Changes in Brain Function Through Repeated Emotion Regulation Training
Phase: NA
Conditions: Depression
Sponsor: Michael Stevens

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 Changes in Brain Function Through Repeated Emotion Regulation Training – Pilot Study
BACKGROUND/SIGNIFICANCE  
Emotion Regulation (ER) involves cognitive processes that change the onset, duration, content, or 
intensity an emotional response 1, 2.  Although emotions can be regulated by avoiding or distracting oneself so 
that the full brunt of emotional reaction is not engaged, in the proposed pilot study, we are concerned with ER 
strategies designed to modify an already-initiated emotional response. Such strategies are arguably the most 
well-studied to date, likely because emotional experience is unavoidable. Further, exaggerated emotional 
reactions, which are reflective of emotional dysregulation, are implicated in numerous psychiatric disorders. 
Thus, understanding how to influence emotional experience likely will have the most immediate translational 
potential.  
Over the past decade, the strategies people frequently use to regulate already-initiated emotional 
responses have been the topic of extensive research.  One way to regulate emotions is through “re-appraisal” 
of a stimulus.  Re-appraisal is often defined as an explicit ER strategy 3 that involves an alternative 
interpretation of a given stimulus or situation (in the form of thoughts or mental images) that alters or 
contradicts one’s immediate spontaneous appraisal 4, 5.  For example, seeing a picture of an angry man 
pointing a gun typically elicits a fear reaction/autonomic fight-or-flight response and prompts negative beliefs, 
action plans, and interpretations.  One can forestall or minimize such responses by reinterpreting the situation 
– e.g., realizing the man is a police officer pointing the gun at a dangerous criminal standing nearby.  
Experimental instructions to deliberately re-appraise the emotional experience of a scene involving threat of 
bodily harm, distress, etc. consistently lead to lower self-reports of negative emotion and lower emotion-related 
physiological reactions (e.g., electrodermal response or heart rate) 6-11.  In general, such re-appraisal tactics 
have been found to be more advantageous and linked to positive mental health compared to alternative 
response-focused ER strategies like suppression of emotional expression 12.
Another effective ER strategy to modify an already-initiated emotional response is called 
“mindfulness.”  Mindfulness emphasizes calm, non-judgmental observation of emotion.  Although it was 
derived from Eastern philosophic traditions, modern Western scientific study has validated its theoretical 
distinctiveness, construct validity, and clinical utility (see reviews; 13, 14).  Mindfulness involves the cultivation of 
emotional awareness, moment-to-moment attention to emotion, and acceptance of all emotional reactions 
(positive or negative) through meditative practice.  At the risk of theoretical over-simplification, while re-
appraisal involves engaging cognitive resources in a different way to lower emotional response, mindfulness 
instead emphasizes non-engagement to accomplish a comparable reduction in emotional reaction.  In terms of 
cognitively-controlled ER processes, they are similar with respect to recognition of negative affect, but differ in 
the need to re-establish stimulus-outcome associations with emotional arousal.  
 Over the past decade, nearly 4 
dozen functional neuroimaging studies 
have been conducted to identify the 
brain regions that are engaged to 
accomplish various forms of ER.  As a 
result, the brain regions engaged for 
cognitive re-appraisal ER are well-
known.  They have been reviewed 
numerous times 4, 15-19 including several 
fMRI 20 meta-analyses.  (See Fig. 1 for 
our fMRI replication of re-appraisal ER activation.  See 21, 22 
for all reappraisal fMRI studies, and 23 for fear-specific 
reappraisal.)  Several prefrontal cortex (PFC) regions, 
including vmPFC, dmPFC, dlPFC, dACC, vlPFC, and 
latOFC, are recruited during re-appraisal-based ER.  (See 
Table 1 to match abbreviations to anatomical regions and a 
brief summary of their specific theorized cognitive/emotional 
processing contributions to ER.).  The findings from these 
studies have prompted a reasonable working theory that 
prefrontal cortex regions exert control over the amygdala to 
Table 1.  ER Brain regions and their theorized cognitive relevance
Amygdala (AMYG)
Personal relevance detection that biases other 
brain region processing, usually seen w/emotion
Ventromedial prefrontal (vmPFC)
Representing the + or - motivational salience of 
S-R contingencies in context/goal-dependent way
Dorsolateral prefrontal (dlPFC)
Generating or holding re-appraisal strategies in 
mind to bias processing in other brain regions
Lateral orbitofrontal (latOFC)
Reward value/prediction representation
Dorsal anterior cingulate (dACC)
Performance and conflict monitoring
Dorsomedial prefrontal (dmPFC)
Interpreting one’s own emotional states
Ventrolateral prefrontal (vlPFC)
Goal-appropriate response selection 
Posterior intraparietal cortex (PPC)
Attention to reappraisal-relevant stimulus features
Fig. 1:  Pilot data showing greater left hemisphere brain 
activation during “re-appraisal” ER in n=24 healthy young 
adults compared to simple viewing of negative emotional 
stimuli (p<.05 FDR). Note, though not depicted, vmPFC 
ER activity was found at p<.001 uncorrected significance.
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reduce emotional reactions.  Strong circumstantial evidence for a possible PFC amygdala control relationship 
comes from the frequent observation of ↑ PFC with ↓ amygdala activity during ER (see 23).  Many studies also 
report inverse “functional connectivity” (i.e., correlation between activation decreases/increases in distal brain 
regions 24) between amygdala and various PFC regions 25-37 (particularly vmPFC and dmPFC 25-27, 30-33, 37). This 
supports a tentative model that successful ER involves prefrontal activation that orchestrates or inhibits 
amygdala/limbic reactivity.
A current NIMH-funded project in the CNDLAB at the Olin Neuropsychiatry Research Center (R01 
MH102854) uses fMRI to better describe the neural architecture of ER.  The goals of that project are to provide 
a validated model of how the causal influences among ER brain regions and to identify any abnormalities in 
how brain regions interact in adolescents diagnosed with Major Depressive Disorder (MDD).  The ultimate 
objective of that project is to identify neural targets for new ER treatment development.  Several major 
psychotherapies have been formulated upon ER core concepts.  Re-appraisal techniques are used in a variety 
of therapies, including new CBT variants still undergoing validation (38-40; see review 41).  Numerous therapies 
also incorporate mindfulness interventions, including Mindfulness-Based Stress Reduction (MBSR), which was 
originally designed to help patients with chronic pain 42, 43, but has since been shown efficacy for various 
clinical groups 14, 44-46.  Mindfulness-Based Cognitive Therapy (MBCT) was developed as a group-format 
therapy intended to prevent depression relapse (Segal et al 2002) by focusing on awareness of and 
relationship to thoughts and emotions instead of changing dysfunctional thoughts 47, 48.  MBCT is validated as 
useful for preventing relapse in treatment-resistant depression 48-52 as well as reducing active symptoms of 
depression 53, 54 and other disorders (55-57;see reviews; 14, 44, 45, 58).  More recently-developed therapies combine 
the strengths of re-appraisal and mindfulness into core intervention features (e.g., Adaptive Coping with 
Emotions), were these traits and the capacity to improve these skills predict patient outcome 59-61 and symptom 
severity 59, 62-64. Perhaps more well-known are the transdiagnostic, ER-focused therapies – such as the Unified 
Protocol for the Treatment of Emotional Disorders in adults 65 and youth 66.
SYNTHESIS AND FORMULATION  
Despite the intense neuroscientific and translational interest, these lines of research do not converge.  
At present, there is little understanding how any ER-based therapies influence ER brain function.  The goal of 
this pilot study is to collect preliminary data that will allow us to formulate theories about ER-related brain 
function change during intervention-like training that will be tested in larger, externally-funded grant proposals 
submitted to the National Institutes of Health.  We are not especially interested in any existing specific, 
manualized therapeutic technique. Existing ER-based psychotherapies typically incorporate a variety of related 
interventions, not all of which focus on the ER component itself.  Instead, we focus on the “building blocks” of 
such therapies, i.e., the ER interventions themselves and their effect on negative emotional reactions.  Here, 
we ask a specific question – How does ER-elicited brain function change with repeated sessions of guided 
practice of various ER techniques?  In other words, does increasing familiarity or mastery of ER skills through 
practice change brain function, and how?  There are logical hypotheses about the likely nature of brain function 
changes that can be gleaned from prior neuroimaging research.  For instance, if prefrontal cortex activation is 
important to ER, perhaps practice simply increases such activation.  However, our pilot work for the current 
NIMH R01 suggests instead that ER practice might work by enhancing the functional integration among key 
frontolimbic regions.  Complicating such hypotheses is the recognition that different ER tactics engage many 
distinct brain regions depending on which approach is employed.  So a person’s individual brain function 
differences prior to ER training might be a potent determinant for what sorts of neural changes result from 
repeated practice.  Because all these issues must be evaluated through the lens of a preliminary study before 
any coherent theoretical model can be formulated, we propose a pilot study here.  Not only will this provide 
empirical data upon which to base future research, conducting such a pilot will showcase any methodological 
hurdles or challenges that need to be overcome in order to successfully conduct a larger-scale study.
APPROACH  
N=40 adults (ages 18-50, approximately equal numbers of males and females) who are pre-screened to have 
mild-to-moderate levels of depression or who will serve as non-depressed/anxious controls will be recruited 
from community resources.  Inclusion criteria are described in detail in the Human Subjects section. In brief, 
we will operationalize inclusion for the depressed group as any participant who scores >1.5 SD from normative 
levels on the Beck-Depression Inventory-II.  Elevated trait anxiety on the Spielberger’s State/Trait Anxiety 
Inventory (STAI) will not disqualify inclusion (as depression and anxiety commonly co-occur).  Any depression 
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symptoms will not have to reach diagnosable levels of Major Depressive Disorder to qualify for the study.  The 
non-clinical control group will have minimal symptoms on the BDI-II and State/Trait scales.  Participants in 
each of these study groups then will be randomized into two conditions (n=10), each of which will receive 2 
assessment/MRI appointments along with 6 sessions of guided practice/training in either 1) Re-interpretation 
or 2) Mindfulness ER techniques.  If randomized into the CBT-based regulation training, subjects will be trained 
to use a cognitive strategy that instructs them to reappraise situations in positive or less negative ways.  If 
randomized into the MBT-based regulation training, subjects will be trained to use a mindfulness strategy that 
instructs them to notice and accept their feelings without judgment or intent to act upon them. In both 
conditions subjects will be shown pictures of negative-affective stimuli and asked to use the strategy over and 
over again.  These appointments will take place ‘virtually’ using a laptop computer that is set up at the 
participants house and is controlled remotely by Olin NRC staff.  The training sessions will then be monitored 
by the research assistant via Skype, so that the participant can be given instructions and supervised while 
completing each session.  “Training” in this context is conceptualized as the cumulative amount of practice, 
with the expectation that greater practice leads to greater mastery.  Immediately after the first and sixth 
practice sessions, participants also will undergo fMRI so that brain function relative to that stage of practice can 
be assessed.  In brief, training will be guided-practice sessions where examples of ER tactics are explained, 
modeled, then practiced with interactive discussion and feedback.  To clarify, the primary outcomes for this 
pilot study will be fMRI-assessed brain function data, examined either to detect any changes in activation 
amplitude (“level of activity”) in a priori ER-linked brain regions-of-interest (ROIs) (Table 1) or changes in the 
degree of functional connectivity among them.  Secondary outcomes will be the BDI-II and STAI. Because we 
have a well-established infrastructure that includes many other datatypes useful for emotion-related 
neuroscience, we also will collect a variety of other individual-level data for exploratory analyses.  By design, 
we propose to cast a wide net to collect information on the most relevant factors suggested by prior research to 
be explored.  All data collection methods are described next, followed by a brief data analysis plan.
The ~15 minute cognitive evaluation (Table 2) will exclude 
subjects with less than an eighth grade-normal educational 
achievement estimate using the Wide Range Achievement 
Test-4 243.  
Clinical Assessment  Information 
will be collected on participants’ 
age, 
race/ethnicity, 
years 
of 
education, handedness 
67, and 
family socioeconomic status 
68.  
DSM 5 psychiatric diagnoses will 
be assessed using the Structured 
Clinical 
Interview 
for 
DSM-5 
(SCID-5 
69) by a trained MA-
educated 
research 
assistant 
supervised in a weekly diagnosis 
consensus meeting by PI Stevens (a licensed clinical psychologist with over 15 years of experience 
supervising clinical assessment and SCID interviewing for several large-scale NIH-funded clinical 
neuroscience projects).  To ensure evaluation standardization and reliability, all assessments will be videoed 
and stored digitally. A thorough use history for major drugs of abuse (including nicotine dependence; i.e., 
Fagerstrom test 70), onset of substance use, quantity and frequency of alcohol/drug use, and number/type of 
treatment episodes (if any) will be obtained using the Olin NRC Healthcare Questionnaire, which includes a 
modified Timeline Follow-Back interview method 71.  This measure also assesses major medical conditions and 
any psychotropic medication use.  Familial psychiatric status will be assessed using the Family History Screen 
for Axis I disorders 72.  Table 3 lists other psychological test measures that will be used to characterize the 
sample.  They will be used in exploratory sub-group analysis of fMRI and other data.
Table 2.  Neurocognitive Test 
Task Name
Domain
Min
WRAT-4 Reading
Achievement
15
Table 3.  Other Psychological Test Measures
Task Name
Domain
Min
Spielberger State/Trait Anxiety Inventory
Self-report of characteristic anxiety
5
Beck Depression Inventory-II 315
Depression: a 21-item self-report     
measure assessing existence and 
severity of depressive symptoms
5
Columbia Suicide Severity Rating 
Scale318
Scale measuring suicidal ideation, 
intensity and behavior
10
Structured Trauma-Related Experience 
& Symptoms Screener319
Assesses  lifetime history of exposure 
to traumatic events and symptoms of 
PTSD
10
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Emotion Assessment (Table 4)  
This 30 minute assessment will 
quantify 
habitual 
use 
of 
re-
appraisal ER strategies, assess 
emotional reactivity and evaluate 
mood state before and after fMRI 
(to confirm there are no changes 
that 
impact 
study 
data 
interpretation).
fMRI 
Assessment 
Overview  
There will be 2 MRI sessions, 
within 5 days of the first and last 
ER training practice sessions.  MR 
images will be collected using the Olin NRC’s Siemens 3T Skyra, requiring ~2 hours in total, with ~1 hour in-
scanner.  All participants will wear hospital “scrubs” to minimize the chance of introducing metal into the MRI.  
Participants will provide a urine sample before MRI to be tested for the presence of drugs metabolites, and for 
pregnancy in females.  Drug testing results may be used as the basis to defer MR data collection and will be 
noted for post hoc analyses.  Caffeine/nicotine intake will be as normal for each individual to prevent any acute 
withdrawal effects on brain activity, restricted to >1 hour prior to MRI.
fMRI Stimulus Delivery/Response Recording  The ER fMRI task will be implemented using E-Prime 
software (Psychology Software Tools, Inc.), which records behavioral ratings for offline analysis.  Visual stimuli 
will be projected via a 5000 ANSI lumens system onto a screen located behind the patients head.  Participants 
view this screen using a mirror attached to the head coil.  An MR-compatible 5-button fiber optic response 
device (Current Designs, Inc.) will acquire behavioral responses. Participants communicate with staff during 
MRI using an MR-compatible auditory sound system delivered by 30 dB sound-attenuating headphones.
ER fMRI Task  Participants are shown pictures of negative-affective or neutral stimuli, and instructed to focus 
on each picture in one of three ways: (a) Reappraise the emotional image in a positive light (‘Reappraise' 
instruction); (b) Notice any feelings that arise in the present moment and attend to it with an attitude of 
acceptance (‘Accept' instruction); or (c) Simply look at the image ('Look' instruction). At the beginning of each 
trial, one of the 3 instruction words is presented in the middle of the screen, followed by a picture (negative-
affective picture if instruction was reappraise or accept; negative or neutral picture if instruction was look; 8s). 
After the picture presentation, participants are asked to provide a self-report rating of the magnitude of 
negative affective on a visual-analogue scale from 1 to 5, where 1 is labeled as ’not at all’ and 5 is labeled as ‘a 
lot’. Participants make responses by pressing on a button using a hand-held, MR-safe button-box. In sum, 
there are four trial types: (1) reappraise-negative image (CBT-based regulation); (2) accept-negative image 
(Mindfulness-based regulation); (3) look-negative image (no regulation); and (4) look-neutral image (non-
affective).  
ER Practice Sessions  During each of the 6 practice sessions, participants will undergo guided instruction 
with research staff.  They will focus only on decreasing emotion to negative stimuli, using whichever of the two 
emotion regulation strategies they are assigned to learn.  In each section, 25% of the stimuli will serve as a 
control similar to the fMRI task when they are simply asked to view the picture and let their emotional reaction 
unroll naturally.  On the other 75%, they will be guided in how to use their re-appraisal or mindfulness strategy.  
Practice will consist of guided instruction, question/answer about their attempted techniques, and use of 
examples for each stimulus if necessary.  An important component of practice is continual reminders and 
examples for how to use the strategy in real-life situations outside the experimental setting with the exact 
stimulus they are practicing with.  Prior research has shown that implementation instructions enhance 
generalization and rapid acquisition of emotion regulation skill.  Each practice session will last between 45-60 
minutes over the three-week practice period.  Importantly, the stimuli used for practice are carefully controlled 
in a way that exposes some pictures repeatedly, while others remain relatively novel.  At the end of the final 
appointment, participants will be tested on each category to tease out any effects of exposure to pictures 
Table 4.  Emotion Test Battery
Task Name
Domain
Min
Cognitive ER Questionnaire 73
Typical ER following negative events
5
Emotion Regulation Questionnaire  74
Habitual use of cognitive re-appraisal 
or suppression for + and - emotions
3
Beliefs of Self-Control Assessment 75
39-item self-report measure of emotion 
sensitivity, intensity, and persistence
5
Five Facets of Mindfullness 
Questionnaire  76
21-item self-report measure of the key 
elements of mindfulness
5
Emotional Reactivity Scale  77
21-item self-report measure of emotion 
sensitivity, intensity, and persistence
5
Profile of Mood States 2 78 Short Form
Mood before and after ER training 
5-10
Beliefs About Will Power Scale
Self-reported traits relating to willpower
3
Emotion Impulsivity Questionnaire
Newly created scale that measures a 
tendency to respond impulsively when 
emotionally aroused
2
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versus a generalized effect of improvement in their assigned emotion regulation tactic. 
Emotional Intensity/Arousal Measurements  Consistent with recommendations to measure ER success with 
convergent measures 83, we use self-ratings, electrodermal response and pupilometry from eye-tracking to 
obtain measures of emotional experience.  Self-Ratings  10 sec after each trial onset, participants will use the 
MR response device to rate on a Likert-scale (Fig. 5) how negative (or positive) each image made them feel.  
Following each fMRI/practice session, participants will indicate the confidence in their successful use of the 
strategy on a Likert-scale of 1-9.  They also will be asked to narratively describe how they approached 5 
sample negative stimuli from the fMRI task, along with confidence ratings of their accurate recall.  These 
responses will be digitally recorded, transcribed into text, then examined via word-frequency decomposition for 
the representativeness of emotion-laden words in their recounting (i.e., an indirect, supplemental means of 
assessing success or failure of ER for the reappraisal-based tasks).  During post-MRI image evaluation (~20 
min), subjects also will rate each stimuli’s valence, arousal, and dominance, using IAPS recommended 9-point 
Likert scales 84.  Skin Conductance Response (SCR)  Electrodermal SCR is an index of peripheral 
sympathetic autonomic arousal whose phasic activity has been linked to fMRI-measured activity in ER-related 
brain regions such as dlPFC, dmPFC, vmPFC, OFC, insula/vlPFC and amygdala 85-89, or amygdala as 
measured by invasive techniques 90, making it a valuable (if indirect) index of emotional arousal.  Although 
physiological measures recorded during ER generally track self-reports 6, 7, 25, 91-96. they do not always change 
in characteristic ways corresponding to simple dimensions of strong negative or positive emotion (see review 
97), or change at the same magnitude 6, suggesting they could provide unique information in ways that will be 
valuable to our hypothesis-testing.  Our recording system (www.biopac.com) is designed specifically for MRI 
acquisition use, incorporating signal processing circuitry to remove spurious MRI-induced artifacts in the 
electrical recording at the source and is integrated with MR paradigm delivery system for task-related data 
analysis.  The EDA100C-MRI amplifier will measure SCR during task trials using a 0.5 constant voltage signal 
across a pair of single-use dry electrodes, to which we will apply Biopac’s biocompatible, isotonic gel (GEL101) 
to ensure maximum electrical conductivity.  After electrode application, we wait >10 minutes while MR 
localizer/structural scans are being done to allow skin conductance level to stabilize.  Acqknowledge software 
will convert digital event pulses from E-Prime to data event markers.  SCRalyze (vb.2.1.7) 98 will parameterize 
phasic changes in SCR amplitude evoked by each event class, using a canonical SCR response function 99.  
Available secondary measures include SCR response latency/rise time, and spontaneous SC fluctuations 
indexing tonic arousal 100, as measured by psychophysiological DCM 101-103.  Exploratory analyses will see if 
they add value to study hypothesis-testing.  fMRI Eye Tracking  Participant gaze fixation will be monitored 
during the fMRI assessment to quantify what stimulus features are being preferentially processed on each trial.  
Although pupil constriction and dilation are more neurobiologically complex than SCR (i.e., are mediated by 
both sympathetic and parasympathetic influences) pupil size has been used as a measure of emotional 
arousal/attention (e.g., 104) and will be used as a secondary dependent variable.  Eye position and pupilometry 
will be monitored throughout fMRI using the Eyelink® 1000 Fiber Optic Core System, integrated with the MR 
paradigm delivery systems via 16-bit digital I/O for TTL-based triggering to synchronize data acquisition for 
offline analysis.  The system includes a non-ferromagnetic optimized infrared camera (1000 Hz) with long 
range mount.  The Eyelink 1000 emits infrared (IR) below the internationally accepted standards for safety 
(1mW per square cm). The IR illuminator and camera are mounted on a non-ferromagnetic stand outside of 
the MRI bore and makes use of the head coil mirror to view the participant’s eye position (the device does not 
touch the participant). Therefore, the distance between the eye and the camera is about 3 ft, far less than the 
distance known to be safe (IR intensity dissipates with distance). Exposure at these levels has been found safe 
for up to 8 hours, as the level of IR used is less than what is found in normal sunlight. In this project, IR 
exposure will be ~ 45 minutes. Setup requires ~5 minutes as each participant’s head is positioned in the MR 
coil so staff can acquire pupil position, refocus camera if needed, manually set pupil and corneal reflection 
thresholds, adjust search field, and perform/validate the nine-point HV9 calibration for tracking.  Only project 
staff who are specifically trained in Eyelink 1000 setup data collection will use the device. Our analysis 
approach will follow that described in 105, where we will define Emotional Areas-of-Interest based upon the 
entire sample’s composite fixation map using Eyelink Data Viewer software combined with participants’ post-
MR ratings of where the positive or negative features lie in each image.  Useful dependent measures will be 
fixation frequency/dwell and pupil size (trial - calibration baseline), segmented into early (1-3 sec), middle (4-6 
sec), and late (7-9 sec) trial phases.  
fMRI Task Length and Head Motion  Although task run length prompts concern over head motion-related 
experimental confounds/data loss, we routinely collect artifact-free data using similar task/scanning lengths.  
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Subjects practice in a mock-scanner, which simulates MRI experience and acclimates participants to the need 
to remain still.  During MRI, heads are secured in a custom-built cushion that reduces movement.  Motion is 
monitored in real time and unacceptably large head movements will prompt MR staff to restart acquisition.  
Data are examined within 12 hours and retained only if translation and rotation estimates are <1 voxel length 
and 3°, and if minor head motion is not significantly correlated with fMRI task conditions.  
MRI Sequences  fMRI gradient EPI (TR/TE 900/35 msec, flip 60 , multi-band AF=7).  Fieldmaps (TR/TE 
8400/62 msec, flip 80, AF=1, 0:25 min, run twice with reversed A>>P phase encoding) (EPI/fieldmap 
sequences have 2.1 mm isotropic voxels, 70 interleaved slices, 228 mm FOV). T1-weighted (3D MPRAGE, 
TR/TE/TI=2400/2.07/1000 msec, flip 8°, FOV=256×256mm, 0.8 mm isotropic vox; 7:02 min). T2-weighted 
(TR/TE=3200/565, FOV=256x256, 0.8 mm isotropic vox; 6:45 min). Structural images will be Radiologist-
assessed to be free of macroscopic pathology. Daily MR stability and QA measurements will ensure scans are 
of equal quality throughout the entire project.
Methods Used To Prepare MRI Data for Study Hypothesis Testing
HCP Pipeline MRI Processing  Our MR sequences were chosen for compatibility with Human Connectome 
Project 106 pre-processing pipelines 107, which provide highly accurate, structural image-guided brain atlas 
normalization for fMRI.  T1/T2 images will be ACPC-oriented, brain extracted, B0 inhomogeneity-corrected, 
mutually co-registered, distortion fieldmap-corrected, and finally MNI152 atlas-registered using FSL 
FLIRT+nonlinear FNIRT algorithms 108.  FreeSurfer-based 109 registration, skull-stripping, and pial extraction on 
1mm downsampled T1/T2 data will create structural volume/cortical ribbon files.  After fMRI EPI data is slice 
timing- and, motion-corrected, it will be registered to FS output, resampled to atlas space, intensity normalized, 
smoothed (volume data @4mm; cortical ribbon surface mapping data @2mm FWHM), and written as 
timeseries.  Both volume and surface-based fMRI data will be compared for hypothesis testing.
Conventional fMRI Analyses FSL FEAT analyses will estimate activation with FMRIB’s Improved Linear 
Model (FILM), using geodesic Gaussian algorithms to estimate autocorrelation and smooth fMRI data.  
Explanatory Variables (EVs) will use a double-gamma HRF convolution to translate event onsets in conditions 
of interest into regressors to be fit to the BOLD timeseries.  EV contrasts (against other conditions or implicit 
“rest”) will isolate task-induced activation, creating activation maps.  The sample data then will be evaluated 
using FSL’s FLAME random-effects models to confirm activation, compare group profiles, etc.  Random-effects 
models for hypothesis-testing will employ PALM 110, a relatively new FSL- and CIFTI-compatible analysis tool.  
Although we will focus on brain ROIs (Table 1), PALM offers both threshold-free cluster enhancement and 
corrections for multiple comparisons following permutation-based statistical inference to explore the possible 
effects of treatment on other brain regions with confidence.
ROI Localization Using Independent Component Analyses (ICA)  
Accurate ROI identification will utilize ICA, in part to reduce the data into a 
small handful of datapoints.  ICA was developed to solve the “cocktail 
party” problem in which many people are speaking at once 111.  ICA 
separates mixed signals into individual sources (e.g., “voices”) based on 
unique temporal characteristics of each signal.  When applied to fMRI 
data, spatial ICA assumes there is a set of non-systematically overlapping 
(temporally independent) brain regions (or small networks) with unique 
time courses.  When ICA fits a high model order to the data (i.e., asking 
for >80-130 components) (e.g., 112-115), components typically depict 
activity in just 1 or 2 brain regions (e.g., Fig 6), or isolate anatomical sub-regions (e.g., basolateral vs. 
centromedial nuclei).  Localization is quantitatively facilitated by correlation with a priori templates (e.g., MNI or 
cytoarchtectonic 116 atlases).  Our whole brain group fMRI ICA methods 117 include a reconstruction step that 
projects the group-derived ICA solution back onto each participant in a way that preserves individual variability 
in each spatial map and timecourse for further analysis.  We have used these ICA methods in many dozens of 
previous fMRI studies (e.g., 115, 118-130), and successfully applied ICA individual timecourses to DCM and other 
effective connectivity applications 119, 121.  Alternatively, semi-blind ICA methods we developed 131 balance 
atlas-based ROI selection (e.g., from ER meta-analysis 5, 23) with subject-specific data-driven influences.  
Finally, ICA is a widely-appreciated “denoising” technique, in fact originally designed to isolate meaningful 
signals from background noise.  Not only does it isolate signal corresponding to head motion artifacts from ROI 
activity, metrics can confirm ROI signals are free from motion-related noise (e.g., comparison of low-frequency 
/ high-frequency BOLD signal oscillations 115).  
Analysis Plan  The core analyses will employ a 4 (ER group) × 2 (training timepoint) mixed-effects ANOVA to 
Fig. 6:  ICA-localization to (A) bilateral 
dlPFC and (B) right BL amygdala 
(t=15 min threshold) from pilot data.
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test a general hypothesis that brain function or functional connectivity will change for the ROIs being studied. 
Because there will be 8 ROIs, we will consider results when using a Bonferroni-corrected α of .00625.  
However, because this is a pilot study, that correction will not be strictly imposed; rather, its use primarily will 
help us to decide whether any preliminary data might be publication-capable.  Supplemental Analyses:  
Because this is a pilot study, we plan to assess the relationship of fMRI-measured ER brain function change 
throughout training to numerous other factors.  Foremost, we need to evaluate whether non-neural measures 
of emotional function are altered by the ER training.  We will use repeated-measures ANOVA to assess 
whether or not self-ratings of emotional intensity, SCR, pupil size/fixation time to emotional stimuli change 
across the fMRI sessions.  We also will assess if POMS-2, BDI-II or STAI scores collected at baseline and in 
the final session differ, as these are clinically-relevant indicators of change that is independent of the ER 
training itself.  Typically in this sort of fMRI experimentation, the degree of change in these measures also is 
correlated with the degree of change in any brain regions found in the primary analysis to bolster the case of 
the potential clinical relevance of the findings in post hoc analyses.  
Power Analysis  Because this is a pilot study, one of its goals is to allow us to assess the size of any effects.  
Therefore, our sample size must be adequate so that the effort of data collection is worthwhile.  A hypothetical 
power analysis for a repeated-measures ANOVA for just one of the 3 experimental conditions can control Type 
II error (1-β=.80) effectively to detect “large” effect size differences (d=0.94) across the 2 fMRI sessions in any 
single brain region (including Greenhouse-Geiser corrections).  If that hypothetical model is expanded to 
include all three ER training groups simultaneously as planned, the total N=40 sample can detect large group 
difference effect sizes.  Changes in brain activity over time common to all groups can be detected if their effect 
size is as small as d=0.48 (or “medium”).  Finally, the planned analyses can detect d=0.56 effect sizes for the 
interaction of group × training timepoint.  This makes us confident that the proposed sample size will be 
adequate for both identifying scientifically-notable effects in the data and allow us to empirically-support their 
magnitude for future study design planning.  
HUMAN SUBJECTS
General Comments The investigators are well-informed regarding proper guidelines for ethical human 
research, and they will insure that all research activities are in full compliance. Furthermore, the investigators 
participate in ongoing training, including continuing education courses, seminars and online education dealing 
with topics specifically of interest to clinical researchers, offered by Yale and Hartford Hospital reflecting a 
commitment to the highest ethical standards in research.  All research participants will be competent adults 
who willingly provide their written consent to participate during a formal informed consent interview prior to 
research participation.  Careful attention is paid to screening of volunteers prior to participation and to 
monitoring them during participation to insure their protection.  All research volunteers with psychiatric or 
behavioral disorders are encouraged to engage in treatment for those conditions and are provided assistance 
in doing so (e.g., referrals).  If danger to self or others is discovered through psychiatric interview, steps will be 
taken to immediately ensure participant safety, e.g., by making appropriate treatment referrals, or involving 
authorities as per Connecticut state law in extreme cases of clear, imminent danger that  cannot be addressed 
in other means.  All volunteers will understand that research participation is voluntary, and that after enrollment 
they are free to discontinue at any time without prejudice whenever they so choose.  The investigators have 
extensive experience in neuroimaging research and have had no instances of significant research-related 
adverse events.  Volunteers generally report that they feel respected and well cared for by research staff.  We 
credit this safe and positive experience to careful attention to ensuring volunteer safety and to following proper 
guidelines for ethical human research.
RISKS TO THE SUBJECTS
Human Subjects Involvement and Characteristics  All potential participants will undergo initial telephone 
screening to ensure they meet general inclusion/exclusion criteria.  Qualified participants will then meet with 
study staff for informed consent.  Following consent, they will undergo psychiatric interview and complete a 
battery of questionnaires to ensure none of the study exclusion criteria are met.  At this point, participants will 
be randomized to one of the two ER training conditions (Reinterpretation or Mindfulness).  The protocol 
requires 6 visits over three consecutive weeks at a time that is convenient for participants, and that they agree 
they can accommodate.  We will attempt to structure an ideal schedule of appointments each week that follows 
a Mon/Wed, Tue/Thu, or Wed/Fri format.  At each appointment, participants will undergo guided training in a 
specific ER tactic.  Also, at the first and last MRI appointments, participants will complete a 60-minute fMRI 
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session.  Prior to fMRI, participants will be tested for the presence of drugs and pregnancy (if female) through 
urinalysis.
Inclusion/Exclusion Criteria  Inclusion: Ages 18-50, right-handed, a high-school level of education (11th 
grade attainment for 18 year-olds is acceptable if they haven’t completed their final year yet), and >8th grade 
English reading level to complete self-report evaluations (most are only available in English).  Exclusion:  
Medical history that will exclude any potential participant includes head injury sufficient to have caused >30 
minutes lost consciousness; past or current CNS disease (e.g., MS, epilepsy, tumor, etc.) or brain lesion 
identified by MR (structural MR scans will be reviewed by a radiologist for the presence of clinical 
neuropathology that may affect cognitive task performance); hypertension or juvenile-onset diabetes (current 
treatment with antihypertensives or insulin); current pregnancy (menstruating females will be tested).  
Psychiatric exclusion criteria include DSM-IV Axis I lifetime history of Bipolar disorder, PTSD, OCD, psychotic 
disorder, Tourette’s disorder, or any Pervasive Developmental Disorder (e.g., Autistic disorder, PDD NOS, 
etc.); current DSM-IV substance dependence, ADHD, Conduct Disorder.  Any participant who currently takes 
psychotropic medications will be excluded, even if psychiatric diagnostic criteria are not met.
Sources of Materials  Participants will be recruited from the community via public notices at community 
centers (i.e., YMCA, rec centers, libraries, etc.), local newspapers, internet-based resources (e.g., Craig’s List 
or Facebook), or through community support groups for families coping with mental illness.  We plan to 
balance our sample by gender and ensure that it will reflect the demographic characteristics of the Hartford 
region.  Research information includes psychiatric interview results, ratings made by participants on self-report 
questionnaires, medical history, family psychiatric history, MRI measures of brain structure and function 
collected during different neurocognitive tasks, electrodermal and eye-tracking responses during fMRI task 
completion, and identifying information such as name, DOB and contact information.
Potential Risks and Protection Against Risks The risks in this study involve procedures performed solely for 
the purposes of research. These risks include: psychological ratings, fMRI involving exposure to emotion-
eliciting images, skin conductance recordings, eye-tracking, discomfort from interviewing, urinalysis, risks 
related to privacy/confidentiality of records, and unanticipated risks. 
Psychological Ratings: Patients may find the battery of psychiatric and neuropsychological tests tedious 
and boring, or intrusive. If the former, brief breaks will be given during assessment. The latter concern is 
addressed through use of trained interviewers who have experience discussing sensitive material with clinical 
research subjects.  However, if a subject finds a topic too uncomfortable or sensitive, those questions will be 
skipped, or with severe reactions, subjects will be debriefed and reminded they can discontinue participation 
without penalty, if necessary.
FMRI:  The FDA has approved the MRI system and the scanning protocols to be used. The MRI scanning 
procedure in a 3 Tesla system is safe in normal healthy humans. However, subjects who could suffer potential 
risks from the MRI scanning procedure include those with metal implants (pacemaker, metal rods, bone 
screws, orthodontics, metal flakes from metalworking).  Subjects are told during the informed consent process 
about the need to carefully screen for metal implants or any other exposure to metal in the body. Subjects who 
could potentially suffer harm will be screened for metal prior to scanning using a HH Radiology Department 
screening checklist, and on the day of the MRI will change clothes prior to entering the MRI suite, be scanned 
for metal using a whole-body ferromagnetic scanner, and be verbally checked by the MR technologist 
performing the scan.  Although there is no proven harm to developing fetuses, women will be screened for 
pregnancy before scanning.  Urinalysis for a one-step pregnancy test is conducted on the scan day to ensure 
that female participants are not pregnant.  Participants with claustrophobia may experience some anxiety 
symptoms, and will be discouraged from participating.  Subjects who feel claustrophobic during the experiment 
will be encouraged to notify experimenters at any time to discontinue scanning.  There are no known risks 
associated with the visual or auditory stimuli presented during recording of event-related fMRI.  All structural 
MR results are reviewed by Hartford Hospital Radiology staff and a brief clinical report prepared if there seems 
to be a need for more in depth evaluation.  Any indication of pathology/abnormality is communicated to Co-PI 
Dr. Pearlson (a licensed psychiatrist), who then will order a full clinical MR for the participant.  If participants 
cannot assume the cost of the MR, one is provided to them by the Olin NRC.  This procedure ensures that any 
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risk to health can be identified and proper clinical planning can be done.
Emotional Stimuli During MRI:  The IAPS stimuli to be used in this project are well-known to include images 
of a graphic or sexual nature.  We have used such stimuli in previous NIH- and foundation-funded projects 
(i.e., R01 MH102854, K23 MH070036 and a recent grant from the American Foundation for Suicide 
Prevention) to probe emotional neural systems.  We do not utilize any erotica stimuli at all.  The informed 
consent procedure carefully and explicitly discusses the issue of image content, and offering several examples 
of stimuli similar to those used in the experimentation.  They also are reminded immediately before fMRI that 
they can discontinue at any time during the experimentation without penalty and still receive compensation for 
that part of the research.  After fMRI, participants are debriefed, which includes initiating a discussion of their 
overall reaction to the experiment, how the stimuli made them feel, whether they have any questions or 
problems that they would like to talk about further, etc.  Note, our previous experience using tasks that include 
IAPS stimuli for fMRI assessment in >200 adolescent and young adult participants have never met with any 
unforeseen, negative reaction after using these procedures.
Skin Conductance Recording During MRI:  Risks of physiological recording during MRI include problems 
related to ferromagnetic equipment being introduced into the MR environment, induced voltages, and 
radiofrequency (RF) heating 132.  The Biopac electrodermal recording devices we use for MR are classified as 
MR conditionally safe by the American Society for Testing of Materials 133.  During scanning, time-varying 
magnetic fields are generated by the gradient coils and RF transmitter, which induce voltages across any 
conductive loops that can possibly stimulate nerves or heat tissue.  These risks are reduced by ensuring that 
MRI-approved electrodes will be placed between 3-5 cm apart in a configuration that avoids loops, “S” or “U” 
shapes.  Electrode leads will not be twisted and none of the radio-translucent, MRI-compatible wires on SCR-
recording electrodes cross on the body.  Electrode wires will be run out of the chamber bore in the straightest 
line possible.  After electrode setup with the patient supine in the MR, all SCR wires will be secured with 
surgical tape at several locations (min 1’ apart) to ensure that casual body movement during the ~1 hour MRI 
session will not displace them.  A blanket will cover each participant to ensure wires do not touch the skin.  All 
electrodes will be checked to ensure close contact with the skin (including use of conductive gel) so that air 
gaps will not break the electric pathway to build up heat at the electrode center and cause burns.  Participants 
are reminded before and periodically during MRI to report any heating or tingling sensations, which will prompt 
MR staff safety checks of the SCR recording equipment setup.
Infrared Eyetracking During MRI:  The Eyelink® 1000 Fiber Optic eyetracking system is approved for use 
within an MRI environment.  The device conforms to numerous international safety standards (e.g., IEC 60825-
1, the Safety of Laser Products standard) which covers laser and LED devices.  The Eyelink 1000 is classified 
as a Class 1 LED device, the highest safety certification, which states that it is “safe under reasonably 
foreseeable conditions of operation, including the use of optical instruments for intrabeam viewing.”  Class 1 
classification is similar to that granted for door motion sensors, TV remotes, etc.  Therefore any risks of IR 
exposure to the eye are minimal.  Moreover, IR camera use will be limited to <1 hour throughout the entire MRI 
experiment.
Videotaped Interview:  Some participants may be uncomfortable with the idea of being videotaped during 
interviews or with research staff having access to their medical files.  These study procedures are explained in 
detail during informed consent procedures so that subjects may make an informed decision whether to 
participate in the study.  To assure the confidentiality and protection of participants with respect to videotaping, 
videos are stored in digital format and are directly uploaded to a secure, HIPAA-compliant database at the Olin 
NRC immediately following interview; any local copies of the file are destroyed.
Audiotaped Task Debriefing Responses: All digitally recorded responses collected during the task 
debriefing procedure will be uploaded onto a computer and saved as an electronic file to be transcribed.. 
Linguistic transcription will be stored on the same secure, HIPAA-compliant database as the videotaped 
interviews. All electronic files will be destroyed within 6 years following completion of the study.
Urine Collection:  Urine specimens are collected to test menstruating females for pregnancy and to monitor 
unreported substance use prior to MRI scanning and therefore serve as safeguards to participants.  It adds no 
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risks other than those normally associated with providing a specimen in a private bathroom.
Psychological status: During the course of the evaluation, it is possible that some participants may express 
either suicidal or homicidal ideation or intent.  During consent, potential participants are advised that these 
issues will be cause to break confidentiality to ensure the safety of the participant or others.  The Olin NRC has 
established procedures to escort participants to the Hartford Hospital Emergency Department psychiatric 
intake if necessary, or to Children’s Hospital ED (which is contiguous with Hartford Hospital/The Institute of 
Living campus on the same city block, roughly the same distance from the Olin NRC as Hartford Hospital’s 
ED).  If study personnel ascertain that participants have increased risk to self or others, the study PI will 
contact the participant’s treatment provider, if any, or police (if necessary).  If an unmet treatment need is 
discovered during the course of psychiatric assessment, study staff under direction of the Principal Investigator 
will recommend further treatment to the participant.  The Olin NRC maintains an updated list of both IOL and 
non-IOL community practitioners for most psychiatric or substance disorder treatment services.  However, 
study personnel cannot break confidentiality to ensure that participants seek treatment for unmet treatment 
needs if there is not an imminent danger to self or others.  The final exception is that researchers are 
mandated reporters of child abuse to the CT Department of Children and Families.  This role also is explained 
to participants during informed consent.
Unanticipated risks: Any experiment may involve risks that we cannot predict or anticipate.  Participants will 
be told of any important new information that might affect their decision to continue in the study.  In addition, 
the IRB at Hartford Hospital will be notified if any new risks are identified. 
Confidentiality: Confidentiality risks will be minimized by using coded records and by storing signed 
consent forms in double-locked file cabinets in a secure area of the Olin NRC.  Electronic records are kept in 
HIPAA-compliant databases, with secure, user account/password-controlled access to specific records.  All 
procedures are fully-HIPPA compliant and approved by HH Research Administration.  All information and data 
concerning volunteers are confidential and never released to anyone outside of the project without the 
volunteer's written authorization.  The identity of participants is never revealed in research reports.
Adequacy of Protection Against Risks
Informed Consent  Informed consent is obtained in writing from all volunteers prior to research participation 
by a trained research staff.  Informed consent meetings will always include a trained research staff member 
and the potential participant.  The detailed consent form will be read to and discussed with these persons.  
They will be given several opportunities to ask questions and will have the option to watch movies of the 
laboratory procedures before signing the consent.  Prior to agreeing to be in study, it is reinforced that 
participants can discontinue at any time without prejudice and that questions at any time are encouraged.  All 
potential participants are offered an opportunity to review Hartford Hospital’s Patient Bill of Rights and given 
phone numbers of Olin NRC faculty and disinterested third-party Hartford Hospital officials who oversee 
research activities or advocate patient rights if there are any complaints. Understanding of study procedures is 
ascertained by the potential participant’s ability to repeat back the procedures and the risks involved in their 
own words.  Any doubts about a potential subject’s willingness to participate and their understanding, the PI 
will opt to exclude the subject.
Potential Benefits of the Proposed Research to the Subjects and Others / Importance of the Knowledge 
to be Gained  Although a substantial investment of time and effort will be required of research participants for 
clinical interviewing, ER training, and MRI scanning, these methods are fairly non-invasive with regard to 
importance of information obtained.  Such information is routinely collected in clinical research.  Appropriate 
safeguards are in place to protect the well-being and confidentiality of subjects who agree to participate in the 
study.  Furthermore, participants will be compensated monetarily for their time to a degree that does not offer 
an undue incentive for their cooperation.  Therefore, we judge the risks and the intrusion to subjects to be 
minimal.  The scientific benefits of the planned study are high, given the scientific importance of understanding 
the neurobiology of emotion regulation network changed following ER training.  With the safeguards in place as 
described above, we believe the risks of the study are low.  The risk-benefit ratio appears, therefore, to be 
favorable.
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Data Safety and Monitoring Plan  Whether or not this study should be considered a clinical trial is debatable.  
On the one hand, we will randomize participants into conditions and train them in how to regulate their 
emotions with separate tactics.  Moreover, we are specifically recruiting a sample who will range from having 
mildly-elevated depression/anxiety symptoms to those who might meet DSM 5 diagnostic criteria for MDD or 
anxiety disorders.  However, we purposefully are not employing any standardized treatment protocol in favor of 
using a “bare-bones” training-based intervention that provides guided practice on specific ways to regulate 
negative affect.  We will not present this study opportunity to potential participants with any expectation that by 
the end of the study they will have achieved any relief for any mood or anxiety problems.  Unless directed by 
the IRB to construct a more formal DSMP, we plan to follow standard Hartford Hospital adverse event reporting 
guidelines, which require reporting within 24 hours to the hospital Research Administration and Institutional 
Review Board any serious adverse event (i.e., serious injury or death related to the investigation) and within 7 
days for minor adverse effects.  However, if a DSMP typically is deemed appropriate by the IRB, we will 
formulate one that is appropriate to the perceived low-risk entailed in this experimental protocol.
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