffering condition, sex, and pubertal stage for all 
outcomes. If the three-way interaction is not significant, we will then 
examine all two-way interactions. If the two-way interactions are not 
significant, we will then examine main effects only models. If the three-way 
interaction is significant, we will not examine any other model. If condition 
is significant as an interaction or as a main effect, we will examine all 
pairwise comparisons using Tukey’s Honestly Significant Difference test. 
 
Multiplicity of testing. We will correct for multiple comparisons using the 
Benjamini-Hochberg (BH) correction. The BH method works by controlling 
the false discovery rate, and relative to the Bonferroni’s correction is more 
powerful, while still adequately protecting against Type I error (Williams, 
Jones, & Tukey, 1999). 
 
Mediational Analysis. When changes in activity of brain regions differ by 
condition or condition by pubertal status and we also see condition or 
condition by pubertal status differences for cortisol, or heart rate, then we 
will examine whether brain activity differences mediate the 
neuroendocrine or autonomic differences. This will be examined using path 
analysis, while controlling for covariates identified in the preliminary 

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covariate analysis, and will be estimated using the lavaan package in R 
(Rosseel, 2012). 
 
The significance of all indirect effects will be assessed using bias-corrected 
95% confidence intervals (Efron & Tibshirani, 1986), where confidence 
intervals that are non-overlapping with zero will be considered significant. 
 
Power Analysis for the Mediational Analysis: To assess power to detect 
mediation, we performed a Monte Carlo-based power analysis using 
simulated data. We varied the parameter values to roughly correspond to 
Cohen’s criteria for small, medium, and large effects. This resulted in a total 
of 27 conditions, which we examined at our target sample size of 200. Each 
condition was replicated 2000 times and we calculated empirical power, 
which we defined as the proportion of times out of 2000 where we 
rejected the null hypothesis at α = .05. Given the exploratory nature of this 
analysis and to simplify the simulation for the power analysis, we omitted 
covariates and did not vary the effects for the other condition contrasts 
(i.e., they were always fixed to 0). Next, we fit a path model using the 
lavaan package in R.  
 
Our findings suggest that in order to detect mediation, the effect of 
condition on the mediator must be large and the effect of the mediator on 
the outcome must be medium or large. In addition, if the effect of 
condition on the outcome is not large, but small or medium sized, then we 
will be underpowered and would likely conclude complete mediation, 
when mediation may only be partial or non-existent. Based on this power 
analysis, it is likely that the MDES for the moderated mediation models will 
need to be at least medium to large as well. 
 
Missing data. Because we will replace participants who do not come in for 
session 2 after doing session 1 (which is rare in our experience), we should 
have 200 completed participants. Missing data, however, could arise from 
problems in obtaining good imaging data. Participants also often fail to fully 
complete questionnaires and/or refuse to provide information on 
household income. If there is missingness in any of the dependent or 
independent variables, multiple imputation will be used (Little & Rubin, 
2014; Peng, Harwell, Liou, & Ehman, 2006; Schafer & Graham, 2002). 
Variables related to the missingness and the variables missing data will be 
included in the missing data model. In the event that missingness is not at 
random (MNAR), we will investigate the use of models for non-ignorable 
missing data. 
15.4 Data Integrity: Questionnaire data will be double-entered to reduce the 
likelihood of errors in data entry. Biospecimen data will be assayed in labs 

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that have standard quality control procedures.  MRI data reviewed for raw 
data quality and artifact detection.   
16.0 Confidentiality 
Data Security: Only authorized research staff will have access to the data, 
and all data will be stored in REDcap databases, secure servers (CMRR and 
Minnesota Supercomputing Institute) or in locked file cabinets. Names and 
contact information, and chid birth date will be maintained in REDcap 
database to avoid enrolling participants more than once, to calculate age at 
test, and to allow us to contact families who have participated with overall 
study findings. IRB and CMRR policies require that we maintain the MRI 
safety screening information for the same duration that we maintain 
consent documents. These will be stored in locked file cabinets and/or 
uploaded to a secure REDcap database.   
A password-protected document linking name and ID number will be kept 
in University of Minnesota Secure Box storage until the conclusion of the 
study in case we must contact a family for any reason, including 
identification of an incidental MRI finding. This document will be destroyed 
at the conclusion of the study. 
All data files are de-identified, password protected, or maintained in secure 
storage locations (REDcap and/or University Box). All paper copies are 
stored in locked cabinets. 
16.1 No documents will be placed in participants’ medical, employment, or 
educational records. 
17.0 Provisions to Monitor the Data to Ensure the Safety of Participants 
17.1 Data Integrity Monitoring: 
All of the planned procedures meet the NIH definition of minimal risk. The 
PI and Study Coordinator will meet regularly to ensure that all study 
protocols are followed and best practices are applied. The Study 
Coordinator will bring any identified protocol violations to the PI in a timely 
fashion and team members will be retrained, if needed. 
17.2 Data Safety Monitoring: 
This project is considered a clinical trial because it involves a) random 
assignment to condition, and b) physiology is being measured.  
There is no risk associated with the assignment to conditions (No Social 
Buffer, Stranger/Experimenter, Parent). All conditions resemble naturally 
occurring social situations that children might experience, and there is no 
inherent danger to one condition over another as there would be for, say, a 
drug vs placebo trial.  

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Further, in two of the conditions the presence of another person is 
expected to provide a social buffer whose stress reduction potency by 
condition is what is being tested. All target youth experience the same 
stressor, the MISTiC. The MISTiC mimics a naturally occurring stressor, as 
when the youth is asked to speak in front of the class and his/her 
performance is being evaluated. This can provoke anxiety.  
The other potential risks of this study to which all of the participants are 
exposed regardless of condition are low, including embarrassment in the 
assessment of pubertal status, claustrophobia or fear of the MRI tunnel, 
loud noises in the scanner, and risk of choking during saliva collection  
 
The steps taken to reduce or prevent these risks include: allowing 
participants to refuse the puberty questionnaire and use of proper saliva 
collection materials, and by completing thorough safety screening prior to 
MRI scanning, and providing youth with a simulated MRI experience first. 
These risks are all managed by the routine processes monitored by our IRB 
and the CMRR Safety Committee. Adverse Events (AEs), including Serious 
Adverse Events (SAEs) such as deaths, hospitalizations, and life-threatening 
events and Unanticipated Problems (UPs), will be managed and reported, 
as required, to the IRB and relevant monitoring authorities. All members of 
the project have received human subjects training and certification in FDA 
Good Clinical Practice through the Collaborative Institutional Training 
Initiative (CITI) curriculum. 
 
The following information will be collected to monitor risk: 
• The number of youth refusing the puberty questionnaire. 
• The number of youth refusing the MRI scan. 
• Any instance of gagging during the saliva collection. 
 
How often will risks be assessed? Every three months we will examine the 
number of youth refusing the MRI scan and adjust procedures, if needed, 
to improve participant tolerance. In our experience, few youth of this age 
who have agreed to come in for testing end up refusing the MRI scan once 
they are here.  
 
The individuals responsible for trial monitoring and advising the appointing 
entity. Because of the low level of risk in this study, the PI, Dr. Kathleen 
Thomas will be responsible for monitoring and reporting events for this 
project. 
 
18.0 Provisions to Protect the Privacy Interests of Participants 

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18.1 
Protecting Privacy: Once the participant has been tested, identifying 
information will be removed, and all materials will be identified with a 
participant number only. A master list with participant names, addresses 
and contact information will be maintained in secure REDcap database, 
separately from the data and study materials. A master list connecting 
study ID to participant name will be maintained in a password-protected 
database on REDCap. Only researchers involved in the study and having a 
legitimate reason for connecting data to an individual participant (e.g., in 
the event of a clinically-relevant incidental MRI finding) will have access to 
the file that merges participant number and personal information. Non-
data study materials (e.g., consent forms, payment forms, MRI safety 
screening forms) will be maintained in a secure REDcap database 
separately from the de-identified data. 
Considerable efforts are made to make participants comfortable, despite 
the nature of the stressor in the second visit. Consent and all study 
procedures will be conducted in private rooms. During consent/assent 
multiple efforts are made to make it clear that participation is optional and 
that the child is in control of refusal; for example, payment is promised at 
the start of the session rather than upon completion. Collection of 
biological measures, placement of electrodes, and administration of 
sensitive questionnaires are all done with a “matter of fact” attitude to 
minimize any feeling of embarrasment. Should a participant need to skip a 
portion or end a session early, experimenters are trained to accept this as a 
standard procedure when working with children rather than a problematic 
ending to the session. We have successfully guided hundreds of youth 
through these very procedures without undue problem, including 
longitudinal studies where the participants continue to return. 
Female participants who have begun menstruation will be verbally 
screened for possible pregnancy. If a child thinks she could be pregnant, we 
will not scan her. By Minnesota Statue, we are not allowed to break 
confidentiality to inform parents of a teen pregnancy. If a child tells us that 
she might be pregnant, we will offer her a urine pregnancy test to give her 
more information but will not require anyone to take a pregnancy test. If 
the test is positive, we will encourage her to tell her parents, and will offer 
to sit with the child while she informs a parent. If a child thinks she may be 
pregnant or has a positive pregnancy test, we will provide her with 
information on services that she can access to receive pre-natal care and 
counseling. 
18.2 
Access to Participants: This study does not involve access to participant 
medical records or other private records. Any personal information 
collected in this study will be provided directly by the participants 

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19.0 Compensation for Research-Related Injury 
19.1 Compensation for Research-Related Injury: All of the proposed procedures 
meet the NIH definition of minimal risk. In the event of an injury, costs will 
be charged to the participant or the participant’s medical insurance plan.  
19.2 Contract Language: N/A 
20.0 Consent Process 
20.1 Consent Process (when consent will be obtained):  
● 
All participants will be children, recruited by contacting parents.  Age 
will be verified with parent.  
● 
Parental permission will be obtained from: 
o 
One parent, even if the other parent is alive, known, competent, 
reasonably available, and shares legal responsibility for the care 
and custody of the child. 
● 
All consent and assent will be collected online via the e-consent 
framework on REDCap. Staff will be present on a video conference call 
with the parent and child for the full consent and assent process and 
will both explain the study and answer all questions. 
● 
On infrequent occasions, a parent requests that a grandparent or 
adult nanny be allowed to accompany the youth to the session.  In 
those cases, we will request the parent sign a consent form to send in 
with the child.  We will conduct the full consent & assent process as 
usual with the grandparent or adult nanny on site.  
● 
All youth will be part of the consent process, will be asked questions 
to verify understanding, including the question of “When are you 
allowed to skip a task or end the session?”. All youth will provide 
assent online. 
● 
Only those individuals listed as personnel on the study will be allowed 
to obtain consent. All listed researchers have completed HIPAA and 
CITI training, and have watched an in-house video of an ICD professor 
explaining and modeling appropriate consenting procedures. Further, 
all consenters have experience consenting/assenting families and 
children in this age range for studies of similar procedures. 
Consenters are trained both to follow a bullet-pointed list of key 
items as well as to sensitively discuss a range of concerns that parents 
or youth commonly raise. The consent process is a regular topic of 
weekly staff meetings, where researchers share experiences (without 
providing identifiable information) in order to help others learn novel 
approaches or gain tips for success.  
20.2 Waiver or Alteration of Consent Process (when consent will not be 
obtained):     N/A 

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20.3 Waiver of Written/Signed Documentation of Consent (when written/signed 
consent will not be obtained):    N/A 
20.4 Non-English Speaking Participants:    N/A 
20.5 Participants Who Are Not Yet Adults (infants, children, teenagers under 18 
years of age): 
● 
All participants will be children, recruited by contacting parents. Age 
will be verified by the parent.  
● 
Parental permission will be obtained from: 
o 
One parent, even if the other parent is alive, known, competent, 
reasonably available, and shares legal responsibility for the care 
and custody of the child. 
● 
On infrequent occasion, a parent requests that a grandparent be 
allowed to accompany the youth to the session. In those cases, we 
will request the parent sign a consent form to send in with the child.  
We will conduct the full consent & assent process as usual with the 
grandparent on site.  
● 
All participating youth will complete an onlinevassent form to 
accompany the online parental consent described above, in 
accordance with Minnesota statutes. The investigator will review the 
consent form with both the youth and the parent, and will ask the 
youth to tell them in their own words what will occur during the 
session (“In your own words, what is going to happen in today’s 
session?”). The investigator will then answer any questions the youth 
has and describe in more detail anything that she/he believes the 
adolescent does not fully understand. Each youth is reminded that 
they can stop at any time. We give them an “out” by saying, “Some 
people change their minds and decide this isn’t something they want 
to do right now, and that’s okay with us. Do you still want to go on?” 
If the youth does agree to participate in the study, s/he will sign an 
assent form. Similar procedures will be followed with the parents. 
Parents will be asked to describe in their own words what the child 
will be asked to do. If the parent seems unsure, the investigator will 
describe the procedures in another way and will provide corrective 
feedback. 
 
Recruitment procedures will make it clear that participation is 
voluntary and that families will still be able to participate in future 
research if they decline to participate in the current project. They may 
also remove their name from the participant database without 
prejudice if they wish. Parents will be mailed or emailed the consent 
forms and directions to the lab so that they have further time to 
make their decision before the session. Investigators will stress that 
parents and children are free to discontinue participation at any time 

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without penalty. Further, compensation will be promised at the time 
of consent/assent, eliminating the chance that someone may feel 
compelled to continue for the compensation. 
20.6 Cognitively Impaired Adults, or adults with fluctuating or diminished 
capacity to consent:    N/A 
20.7 Adults Unable to Consent:    N/A  
21.0 Setting 
21.1 Research Sites:  
● 
Participants will be recruited from the ICD registry of potential 
participants, or through direct response to posted advertisements.  
● 
Research will be conducted online via video conference, and at the 
Center for Magnetic Resonance Research (CMRR). 
21.2 International Research:    N/A  
 
22.0 Multi-Site Research:    N/A 
 
23.0 Resources Available 
23.1 Resources Available:  
● 
There are sufficient potential participants on the registry to meet the 
needs of the study design/cell count. In the age range we would need 
over the years of this proposal there are 14,529 girls and 15,284 boys 
from which to draw. 
● 
We anticipate that it will take approximately 2.5 years to complete 
recruitment and testing. We anticipate an additional 12 months 
needed to complete primary analyses of the MRI and physiological 
data. 
● 
The Center for Magnetic Resonance Research are ideal facilities to 
collect these data, with appropriate rooms, parking, private consent 
areas, and sibling/family-friendly waiting areas. The CMRR has a 
realistic MRI simulator that will be critical for this study. The CMRR 
houses 7 research-dedicated scanners, three of which are 3-Tesla 
scanners appropriate for the current study. Full-time staff maintain 
the scanners to ensure high-quality data acquisition, as well as linen 
services to provide necessary sheets, blankets, and hospital scrubs for 
participant safety and comfort. The CMRR has a -80 degree freezer 
for secure storage of biospecimen samples until samples can be 
batched for shipping to the assay labs. 
● 
We do not anticipate the need for either medical or psychological 
resources as risk is minimal in the current study. However, the CMRR 

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has First Aid and AED kits on-site, and islocated at on the medical 
campus of the University of Minnesota, providing close proximity to 
emergency medical services.   
● 
All staff & lab personnel, including undergraduate students working 
with de-identified data, undergo required and available ethics 
training. The study coordinator has over 25 years of experience 
training staff and conducting behavioral and psychophysiological 
research with children and adolescents. Dr. Thomas has over 20 years 
of experience in designing and running MRI studies with child 
participants, and in mentoring junior scientists in MRI methods. The 
staff scientist has 16 years of experience scanning children and 
supervising MRI data processing and analysis in Dr. Thomas’ lab. 
Weekly project meetings keep us adequately informed about any 
changes in protocol, updates to research procedures, or distribution 
of staff duties and functions. 
24.0 References 
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childhood and adolescence: Effects on HPA and oxytocin activity. Social 
Neuroscience, 25, 1-14. 
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puberty and age in explaining the diminished effectiveness of parental buffering of 
HPA reactivity and recovery in adolescence. Psychoneuroendocrinology, 59, 102-11. 
Efron, B., & Tibshirani, R. (1986). Bootstrap methods for standard errors, confidence 
intervals, and other measures of statistical accuracy. Statistical Science, 1(1), 54–77. 
Gunnar, M. R. (2017). Social buffering of stress in development: A career perspective. 
Perspectives in Psychological Science, 12, 355-373. 
Hostinar, C. E., Johnson, A. E., & Gunnar, M. R. (2015). Parent support is less effective in 
buffering cortisol stress reactivity for adolescents compared to children. 
Developmental Science, 18, 281-297. 
Hostinar, C. E., Sullivan, R. M., & Gunnar, M. R. (2014). Psychobiological mechanisms 
underlying the social buffering of the hypothalamic-pituitary-adrenocortical axis: A 
review of animal models and human studies across development. Psychological 
Bulletin,140 (1), 256-282. 
Kaplan, B. H., Cassal, J. C., & Gore, S. (1977). Social support and health. Medical Care, 15, 
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Laursen, B., & Hartl, A. C. (2013). Understanding loneliness during adolescence: 
developmental changes that increase the risk of perceived social isolation. Journal of 
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Little, R. J., & Rubin, D. B. (2014). Statistical analysis with missing data. John Wiley & 
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Rosseel, Y. (2012). lavaan: An R Package for Structural Equation Modeling. Journal of 
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Uchino, B. N., Bowen, K., Carlisle, M., & Birmingham, W. (2012). Psychological pathways 
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Williams, V. S., Jones, L. V., & Tukey, J. W. (1999). Controlling error in multiple 
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