Brain and Genetic Predictors of Individual Differences in Pain and Analgesia
Conditions: Pain, Acute
Sponsor: Trustees of Dartmouth College

Brain and Genetic Predictors of Individual 
Differences in Pain and Analgesia 
(Pain Genetics) 
 
NCT03781570 
 
Study Protocol 
 
4/11/2023 
 
 

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TITLE:  Brain and Genetic Predictors of Individual Differences in Pain and Placebo Analgesia 
 
PROTOCOL VERSION DATE: 11 April 2023 
VERSION: 16 
PRINCIPAL INVESTIGATOR (PI):  
Name: Naomi Friedman  
Address: Institute for Behavioral Genetics, Campus Box: UCB 447 
Telephone: (303)-735-4457 
Email: Naomi.Friedman@Colorado.edu 
KEY PERSONNEL 
Name: Corinne Gunn 
Role in project: Research Coordinator 
 
 
Name: Marta Ceko 
Role in project: Co-Investigator 
  
Name: Matt Keller 
 
Role in project: Co-Investigator 
 
Name: Tor Wager, Dartmouth College   
Role in project: Co-Investigator 
 
Name: Andrew Smolen 
Role in project: Co-investigator 
 
Name: Bogdan Petre, Dartmouth College 
Role in project: Research Coordinator 
 
Name: Patricia Townsend 
Role in project: Research Coordinator 
 

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Name: Daniel Ryan 
Role in project: Research Coordinator 
 
 
I. 
OBJECTIVES 
The goal of this study is to characterize the neurophysiological mechanisms that make an individual a 
strong or weak placebo responder, and provide behavioral and genetic markers for these individual 
differences. This would provide a tool for characterizing likely placebo responders in clinical settings and 
clinical trials, with two benefits. First, it could be used to better separate placebo vs. verum treatment 
effects, allowing for better targeting of the mechanisms of each type of effect.  Second, it could be used 
to eliminate or control for placebo responses in clinical trials, increasing efficiency and trial success 
rates. 
II. 
BACKGROUND AND SIGNIFICANCE  
Throughout history, placebo effects have been variously considered as tricks played upon the gullible by 
medical practitioners and powerful but mysterious healing forces.  With the advent of direct 
measurements of human brain function, modern science has shown that placebo effects are neither of 
these. Rather, they reflect the principled impact of psychological and brain processes on diseases of the 
brain and body. Placebo effects represent an opportunity because they provide a window into internal 
brain processes that influence health, and a challenge because many clinical trials have now failed due to 
large and durable placebo responses, at great cost to health care providers and consumers.  
Definitive studies of the brain pathways involved in placebo responses—and the genetic, 
environmental, and neural factors that lead some individuals to respond more strongly than others—are 
critical to harnessing placebo effects, eliminating or controlling placebo responses in clinical trials, and 
understanding the psychological and brain factors that predispose one to successful treatment and 
“spontaneous” improvement. Placebo analgesia is the best-studied type of placebo effect, with well-
developed paradigms and preliminary data on its brain mechanisms.  This background provides a 
foundation for larger-scale, definitive studies.  
In this project, we propose the first such large-scale study of brain mechanisms of placebo 
analgesia, combining neuroimaging, behavioral, and genetic approaches.  It builds on 15 years’ experience 
in PI Wager’s laboratory on fMRI and placebo analgesia and 40 years of genetics research at the Institute 
for Behavior Genetics (IBG) at the University of Colorado, Boulder.  We will use fMRI to characterize the 
neural bases of placebo effects in 600 twins recruited from the Colorado Twin Sample and predict 
individual differences in placebo effects across two forms of pain. In Aim 1, we will develop models that 
predict the magnitude of individuals’ placebo effects in pain and pain neurophysiology based on a) fMRI 
activity, b) brain structure, and a combination of personality, behavioral, and cognitive measures that can 
be deployed clinically. In Aim 2, we conduct the first analyses of heritability of placebo effects and their 
neural predictors, and genetic correlations that can identify brain features whose relationships with 
placebo effects are genetic in origin. In Aim 3, we leverage the >50,000 person Enhancing Neuro-Imaging 

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Genetics through Meta-analysis (ENIGMA) consortium to identify genome-wide associations with 
placebo-linked brain features and develop polygenic risk scores for placebo effects. The research products 
from this endeavor will include data and models useful for characterizing and screening participants in 
clinical trials, assessing interactions between placebo responses and other treatments, and assessing 
placebo effects across disorders.  
III. 
PRELIMINARY STUDIES 
Large placebo responses occur in clinical trials of multiple mental health and neurological disorders, 
including Parkinson’s1-3, autism4-7, epilepsy8, schizophrenia9, 10, migraine11-13, other forms of chronic pain14-
17, and other brain disorders18-21. Fifteen years of research on brain mechanisms of placebo by our lab and 
others has found that placebo effects are reliably caused by active neurobiological processes that must be 
studied and understood in relation to health and disease20, 22-26. Placebo analgesia is the best studied area, 
but evidence suggests mechanistic overlap with placebo effects in Parkinson’s disease27-30 and 
depression31-35. These include prefrontal-striatal-brainstem interactions and involvement of endogenous 
neurochemicals (opioids, dopamine, cannabinoids, serotonin, and others) that may form a common 
substrate for influences of affective systems and context across disorders22-24.  Understanding the 
neuroscience of placebo is a key to understanding endogenous brain contributions to disease vulnerability 
and resilience, and maximizing clinical benefit. It is also a key to increasing success rates in clinical trials. 
A growing number of trials are failing because of large placebo responses9, 16, 18, 36, with large economic 
and public health costs. The ability to eliminate or control for placebo responses in clinical trials would 
help bring successful drugs to market. 
IV. 
RESEARCH STUDY DESIGN  
 
 
We will recruit 600 twins (300 same-sex pairs; 150 monozygotic and 150 dizygotic; ~50% female) from 
the Colorado Community Twin Sample (CTS). The CTS includes 727 same-sex twin pairs between 26-38 
years of age (as of Spring 2017), originally derived from the Colorado Twin Registry, a population based 
registry, housed at the Institute for Behavioral Genetics (IBG) at the University of Colorado since 1984, 
with three previous waves of data collection (1997-2002, 2002-2008, and 2009-2014)380. The CTS 
participants are on average 37.8 years old (SD 2.5), ~50% female, mean education 14.5 years, 80% non-
Hispanic Caucasian, 14% Hispanic, 4% African-American, 2% Asian, and < 1% Native American. ~75% of 
the sample still lives in Colorado. Twin recruitment: CTS pairs will be recruited via direct contact by IBG 
staff Corinne Gunn, who has worked with this sample for 15 years. The recruitment rate for repeat visits 
has been 87%; we need ~40% for the proposed study to be viable. Thus, we will be able to meet 
recruitment needs. An ongoing fMRI study of a separate sample of twins (target ~320 adult twin pairs) 
from our group (studying cognitive control; MH063207, PI Friedman, Co-I Wager) additionally 
demonstrates recruitment feasibility: to date we have tested 356 individuals and only 7 have declined to 
participate, 29 are not eligible for MRI, and 10 are deceased. Recruitment for pain studies: Extensive 
published and preliminary work by our group demonstrates that we can deliver intensities in the painful 
range that are safe and tolerable for virtually all participants. In fMRI studies we have run over the past 

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several years (N = 495 for heat and N = 81 for pressure), only 2 participants have dropped out due to 
pain intolerance.  
A.2 Experimental design  
A.2.1. Behavioral assessments. Before the main neuroimaging session, participants will complete a 
battery of online behavioral measures, including measures of (a) personality and (b) cognitive control 
(Fig. 3). At a six to twelve-month follow-up, participants will be contacted to complete a series of tasks, 
including tasks that measure (a) perceptual/decision biases and (b) affective biases. Personality and 
affective state measures include those with previous relationships with placebo effects in smaller 
studies (reviewed above), particularly the Life Optimism Test Revised (LOT-R381), Behavioral 
Inhibition/Behavioral Activation (BIS/BAS382), State/Trait Anxiety Inventory (STAI59, 240, 383), Beck 
Depression Inventory (BDI384), Fear of Pain (FOP385), Ego Resilience (ER-89386), and the “Big 5” brief 
inventory (neuroticism, openness, agreeableness, extraversion, conscientiousness387). 
Perceptual/decision bias tasks are adaptations of sensory suggestibility tasks predictive of placebo 
effects in past studies388-390. A series of perceptual judgments are made of the color, size, etc. of visible 
objects, and the test assesses whether judgments are biased by the identities of the objects. Affective 
bias measures use a well-validated task that assesses whether individuals consistently identify 
ambiguous faces and images as positive or negative in valence390, 391. Cognitive control measures include 
tasks studied extensively by our group392-395, including measures of cognitive flexibility (letter/number 
task switching), response selection (go/no go, stop-signal, and antisaccade), and working memory (N-
back and keep-track tasks). These tasks will be administered online in the month before scanning 
through the study website (www.paingen.science) in which all data will be stored in the secure RedCap 
system396, with reaction time-based tasks implemented using Javascript embedding. Prior data collection 
waves on the CTS sample include neuroticism and extroversion397, reward-dependence398, harm-
avoidance398 and behavioral disinhibition399, and standardized  cognitive tests (PIAT-R, WISC-III), which 
we will also correlate with placebo outcomes.  In order to test functionality and gather preliminary data 
for these online measures, we will recruit a pilot sample of 1000 ‘online only’ participants using 
Amazon’s Mechanical turk system. The link for mturk participants will be: www.paingen.science/mtruk.  
An additional behavioral assessment titled ‘Skin Prick Test (Allergy test)’ will be administered after 
the fMRI tasks on the day of testing at the Center for Innovation and Creativity (CINC). This task is 
modeled after previous research done in the Stanford Psychology department (Howe, Grover, Crum, 
2017) in which 164 participants received the skin prick test with no adverse side effects. All subjects who 
are cleared for this task (based on a short in person screening procedure on the day of testing: see 
attachment ‘skin prick test screening and allergy task instruction’ document) will participate after the 

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fMRI scanning procedures. The allergy test will involve participants experiencing an induced itch through 
a standardized ‘skin prick test’ involving administration of a drop of 1mg/ml histamine gel (1% histamine 
dihydrochloride in 2.5% methylcellulose). Skin prick tests involving histamine are used as controls in 
routine allergy diagnosis tests. Typical allergy tests are done with actual (food or environmental) 
allergens. However, we will not be using any allergens in this procedure. Histamine is an organic 
compound naturally produced by the body and is classified as a monoamine neurotransmitter.  
 This test has been used in numerous studies to induce itching (e.g., Bromm, Scharein, Darsow, & 
Ring, 1995; Darsow, Ring, Scharein, & Bromm, 1996; Pfab et al., 2006, 2010). Further, this procedure 
induces an itching sensation, but did not cause pain or any other sensations to any participants in 
previous studies. Histamine will be applied to two skin sites on the participant’s non-dominant volar 
forearm. Two inert creams will be administered to the participant with the suggestion that one will 
‘decrease the allergic reaction and itching’ and the other will ‘increase the allergic reaction and itching’ 
(counterbalanced between subjects). Allergic reactions or ‘wheals’ will be measured for 30 minutes after 
application. This task is designed to measure participant’s placebo response(s) in a non-painful scenario. 
This will allow us to compare different types of placebo responses, expectations, and genetic factors 
that contribute to the placebo effect.  
 A.2.2. fMRI study design. The design of the proposed fMRI experiment is based closely on our work 
on fMRI of placebo analgesia over the last 15 years221, 400-409, including recent studies comparing 
intensity-matched thermal and mechanical pain (N = 81 fMRI subjects run to date). There are two main 
phases during fMRI: In the placebo administration phase, participants are given two identical creams 
with different instructions: “Prodicaine, an effective pain-relieving drug” (Placebo) and “a control cream 
with no effects” (Control) applied to 4 fingers each on the left hand, with  
locations counterbalanced across subjects. Disclosure of side effects are presented on forms with 
realistic-looking drug company logos (see attachment ‘drug_info_packet_script_PAIN-GEN’), and testing 
is done in a medical environment with medical equipment and related context cues. A 2-minute delay 
follows for the “cream to take effect”, during which we will ask the participant to complete the survey 
“side effects questionnaire”.  Structural (T1-weighted) images will be collected prior to any of the 
placebo tests. A post-conditioning test phase tests both pain types on and off placebo treatment after 
reinforcement. We have successfully used similar procedures414, as have others222 (e.g., C. Büchel, N = 
700 with suggestion-alone and post-conditioning, no imaging) Rating scale: Ratings of pain intensity and 
unpleasantness will be made after each trial on a 100-point generalized visual analogue scale415-418 with 
anchors of “no pain”  or “no unpleasantness” and “most intense sensation imaginable,” or “Most 
unpleasantness imaginable”, using an fMRI-compatible trackball221.  
Stimulation and trial details. A Control-Placebo-Control design (e.g., 222, 414) in each stimulation 
phase permits assessments of placebo effects within individuals, without confounding placebo with 
habituation (or sensitization) to pain and avoiding order effects. Based on prior work, we expect little to 
no habituation of pain responses with this protocol221, 288, 414, 419, 420. Each trial will consist of: (1) A 
warning cue, followed by a variable-duration anticipation interval (4-16 sec with exponential 
distribution, 6 sec mean 73, 221); (2) thermal or mechanical pain  (10 sec plateau with 2 sec ramp 
up/down); (3) a 4-16 sec jittered delay followed by rating scales (4 sec each); (4) a 4-16 sec inter-trial 
interval. Ramps minimize head movement during stimulation to very low levels (0.07 mm average37). 
Standard stimulus intensities will permit assessment of individual differences in brain and behavior 
holding objective stimulus intensity constant144, 421. Based on previous calibration studies in our lab (heat 
N = 495, pressure N = 81), we will select heat and pressure intensities matched for pain in the 

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population. Max “high pain” intensities of 49 C and 7 kg/cm2 are expected to be “very painful” for ~83% 
of the sample but tolerable for nearly all (98%) of participants144, 270, 421. A variable-duration anticipation 
period separates anticipatory activity from stimulus processing. 
 
For the thermal stimuli, the temperatures will range from 43 to 49 °C, and the pressure will 
range from 4 to 7 kg/cm2. As part of the screening process, we will perform a pain calibration procedure 
with the thermode (Medoc, Inc.) on each participant in order to ensure that all temperatures delivered 
during any subsequent procedures are within participants’ tolerable range. Pain stimulation will be 
performed using a contact thermode (Medoc, Inc.) that is placed against the skin. The thermode is 
actively heated and cooled by the hardware of the device. Temperature values are controlled to within 
.1 degrees Celsius by a computer, with a safety shutoff at a level tolerable to some participants, and 
nondamaging to skin (the hardware will not allow stimulation at 50 degrees Celsius for longer than 6 
seconds). Stimulation will not exceed 50 degrees Celsius in any of the studies covered under this 
protocol unless explicitly noted in a separate protocol. 
 
The pain calibration procedure consists of applying temperatures to the subject's right index 
finger. Participants rate each stimulus on a visual-analog scale. Eligibility for participation in the 
behavioral or fMRI portion of this study involving pain requires that participants have pain tolerances 
that are not too high or too low—i.e., those that would require delivery of temperatures above the 
boundaries stipulated by the guidelines detailed below, or that do not allow for sufficient experimental 
variation of the temperatures delivered. 
 For pressure stimuli, the newly developed pressure pain device (Attachment 13) will be used within 
the safe range based on our pilot data and previous studies (Attachment 14: “Pressure pain pilot study 
results” and Attachment 15: “Pressure pain stimulation guideline”). 
During post-conditioning test phases, participants experience 32 medium-intensity trials divided 
equally among 4 conditions: Tests on Placebo and Control sites crossed with heat (47 C) vs. pressure 
(5.5 kg/cm2), all on separate fingers. Our previous analyses of NPS “pain signature” responses indicate 
that 7 trials or more per condition will yield 90% classification accuracy for discriminating moderate (1 
s.d.) differences in pain intensity37, 38, 270; thus, 8 trials per condition here is adequate. The conditioning 
phase is similar, except that stimulation will be applied at high intensities (49 C or 7 kg/cm2) on Control 
sites and low intensities (45 C or 4 kg/cm2) on Placebo sites, to associate placebo treatment with 
reduced pain. We will test different fingers during conditioning to avoid skin site-specific habituation 
effects from influencing test phases.  
A2.3. Genetic assessments. IBG has over 40 years’ experience as a leading center for behavioral 
genetics, and extensive experience collecting and analyzing Genome-Wide Association Study (GWAS) 
data. Our team also has substantial expertise in neuroimaging genetics (co-Is Friedman) and analysis of 
GWAS data (co-Is Keller). We will collect saliva samples during the fMRI scan day and genotype using the 
same facility (same laboratory, personnel, platform, and protocols), following standard GWAS quality 
control procedures422 (e.g., for minor allele frequencies, Hardy-Weinberg equilibrium, call rates, etc.).  
A.3 Analysis plan and hypotheses 
Aim 1: Analyses 1a-1c identify brain and psychological predictors of placebo analgesia, respectively. 
Placebo effects will be defined as Control vs. Placebo ([C – P]) differences in pain report (“placebo 
analgesia”) and two validated, pain-related brain measures: the NPS and SIIPS. These analyses will allow 
us to identify common and differential brain predictors of placebo effects on behavioral (pain report) 

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and neurobiological outcomes. They will permit three other novel tests: (1) Whether predictive patterns 
generalize across thermal and mechanical pain; (2) Whether they generalize across placebo effects in 
suggestion alone or post-conditioning; and (3) Whether we can identify latent factors across outcomes 
(thermal/mechanical, suggestion/conditioning), and develop predictors of those domain-general 
outcomes.  Such factors are more likely to be genetically determined and useful in broader clinical 
contexts.  
Multiple comparisons and power: 
This study is the first large-scale study 
powered for whole-brain analyses of 
structural and functional correlates of 
placebo analgesia with Family-Wise Error 
Rate (FWER) map-wise correction. We 
will use FWER correction as 
implemented in Statistical 
Nonparametric Mapping (SnPM)423. This 
correction is valid, makes minimal 
assumptions, and is not affected by 
recent findings of inflated false positive 
rates with some correction methods424. 
With our sample size (N = 600), we have 
80% power with whole-brain FWER 
correction to detect correlations with 
placebo analgesia of r ≥ 0.22, which is 
within the range of our previous cross-
validated estimates 37, 268, 269, 353, 354, 369. 
Previous studies have been substantially 
underpowered; with N = 50, which is larger than most placebo studies, correlations must be r = 0.65 or 
larger to be detectable with 80% power. Though this is the first study adequately powered for whole-
brain correction, we will also focus on testing hypotheses derived from the previous literature, as 
described below.  
Analysis 1a identifies functional brain predictors of placebo analgesia. We focus on two systems 
related to placebo analgesia35, 409 (Fig. 1a): A fronto-parietal system involved in executive control and 
cognitive appraisal of emotion425-431, and a medial cortical-striatal-brainstem system—including mid-
lateral OFC, vmPFC, VS/nAC, and PAG. We hypothesize that behavioral analgesia will be related to 
anticipatory fronto-parietal activity and down-regulation of SIIPS responses (related to endogenous 
brain contributors to pain), but not modulation of the NPS (related to nociceptive pain), indicating that 
reported analgesia is influenced by decision bias and providing brain correlates of this bias. 
Alternatively, analgesia may be correlated with NPS modulation and reductions in local targets of 
nociceptive afferents (S2, dpINS, mid-INS, aMCC), validating pain report as a predictor of neurobiological 
placebo effects. Increases in the medial system may also predict larger placebo effects on the NPS, 
linking this system to strong control of pain in placebo-responsive individuals. 
Analysis 1b identifies structural brain predictors of placebo analgesia. We hypothesize that gray-
matter density in the medial cortical-striatal-brainstem system, particularly nAC and vmPFC, will predict 
larger placebo responses in pain report, NPS, and SIIPS responses. Prior work suggests that gray-matter 

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density in the hippocampus432, 433 and fronto-parietal system273 may also be related to placebo effects. 
Diffusion-weighted imaging (DWI) will be used to assess the size/integrity of fiber tracts. We expect 
placebo effects to be associated with tracts connecting the vmPFC and OFC with the PAG261; but 
alternatively, connectivity in the fronto-parietal system (esp. dlPFC) and lateral frontal-brainstem 
connectivity may be more predictive. Such findings would connect placebo effects to systems involved 
in value and motivation, on one hand, or cognitive control on the other. This would permit evaluation of 
the parallel between pain regulation and other forms of cognitive regulation, and connect placebo 
effects with systems that degrade with age and dementia. 
Analysis 1c will test whether placebo effects on behavioral and brain measures of analgesia are 
related to personality and behavioral measures that can be easily assessed clinically. We will apply 
confirmatory factor analysis to web-based assessments to identify latent factors434 related to (a) 
susceptibility to perceptual/decision bias (i.e., influences of instructions on judgments of shape, color, 
etc.); (b) positive vs. negative interpretations of ambiguous events; (c) latent variables related to task 
switching, inhibition, and working memory identified in our previous work331-336; and (d) personality 
measures, focusing on an “optimistic/pro-engagement” style and an “anxious” style. We will conduct 
five-fold cross-validated analyses435-437 developing an optimal combination of behavioral variables for 
predicting placebo analgesia. We expect that placebo analgesia will be related to (a) larger 
perceptual/decision biases282, (b) high optimism438, and (c) low anxiety and fear of pain291-293. We are 
agnostic about whether cognitive control measures will predict placebo analgesia, but it is important to 
know either way; some studies suggest placebo effects are related to cognitive control438, 439, whereas 
others suggest they are distinct forms of motivation-based regulation257, 440. Positive results from 
Analysis 1c will provide a behavioral phenotype that can be readily assessed in clinical settings. We can 
then relate these behavioral measures to brain measures, as in Analysis 1a, to link them to 
neurobiology. Negative results will also be very important, as several companies are currently investing 
in personality-based predictors for clinical settings. The field needs to know whether or not such 
measures are viable predictors of placebo response magnitude. 
Extensions and alternative plans. (1) We are developing new large-sample predictors of pain, and will 
test those as they become available. (2) Another important extension relates to individual differences in 
pain sensitivity. While not the main focus here, this dataset and our approaches are well-suited to 
predicting individual differences in pain sensitivity—a core feature of many pain conditions441-443 and a 
risk factor for later chronic pain 444-446. Models of risk for chronic pain following surgery (unfortunately 
common) and other procedures would be a crucial advance in precision medicine447-452. Risks can be 
assessed at both phenotypic (predicted by evoked pain sensitivity) and genetic453 levels. Exploratory 
analysis will allow us to develop a foundation for understanding the neurobiology of individual differences 
in pain sensitivity. 
Aim 2: Analyses 2a-2c will assess the heritability of placebo effects and placebo-predictive brain 
features, and relate these features to candidate gene families. Analysis 2a (Aim 2.1) identifies the 
heritability of placebo effects in pain report and brain responses for the first time. We hypothesize that 
heritability of pain reports will be modest, but heritability of placebo modulation of specific pain-related 
brain measures (the NPS and SIIPS) will be stronger, as these reflect component systems that are less 
etiologically complex and less context-sensitive that overall pain report. We also expect heritability to be 
high for gray-matter density in nAC and hippocampus454, and mPFC-brainstem pathways. The model for 
analysis 2a is a standard univariate ACE model455,456, which decomposes twin covariances for a trait of 
interest into additive genetic (A), shared environmental (C), and non-shared environmental (E) variance 

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components (Fig. 5). This model will be applied to individual placebo measures (e.g., thermal and 
mechanical pain), and also to latent variables (i.e., using a common factor model with thermal and 
mechanical pain loadings constrained to equality to identify the factor).  
Analysis 2b (Aim 2.2) identifies genetic 
correlations between placebo effects and brain 
features that predict them. It tests whether 
individuals who are more genetically related show 
higher cross-twin, cross-trait correlations. The 
model for this analysis is a bivariate extension of 
the ACE model455, 456 (Fig. 5b). The covariance 
between two measures is decomposed into 
genetic and environmental components with a 
Cholesky decomposition. The ACE variables for the 
first measure (e.g., placebo analgesia) are allowed 
to predict the second trait, and the second trait is 
allowed to have unique ACE influences. Of primary 
interest for Aim 2.2 is the genetic correlation (rA) 
of the two phenotypes, which indicates the extent 
to which the genetic influences overlap across the 
two traits: rA = a12/√(a122 + a22); rC and rE can also be computed analogously. We will apply this model 
to relationships between analgesia and pain neurophysiology (i.e., NPS and SIIPS), and functional and 
structural features that predict placebo effects (e.g., nAC density, fronto-striatal activity). We expect the 
strongest genetic correlations in structural MRI measures that are likely highly genetically determined457. 
Structural features that are both placebo-linked and genetically associated with placebo (e.g., nAC gray-
matter density) are good candidates for polygenic risk scores in Aim 3. Power: Assuming a moderate E 
variance of 30%, we will be able to detect genetic variance as low as 34% and shared environmental 
variance as low as 36% with >80% power. With an E variance of 40%, we can detect an A of 45% and C of 
40% with >80% power. Genetic correlations: We will have power to detect either an rA or rC in the 
presence of an rE: Assuming 40% A and 30% C and E variances for both variables, we will be able to 
detect rAs of at least .36 and rEs as low as .22 with >80% power. Assuming 40% C and 30% A and E 
variances for both variables and correlations between both Cs and Es, we will be able to detect an rC of 
.34 and rE of .19 with >80% power. 
In Analysis 2c, we will attempt to replicate previously identified associations between candidate 
genes and placebo effects (reviewed in 376). We expect the most meaningful results to come from 
polygenic risk scores (see below), but it is important for the field to know whether the candidate genes 
already identified predict placebo responses or not. Based on previously reported associations, our study 
is well powered to determine this. We focus on SNPs within (+/- 10 kb of gene boundaries) several 
neurotransmitter/ neuropeptide gene families previously linked with placebo analgesia in 
pharmacological and molecular imaging studies, including catecholamine and monoamine clearance 
(COMT, MAO-A), opioids (OPRD1, OPRM1, OPCML, SIGMAR1, OPRK1, PDYN, OGFR), dopamine (DBH, 
DRD5, PPP1R1B, DRD1, SLC6A3, DRD4, DRD2, DRD3, CALY), serotonin (5-HT; HTR[1-3]A/B, HTR[1-3]B, 
HTR1[D-F], HTR2C, HTR3C/D/E, HTR5A/B, HTR[4, 6, 7], MAOA, SLC6A4), cannabinoids (CNR1, CNR2, 
CNRIP1), and cholecystokinin (CCK; CCKAR, CCKBR, CCK, DBI), and BDNF. We will test for statistical 
enrichment 
in 
SNP 
associations 
within 
these 
gene 
families 
using 
the 
MAGMA 

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[doi:10.1371/journal.pcbi.1004219] and VEGAS [http://dx.doi.org/10.1016/j.ajhg.2010.06.009] software 
tools. This set comprises 48 genes with an estimated 320 genotyped SNPs and 4800 imputed SNPs with 
MAF > .005. Most of these SNPs do not exist on the Affymetrix Axiom Precision Medicine chip, but they 
can be imputed with excellent fidelity from genotyped SNPs using the Haplotype Reference Consortium 
reference sequence database [doi:10.1038/ng.3643], which has replaced the 1kG reference database as 
the gold standard. This approach is now standard for GWAS. We estimate imputation of ~97% of candidate 
SNPs with ~99% accuracy using the MACH program458. Power: Assuming correction across ~50 candidate 
genes, we will have 80% power to detect effects of d = 0.24 (“small”) or greater, substantially smaller than 
most previously reported associations. Hypotheses: Previous studies reported that opioid genes were 
related to fMRI signal in PAG and vmPFC459-461; dopamine genes to enhanced placebo after conditioning, 
compared to suggestion alone462, 463; and serotonin and CCK genes to (a) anticipatory activity in vmPFC, 
which correlates negatively with placebo analgesia63, 70, 464 and positively with anxiety408, and (b) activity 
in the serotonergic dorsal raphe nucleus (adjacent to the PAG), which is activated in placebo studies162, 403, 
465, 466) and is linked to threat and analgesia in animal models467-469. 
Aim 3: Analysis 3. These analyses will develop Polygenic Risk Scores (PRSs) for placebo-linked brain 
structural features in the ENIGMA consortium dataset (Fig 6). The ENIGMA consortium is the world’s 
largest study of relationships between brain structure and GWAS470 . ENIGMA contains no personally 
identifiable information; investigators share summary statistics from analyses back to the requestor, in 
this case the Wager Lab. As such, it is a meta-analysis and does not involve sharing of identifiable data 
which could be linked to participants. In Aim 3, we leverage the information provided by the smaller, pain- 
and placebo-phenotyped twin sample to take advantage of the information in the larger, but less 
extensively phenotyped, consortium sample. Structural brain features with the highest genetic 
correlations with placebo response in our study—e.g., gray-matter density in nAC, vmPFC, and 
hippocampus—will be extracted from T1 data and regressed on GWAS SNP data in the ENIGMA sample., 
We estimate including at least N = 10,000 individuals, with a target of N = 30,000. PRSs will be developed 
by regressing individual SNPs on structural features using linkage disequilibrium (LD) regression471, 472 as 
implemented in the PLINK package 472. Regression weights on SNPs comprise a model that can be applied 
to new GWAS data to calculate an individual’s aggregate genetic risk130. We employ several choices shown 
to be advantageous, including using a wider set of SNPs in the model472, 473 and pruning SNPs in linkage 
with the strongest signals (e.g., r2>0.05)130, 474. Finally, to estimate the effects of this PRS on placebo 
responses, we will correlate PRSs with clinical placebo responses in the Women’s Health Study (N = 5,800 
with GWAS data) in collaboration with Consultant K. Hall. Testing aggregate risk involves only a single test, 
with >80% power to detect even extremely small relationships (e.g., r2 = 0.002) between PRS scores and 
placebo responses.  
Extensions and alternative plans. (1) While we expect ENIGMA to be an outstanding resource for 
these analyses, other options exist. The UK Biobank recently released its first 10,000 participants475, and 

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it includes structural and diffusion imaging and GWAS; similar to the ENIGMA consortium, UK Biobank 
data is de-identified and cannot be linked to individual participants. The ENIGMA consortium have 
access to this data. (2) In addition to LD models for PRS development, we will explore using the machine 
learning models used for the main fMRI analysis, adapted for binomial allelic outcomes (5-fold cross-
validation stratified by data-collection site, modeling covariance across twins).  This will allow us to train 
models on a subset (4/5) of the twin sample, train PRSs on the ENIGMA sample, and then “back-
validate” the relationships with placebo effects on an independent (1/5) of the twin sample.  This will 
also let us identify psychological/behavioral correlates the PRSs in the twin sample. (3) Finally, new 
informatics methods are actively being developed to conduct more elaborate analyses on the ENIGMA 
data. As these methods become available, another extension is to run cross-validated analyses 
identifying more complex patterns of structural changes in the twins, identifying PRSs in ENIGMA, and 
back-validating in independent subsets of the twin data. (4) We will conduct standard GWAS with 
whole-genome correction (p < 5e-8) further corrected for number of brain variables, with 80% power to 
detect SNPs correlated at r2 > .006, as well as the 6 gene families discussed above, with virtually perfect 
power to detect SNPs with even extremely small effects (r2 > .006).  
A.4 Data acquisition and model specifics  
fMRI acquisition and analysis. Images will be acquired using the University of Colorado’s 3T Prisma 
scanner with 32-channel parallel imaging. Structural T1 (0.7 mm isotropic) will follow the 10,000-person 
Adolescent Brain Cognitive Development (ABCD) study protocol; CU Boulder is a project site and PI 
Wager and Co-I Friedman are members of the study team. Functional T2*-weighted echo-planar imaging 
(EPI) data will be acquired using a multiband sequence (MB factor 8, TR=460ms, 56 slices, 2.7 x 2.7 x 2.7 
mm, TE=29 ms, FA=52˚) aligned to the plane of the OFC. Head movement will be minimized by soft 
restraint as in the ABCD study; we are the study site with the best quality control measures among the 
18 sites in tests to date. We anticipate <1 mm displacement / 1.5° rotation within-run in ~95% of 
participants. State-of-the art image preprocessing techniques (SPM12; Wellcome Department of 
Cognitive Neurology, UCL), use procedures detailed in our published work 406, 408, 476-480, with 
improvements described below. Diffusion-weighted imaging (DWI) for tractography will follow the UK 
Biobank protocol (100 diffusion-encoding directions, 2 shells, 7-min acquisition; 481, 482).  Preprocessing of 
structural images: inhomogeneity correction, coregistration to the mean realigned functional image, 
enhanced nonlinear normalization to standard brain space using SPM12 plus a custom genetic 
algorithm406. DWI images: DWI analysis will follow the UK Biobank pipeline protocol483 implemented in 
FSL484, 485, including distortion correction, fractional anisotropy, tensor mode and mean diffusivity 
estimation (DTIFIT), Neurite Orientation Dispersion and Density Imaging modelling(NODDI)485, 486, 
registration to standard space using Tract-based Spatial Statistics (TBSS), and probabilistic tractography 
with crossing fiber modelling (bedpostx/probtrackx)487-490.  Outputs will be used to run tractography 
from seed regions of interest (dlPFC, vmPFC, PAG, mid-lateral OFC), and from each brain region in 
standard parcellations491, 492 for pattern-recognition based analyses. Preprocessing of functional images: 
Time series outlier/artifact assessment; realignment/motion correction; application of normalization 
parameters, high-pass filtering with an optimized cutoff. Head movement: High motion (0.3 mm) and 
high image-to-image root mean square successive differences (q < 0.05 FDR) modeled as nuisance 
covariates. Additionally, 24 movement-related regressors per run in first-level GLM493, 494. First-level 
functional models: Two-level General Linear Model (GLM). First level: locally smoothed AR(2) model, 
linear and higher-order movement-related covariates493, 494, flexible hemodynamics optimized for 

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pain406, 495-498. Event timing optimized using a genetic algorithm (GA)499, design efficiency evaluated for 
each individual. Second level: (1) Voxelwise hierarchical mixed-effects modeling with restricted iterative 
generalized least squares (rIGLS)500 accounting for correlated errors across DZ/MZ twins; or (2) 
multivariate machine learning models.  
The scanning portion of the study will take place at the Intermountain Neuroimaging Consortium at 
the Center for Innovation and Creativity at 1777 Exposition Dr., Boulder, CO 80301. The MRI device for 
these scans is FDA approved for research with human subjects and has all the safety inherent in a clinical 
MRI scanner.  The radio frequency fields conform to guidelines determined by the FDA and the FDA has 
designated MRI scanners to be a non-significant risk device. MR techniques non-invasively produce 
images and measurements from tissues in the intact, living human.   
 
Statistical learning framework for fMRI and molecular genetic analyses. In contrast to typical voxel-
wise mapping fMRI analyses and SNP-by-SNP GWA analyses, multivariate predictive models predict 
outcomes as a combination of (pattern across) multiple voxels/SNPs. This model yields an aggregate 
predictive score based on all available brain/SNP information, which is validated by applying it to new 
individual test subjects. Placebo analgesia ([C – P] in pain report) and changes in pain-related NPS and 
SIIPS responses ([C – P]) are the outcomes to be explained. We use LASSO-PCR408 or Least Absolute 
Shrinkage and Selection Operator501 Principal Components Regression (Fig. 2; this compares favorably 
with popular support vector regression). Data reduction: principal components analysis retaining N-2 
orthogonal predictors; Penalized regression of component scores onto the outcome uses LASSO 
shrinkage, simplifying the model. Cross-validation is five-fold, balanced cross-validation502 in discovery 
sample (N = 500), with  N = 100 reserved as a final test set435. These methods provide stable, robust 
estimates when there are more predictor variables (voxels or SNPs) than observations, and account for 
covariance among predictors (i.e., linkage disequilibrium in SNPs503). In addition, feature (variable) 
selection using our prior work on placebo analgesia408 will simplify the models and increase 
interpretability. Tests of voxel and SNP importance will be accomplished using a null-hypothesis 
permutation test408, 423, 504 (p < 0.05 FWER). In molecular genetic analyses, feature selection will be 
accomplished by (a) focusing on polymorphisms related to six key placebo-linked gene families, or (b) 
optimizing the inclusion threshold and sparsity503 via nested cross-validation. 
Study timeline  
Data collection and analysis will take place in each year of this 5-year project. We anticipate testing 
~10 subjects/month, for target totals of 115 in Year 1, 135 in Year 2, 120 in Year 3, 120 in Year 4, and 
115 in Year 5. We project 3 months of development and pilot fMRI scanning time to ensure that the 
paradigm and image acquisition are optimized for the main study. Prior arrangements with the Brain 
Imaging Center ensure that adequate time will be available for testing. fMRI and genetic data analysis 
will be conducted on a rolling basis. Preliminary reports will be written and submitted for publication in 
Years 2-4 that focus on group fMRI effects of placebo and methodology of machine learning applications 
to fMRI and genetic data. Final analysis and a report on the full sample will be prepared in Months 7-12 
of Year 5.  
 

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V. 
ABOUT THE SUBJECTS  
 
Subject Population(s) 
Number to be enrolled in each group  
Pilot Participants  
35 (30 behavioral only + 5 fMRI)  
Colorado Community Twin Sample (CTS) 
~700 
MTurk Pilot Participants 
1000 
 
All CTS participants will be screened over the phone by Corinne Gunn. Corinne will walk through the 
attachment ‘fmri twin recruitment script’ which will lead to the ‘fmri safety screening’ (attachment 
‘18_twin_fmri_safety_screening (1).doc’) questions if the potential participant is willing to be in the study. 
We will primarily recruit individuals aged 18-55 years who are fMRI compatible (passed the ‘fMRI safety 
screening’), capable of performing experimental tasks (e.g., are able to read), are fluent or native speakers 
of English, and have no current or recent history of reported neurological disorders. Participants less than 
18 years old will be excluded because of population vulnerability issues. Participants over 55 years of age 
will be excluded based on a diminished sensitivity to pain and changes in brain structure that require 
special studies of older populations, which is outside the scope of this study.  
 
In addition, we will also exclude people who cannot tolerate the maximum level of thermal pain stimuli 
(for thermal stimuli: 49 °C). 
 
Participants who have any contraindication to magnetic resonance scanning (e.g., metal in body, 
claustrophobia, pregnant) will be excluded from the study. These exclusions are specific to MRI and are 
consistent with most studies involving MRI. Potential participants will be screened for the presence of 
any of these exclusion criteria prior to participating in this MRI study (see attachments 18 and 19). 
 
For M-Turk Participants: Recruitment will only target workers who are above the age of 18 (all Mturk 
works are already required to be fluent in the English language).  We will also only recruit MTurk 
workers with the “master worker” designation and with a 95% previous work approval rate, to increase 
the likelihood of obtaining good quality data.  No other inclusion/exclusion criteria will be used. The 
website link for Mturk participants will be www.paingen.science/mturk. 
 
VI. 
VULNERABLE POPULATIONS 
We will not exclude anyone based on class or income so there is a possibility that economically 
disadvantaged individuals will be enrolled in this study, but no other vulnerable populations will be 
included, and recruitment will not target vulnerable populations. 
 
VII. 
RECRUITMENT METHODS 

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We will recruit 700 twins (350 same-sex pairs; 175 monozygotic and 175 dizygotic; ~50% female) from 
the Colorado Community Twin Sample (CTS). The CTS includes 727 same-sex twin pairs between 26-38 
years old (as of Spring 2017), originally derived from the Colorado Twin Registry, a population based 
registry, housed at the Institute for Behavioral Genetics (IBG) at the University of Colorado since 1984, 
with three previous waves of data collection (1997-2002, 2002-2008, and 2009-2014)380. The CTS 
participants are on average 37.8 years old (SD 2.5), ~50% female, mean education 14.5 years, 80% non-
Hispanic Caucasian, 14% Hispanic, 4% African-American, 2% Asian, and < 1% Native American. ~75% of 
the sample still lives in Colorado.  
 
Twin recruitment and screening: Colorado subjects are drawn exclusively from the samples of past 
participants in the CTS. They will be contacted by telephone and invited to learn more about the study 
(See Attachment #17). If they agree to participate, the interview will be scheduled and instructions for 
filling out the online consent form and questionnaires will be conveyed over the phone. CTS pairs will be 
recruited via direct contact by IBG staff Corinne Gunn, who has worked with this sample for 15 years. 
Corinne will also conduct the fmri safety screening (attachment 18) over the phone. All participants who 
express willingness to participate in this study will then be given a Docusign consent form by Corinne via 
email. Once a participant has signed the consent form, Corinne will mail them a brochure (see 
document: ‘pain-mailer10.02’). This brochure will be used as additional recruitment material. The 
brochure contains information about the study (includes some deception to maintain the notion that 
this is a drug study), Corinne’s contact information, and a link to the website which they will be filling 
out the online tasks (www.paingen.science). Since participants are able to stop and start the online 
surveys at any time, we hope that this brochure will serve as a physical reminder for participants.  
 
MTurk recruitment: Mturk participants will be recruited through Amazon’s Mechanical Turk. Workers 
that fill out surveys will be able to see the web based task as a HIT (Human Intelligence Task; title: Brain 
and Genetic Predictors of Individual Differences in Pain and Placebo Analgesia) on a posting board 
including thousands of other HITs.  
Pain study screening: Pain screening will be conducted when participants arrive at CINC (see 
Procedures: pain calibration task).  
 
 
VIII. 
COMPENSATION  
Laboratory participants will be given $200, in cash or electronically, at the end of the study. We will also 
give participants an image of their MRI scan, and a 3-D model of their brain created on a 3-D printer. 
Participants who withdraw prior to study completion will be paid pro-rated for their time to the nearest 
hour.  
List recruitment methods/materials and attach a copy of each in eRA 
1. 17_fmri_twin_recruitment_script.docx 
2. Pain-mailer10.02 

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Some participants in the CTS may be residing outside of the state of Colorado or the city of Boulder. 
Participants will be reimbursed for all travel costs (airfare or car travel over 1 hour) and a room will be 
purchased at the Best Western Hotel in Boulder, CO (770 28th St, Boulder, CO 80303) by the Wager lab 
for the time needed to complete this study.  
 
Twin participants who participate in completing the follow up surveys will be paid an additional $25, which 
will be sent to them via mail. 
 
M-Turk Participants will be compensated with $0.1 per minute.  We worked out this pay rate because 
higher pay rate makes respondents repeat the survey, which ends up creating useless data. Participants 
who do not complete the study will not be paid.  Participants who provide poor quality data 
demonstrating careless responding (i.e. too many surveys open at the same time; or improper data 
responding using certain data quality question checks) will not be paid.  Participants will be notified in 
advance that data quality measures are in place, and participants with poor data will not be paid. 
 
IX. 
CONSENT PROCESS 
Corinne Gunn will contact participants on the CTS list to describe the study (attachment 17) over the 
phone and check for interest in participation, then conduct an fMRI safety screening if they agree to 
participate. Then, we will obtain an additional experiment specific consent (Attachment 2), via Docusign, 
provided by Corinne after the initial phone screening. The online consent form will have the Wager lab’s 
email and phone number contact information if they have any questions. When the participant arrives at 
CINC the day of the fMRI scan, they will meet a research coordinator who will remind them of the study’s 
procedures. The research coordinator will have a copy of the consent form to re-confirm the participant’s 
willingness to participate and go through the procedures of the study. The participant’s voluntary 
participation is stressed in that they are informed, both verbally and in writing, that they can discontinue 
the study at any time.  
 
To study the effects of individual differences in placebo responding, some deception is required.  
Specifically, we will inform participants that a cream will be applied to the skin sites that will receive heat 
and pressure pain stimulation in order to test the effects of a pain relieving topical treatment, Prodicaine. 
We believe that our experiment qualifies for a Waiver of Some Elements of consent.  As is detailed in the 
“Risks to Participants” section, substantial regular testing of our laboratory equipment and standardized 
safety procedures for the use of stimulus delivery systems ensure that the risk of injury to participants is 
minimal.  The use of deception in this way will not adversely affect the welfare of the subjects in this 
experiment, as our use of placebos here is designed to attenuate the experience of pain, not increase it.  
Deception is inherently necessary to induce placebo effects, and research in this area cannot be 
performed without it.  A debriefing session will be held (Attachments 11 and 12) upon termination of the 
study, where participants will be informed of all aspects of the deception used, and be able to voice any 
questions or concerns they have.  
 

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As a part of the consent form, participants will indicate whether they wish to be contacted for future 
studies.  If so, a member of the research team will store contact information for this participant in a secure 
location, outlined in the “Data Management” section, so that this participant may be contacted in the 
future.   
 
For M-Turk Participants: Informed consent will be administered as the first page of the survey.  
Participants will be required to answer a yes/no question, “Do you consent to being part of this study?”.  
Only those who answer ‘yes’ will be advanced to the next page of the survey.  We reformatted the 
consent form template provided by the IRB to be more suitable for the online environment.  All the 
information from the IRB template was included in the reformatted (condensed) consent from attached 
to this protocol (see attachment - Mtruk consent online). Mturk participants will see this consent form 
after being directed to their specific link: www.paingen.science/mturk.  
X. 
PROCESS TO DOCUMENT CONSENT IN WRITING 
As noted above, digital documentation of consent will be obtained for laboratory and online sessions via 
docusign (or through the study website for Mturk participants), and a trained experimenter will review 
the consent document and procedures with the prospective participant before informed consent is 
obtained.  
XI. 
PROCEDURES  
1. Online Behavioral Assessments through the study website (www.paingen.science or 
www.paingen.science/mturk  ) –all data stored on Redcap: After agreeing to participate in the 
study and completing the consent form (to be completed within one month of fMRI scanning). 
a. Personality and affective state measures (Surveys a-l in the table below)  
b. In the event that an Mturk participant comes across an issues they will be given the 
following email to contact: paingene.canlab@gmail.com.  Only research personnel listed 
will have access to the email account. Xiaochun Han, co-investigator, will be managing 
the email and responding to participants’ inquiries.  
2. Online Cognitive Tasks (also via study website): Participants will perform the following tasks 
implemented in an online system (i.e., using JavaScript) that will collect reaction times and error 
rates.  Protected information (e.g., names and contact information) will not be collected in this 
system. Instead, participants’ data will be coded with a unique alphanumeric code.  Data will be 
collected on and stored in Redcap. 
a. Cognitive Control Measures: 
i. Letter/number task: Participants will be presented with a set of numbers and 
letters. Their task is to classify the letter as either consonant or vowel and whether 
the number is even or odd.  
ii. Go/no-go task: Participants will be presented with the word “go” or “no-go” and 
will be asked to press a certain key when the “go” signal appears and to avoid 
pressing the key when the “no-go” signal is present.  

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iii. Stop Signal task: Participants will perform a choice reaction time in which they have 
to respond as quickly as possible to a particular stimulus feature (e.g. colour, shape, 
identity, or location). On a minority of the trials, the go stimulus is followed by an 
additional signal (e.g. an auditory tone), which instructs participants to withhold 
their planned response.  
iv. N-back Task: Participants will be presented with letters one at a time and they must 
identify each item that repeats relative to the item that occurred "n" items before 
its onset.  
v. Keep track task: Participants are first shown a set of categories to keep track of for a 
particular trial (e.g., animals, colors, and countries). They are then presented with 
words (including words from each category), and must remember the last word that 
was presented from each of the categories and recall those words at the end of the 
trial. 
 
3. Intro Survey (CINC room 174): 
Within one month of completion of the online behavioral/cognitive tasks (and fMRI eligible) the 
participant arrives at CINC they will meet a research coordinator who will have a copy of the 
consent form in order to walk them through the experiment details and remind them of 
procedures for this study. Then, they will be asked to complete the Pain Genetics intro survey in 
room 174. 
i. Antisaccade task: After the intro survey, in room 174 participant will complete the 
anti-saccade task in the same room. Participants view a fixation point and a visual 
target (e.g. a black circle) is presented. Subjects are instructed to look away from 
the target (antisaccade) and respond with what number they saw on the screen. 
ii.  Saliva Sampling: Prior to the fMRI scanning session, participants will be asked to 
give a saliva sample. This will involve spitting into a tube to the desired amount of 
fluid needed. 
 
4. Pressure Pain Calibration task (Room 174 and room 173): Then, they will be asked to conduct 
the ‘pressure pain calibration’ task. Participants will receive mechanical pain stimuli (between 4 
kg/cm^2 and 8 kg/cm^2, max duration = 10 seconds), and will provide their pain ratings verbally 
between 0 and 10. If the participant is unable to tolerate the pressure stimulations, they will be 
deemed ineligible for this study. 
 
5. Intensity and Unpleasantness scale practice (Room 173): Participants will then complete the 
“Scale-practice survey” in order to get more familiar with using our rating scales.  
 
6. Thermal Pain Calibration task (room 173): Then, they will be asked to conduct the ‘thermal pain 
calibration’ task. Participants will receive different levels of thermal stimuli in a random order 
(between 45.5 and 48.5 °C, max duration = 12 seconds), and will provide their pain ratings 
verbally between 0 and 10. If the participant is unable to tolerate the maximum thermal 
stimulations, they will be deemed ineligible for this study. 
 

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7. Pain tasks outside the fMRI scanner (Room 173): There are three main phases during 
behavioral testing: In the placebo administration phase, participants are given two identical 
creams with different instructions: “Prodicaine, an effective pain-relieving drug” (Placebo) and 
“a control cream with no effects” (Control) applied to 2 fingers on the left hand (two sites for 
thermal stimuli only), with locations counterbalanced across subjects. Disclosure of side effects 
are presented on forms with realistic-looking drug company logos (see attachment 
‘drug_info_packet_script_PAIN-GEN’), and testing is done in a medical environment with 
medical equipment and related context cues. A 2-minute delay follows for the “cream to take 
effect”, during which we will ask the participant to complete the survey “side effects 
questionnaire”. A suggestion-only test phase in which participants will view pseudo-randomly 
generated pain ratings given by “prior” participants. The suggestion only test does not use any 
thermal or mechanical stimulation but tests participant’s ability to track images of “pain” ratings 
in order to generate an expectation that the placebo cream will work. Next, is a conditioning 
phase in which delivery of high- and low-intensity stimuli (thermal only) on placebo- and 
control-treated skin sites, respectively383, 410-412. This reinforcement strengthens expectations383, 
410-412 and induces non-conscious learning24, 234, 413. Ratings of pain intensity and unpleasantness 
will be made after each trial of the conditioning phase on a 100-point generalized visual 
analogue scale415-418 with anchors of “no pain” or “no unpleasantness” and “most intense 
sensation imaginable” or “Most unpleasantness imaginable”, using an fMRI-compatible 
trackball. 
 
8. Preparation for MRI scanning: Following the calibration task, but prior to going into the MRI 
scanner, the MRI technologist on duty will ask participants to remove all jewelry and metal objects 
from their pockets. Participants will be required to change into scrubs to prevent any possible risk 
from metallic objects or decorations in their clothing. Participants will complete the required fMRI 
and CINC safety screening forms (Attachments 18-19) prior to entering the scanning room.  
 
In an MRI scan the subject lies down on a table and is placed into a long donut-shaped magnet.  A 
specially designed coil will be placed around the head to provide better images (as is done with 
standard clinical examinations).  As the MRI scan is performed, the subject will hear loud rapping 
and knocking noises that are normal for a MRI scan. 
 
9. Pain tasks in the fMRI scanner: During the MRI scan, multiple pain tasks described in the 
“Research Design” section will be conducted. All scanning will be conducted on a research-
dedicated Siemens 3T MRI scanner at the Center for Innovation and Creativity at the University 
of Colorado at Boulder. Scanning will include structural and functional scans; total in-scanner time 
will be approximately 1.5 hours.  
 
In the fMRI control room, participants will go through a second placebo administration phase, 
where they are given two identical creams with different instructions: “Prodicaine, an effective 
pain-relieving drug” (Placebo) and “a control cream with no effects” (Control) applied to 4 
fingers on the left hand (two sites for thermal stimuli and two sites for mechanical stimuli), with 
locations counterbalanced across subjects. A 2-minute delay follows for the “cream to take 
effect”, during which we will ask the participant to complete the survey “side effects 

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questionnaire”. The participant then enters the scanner with the MRI tech and researcher. After, 
the participant lies down and is connected to the physiological monitoring devices and is set up 
to scan. Structural (T1-weighted) images will be collected prior to any of the pain tasks and the 
participant will have a chance to practice using the trackball device while in the scanner during 
this time. Resting state images will be collected prior to the first pain task. Participants are 
instructed to look at a fixation cross (visually looks like a plus sign on the screen) for five minutes 
during this scan.  Next, A post-conditioning test phase tests both pain types on and off placebo 
treatment after reinforcement. We have successfully used similar procedures414, as have 
others222 (e.g., C. Büchel, N = 700 with suggestion-alone and post-conditioning, no imaging) 
Rating scale: Ratings of pain intensity and unpleasantness will be made after each trial on a 100-
point generalized visual analogue scale415-418 with anchors of “no pain” or “no unpleasantness” 
and “most intense sensation imaginable” or “Most unpleasantness imaginable”, using an fMRI-
compatible trackball221. 
 
10. Next, participants will complete a perspective taking task (10 minutes) while in the scanner. 
During this task, participants will view an image of either a “tough” actor or a “sensitive” actor 
(see “perspective taking instructions”) and try and take their perspective during medium heat 
stimulations (47 and 47.5 Celsius). They will also experience the thermal stimulations taking no 
perspective, or being yourself. Participants will make intensity and unpleasantness ratings after 
each heat stimulus.  
 
11. Finally, participants will lie still with eyes closed or open in order to take Diffusion Tensor Images 
(DTI). These images take about 5 minutes and allow researchers to examine functional 
connectivity in the brain.  
 
 
There are four main phases during fMRI: In the placebo administration phase, participants are 
given two identical creams with different instructions: “Prodicaine, an effective pain-relieving 
drug” (Placebo) and “a control cream with no effects” (Control) applied to 2 fingers each on 
each hand, with locations counterbalanced across subjects. Disclosure of side effects are 
presented on forms with realistic-looking drug company logos (see attachment 
‘drug_info_packet_script_PAIN-GEN’), and testing is done in a medical environment with 
medical equipment and related context cues. A 10-minute delay follows for the “cream to take 
effect”, during which we will collect structural (T1-weighted) images. A suggestion-only test 
phase will test thermal and mechanical pain delivered to fingers on and off treatment with a 
placebo analgesic cream. Placebo effects will be defined as within-person Control vs. Placebo ([C 
– P]) differences in pain report (“placebo analgesia”) and brain activity. A conditioning phase 
follows, with delivery of high- and low-intensity stimuli on placebo- and control-treated skin 
sites, respectively383, 410-412. This reinforcement strengthens expectations383, 410-412 and induces 
non-conscious learning24, 234, 413. To limit testing to one session, structural (T1 and diffusion-
weighted) images will be collected during behavioral conditioning. A post-conditioning test 
phase tests both pain types on and off placebo treatment after reinforcement. We have 
successfully used similar procedures414, as have others222 (e.g., C. Büchel, N = 700 with 
suggestion-alone and post-conditioning, no imaging) Rating scale: Ratings of pain intensity and 

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affect will be made after each trial on a 100-point generalized visual analogue scale415-418 with 
anchors of “no pain” and “most intense sensation imaginable,” using an fMRI-compatible 
trackball221.  
 
 
12. Physiological measurements and visual eye tracking: In addition to MRI scans, we will passively 
record a number of physiological variables, including (a) heart rate, (b) skin conductance, (c) pupil 
diameter, (d) eye gaze, (e) respiration, and (f) blood pressure. These recordings will be entirely 
passive and non-invasive, and will not require any additional effort on the part of the participants 
(except for having sensors attached to one’s hand and torso for physiological recording). 
Physiological data will be recorded using the BioPac Acquisition system and the Siemens 
Physiological Monitor system. Visual eye tracking and pupillometry will be conducted using an 
integrated Tobii eye-tracker. The eye-tracker does not record the video of participants, but only 
a record of their pupil diameter and the on-screen location at which they are currently fixating. 
 
13. Skin Prick Task/Allergy test (see ‘skin prick testing procedures and allergy task instructions’ for 
details of procedure): Prior to the start of this task, participant will fill out the document: ‘skin 
prick test screening’ to ask about any criteria that would exclude them from this task. If the 
participant marks any of these questions with an ‘X’, they will be excluded from this task. Although 
the ‘skin prick test screening’ asks about regular intake of antihistamine medication, we will not 
exclude participants from the allergy test if they indicate any allergies or regular intake of anti-
allergy medication. This information regarding allergies, will be documented and referred to if 
necessary. The experimenter will conduct a blood pressure test to check for excessively high/low 
blood pressure. Only participants who fall within the normal blood pressure range will be eligible 
for this task. Participants with excessively high (over 150 Systolic and over 100 Diastolic) or low 
(Under 80 Systolic and under 60 Diastolic) will be excluded from the test. These are the blood 
pressure levels appropriate for testing as advised by the allergist.  The histamine gel is applied to 
the skin on the lower left arm and skin is punctured superficially using a standard blood lancet 
(UniTest PC produced by Lincoln Diagnostics; Decatur, IL). The UniTest PC testing system includes 
three parts: 1) the blood lancet used for delivering the prick, 2) a reservoir filled with the 
appropriate allergen (in this case histamine gel) and 3) a tray containing 40 sterile pouches for 
storing the reservoirs (for more details see attachment: ‘ Dipwell_instructions’ and ‘unit-pc-ad’ 
pdf). Reservoirs will be filled with 12 drops of histamine gel and stored in the trays. The UniTest 
PC blood lancets are then placed individually in each reservoir and stored in a refrigerator at 2-8 
degrees Celsius.  When the allergy test needs to be conducted, the researcher will first swab the 
forearm site with an alcohol wipe and allow to dry. Then, they will take a single use UniTest PC 
blood lancet out of the tray (loaded with histamine) and prick one site on the participant’s 
forearm. The UniTest PC prick lancets will be applied with medium pressure to the skin site in 
order to apply the histamine gel. Researchers will follow the UniTest PC instructions as well as 
training from an allergist (see ‘Allergist training instructions’). This process will be repeated with 
a new UniTest PC blood lancet for the other site on the forearm (two total sites pricked using two 
different blood lancets). The blood lancets will be disposed of in a sharps container after the 
allergy test. Two inert creams will be administered to the participant with the suggestion that one 
will ‘decrease the allergic reaction and itching’ and the other will ‘increase the allergic reaction 

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and itching’. Allergic reactions will be measured using a ruler and and a picture of the reaction will 
be recorded for a blind experimenter to rate afterwards. Participants will rate how itchy their skin 
feels and how comfortable/anxious they feel at 5 different time points (3, 6, 9, 12, 15 minutes 
post-histamine application) during the allergy test using the Allergy (skin prick test) survey..  
 
 
14. Debriefing: Participants will be given a short debriefing form (attachment 11) to fill out. This will 
ask generally about their thoughts during and after the experiment. Next, the experimenter will 
inform the participant that Corinne Gunn will contact them within the next six to twelve months 
in order for them to complete follow-up surveys and tasks. They will also be informed that they 
will be compensated $25.00 following completion of the follow-up surveys. After enrollment is 
complete, Corinne Gunn will contact previous participants to do a final debriefing using the 
‘Debriefing form’. Participants will be fully informed of the deception that was necessary for the 
experiment and also be given Prof. Wager’s contact info in case of further questions or concerns 
(see attachment 12: debriefing form).  
 
15. Follow up surveys (online via study website ~6 to 12 months after scan session): Participants 
will be sent a link by Corinne Gunn, six to twelve months after their scan session, and be asked 
to complete: The Perceptual/decision and Affective bias measures (Attachment 10: Perceptual 
bias stimuli), “Canlab pain survey”, “Phenx substances”, and the “Jessor demographics survey”.  
 
 
Name of instrument/tool/procedure 
Purpose (i.e. what data is 
being collected?) 
Time to Complete 
Online Behavioral Assessments (a-
s_ 
The following measures 
will be used assess 
personality, mood, 
Perceptual/Affective 
Bias, and Cognitive 
Control 
80 min total 
a. Life Optimism Test Revised 
(LOT-R, Attachment 3)  
Trait levels of optimism and 
pessimism 
3 min 
b. Behavioral 
Inhibition/Behavioral 
Activation (BIS/BAS, 
Attachment 4)  
Trait levels of approach and 
avoidance behavior 
3 min 
c.  PROMIS–57 Profile v2.1  ( 
Attachment 5) 
Measures Physical 
function, anxiety, 
depression, and pain.  
5 min 
d. Marlowe-Crowne Social 
Desirability Scale 13-Item 
Short Form 
Measures Social 
Desirability  
2 min 

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e. Fear of Pain (FOP, 
Attachment 7)  
The FOP measures fear 
and anxiety associated 
with pain 
2 min 
f. Ego Resilience (ER-89, 
Attachment 8)  
Ability to respond 
adaptively and 
resourcefully to new 
situations 
3 min 
g. The “Big 5” brief inventory 
brief version (Attachment 9)  
Survey designed to 
measure various 
personality traits 
1 min 
h. Multidimensional 
Assessment of 
Interoceptive Awareness 
(Attachment 20) 
Measures Interoception 
3 min 
i. 
Jessor Demographics 
(Attachment 21) 
Measures 
Demographics 
5 min  
j. 
Canlab Pain Survey 
(Attachment 22) 
Records any pain 
disorders or sensitivities 
4 min 
k. Adverse Childhood 
Experience (ACE) 
Questionnaire (Attachment 
23a/b) 
Measures childhood 
trauma (if any) 
2 min 
l. 
Perceptual Bias Stimuli 
(Attachment 10)  
A series of perceptual 
and emotional 
judgments to assess 
bias in specified areas 
(color, size, emotion, 
etc.)  
15 min 
m. Letter/number task 
switching 
Measures cognitive 
flexibility 
5 min 
n. Go/no-go task 
Measures response 
selection 
5 min 
o. Stop signal task 
Measures response 
selection 
5 min 
p. Antisaccade task 
Measures response 
selection 
5 min 
q. N-Back task 
Measures working 
memory 
5 min 
r. Keep-track task 
Measures working 
memory 
5 min 
s. Side effects Questionnaire 
(Attachment 13)  
Used to record any 
reported side effects 
1 min 

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t. Debriefing Survey 
(Attachment 11) 
Used to assess 
participants’ beliefs 
after the experiment 
5 min 
u. Debriefing form 
(Attachment 12)  
Given to participants 
post-experiment if they 
have additional 
questions or concerns 
1 min 
v. Twin recruitment script 
(Attachment 17) 
Sample script for 
contacting participants 
5 min 
w. Twin fMRI safety screening 
(Attachment 18) 
Used to confirm fMRI 
eligibility  
1 min 
x. CINC safety screening 
(Attachment 19a/b)  
Used to confirm fMRI 
eligibility 
2 min 
y. Skin prick test screening and 
blood pressure 
measurement 
Screen for eligibility to 
take part allergy (skin 
prick test) task 
2 minutes 
z. Pain Genetic intro survey 
Basic demographics, 
medications, and 
current mood 
3 minutes 
aa. Allergy (skin prick test) 
survey 
Measure itch, anxiety, 
and comfortability from 
skin prick test 
5 minutes 
bb. Perspective taking 
instructions 
Instructions for fMRI 
task (Perspective 
taking)  
2 minutes 
cc. Scale practice survey 
Instructions for making 
pain intensity and 
unpleasantness ratings  
5 minutes 
 
 
Total time to complete experiment: ~7 hours  
Visit # 
Procedures/Tools 
Location 
How much time the 
visit will take 
NA 
● Surveys/tasks a-s 
(except perceptual 
bias and anti-
saccade) 
At home/online 
2 hours 
Visit 1 
● Intro survey 
● Anti-saccade task 
● Saliva Sample 
● Pressure pain 
calibration 
Room 174 
30 minutes 
Visit 1 
● Scale practice survey 
Room 173 
1.5 hours 

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● Thermal pain 
calibration 
● Drug info packet 
● Cream application 
● Side effect 
questionnaire 
● Test: Suggestion 
Only 
● Test: Conditioning  
● Pain rating scales 
Visit 1 
● Prep for scan 
● Re-administer 
creams 
● Side effect 
questionnaire 
● T1 structural scan 
● Resting state scan 
● Test: Post-
Conditioning 
● Perspective taking 
task 
● DTI scan 
fMRI control room 
and fMRI scanner 
1.5 hours   
Visit 1 
● Skin prick test 
● Allergy survey 
CINC room 174 
30 min 
Visit 1 
● Debriefing 
CINC room 174 
5 min 
Follow-up 
● Jessor demographics 
● Pain symptoms 
● Phenx substances  
At home/online 
1 hour 
XII. 
DATA MANAGEMENT 
Strict standards of confidentiality are maintained for each experiment and any follow-up procedures. 
MRI data and questionnaire data will be electronically stored and analyzed using ID codes. If the data 
are published subjects will remain anonymous in all publications. All MRI and behavioral data 
will be submitted to the NIMH Data Archive (NDA) according to the terms and conditions outlined on 
their website (https://ndar.nih.gov/contribute_data_sharing_regimen.html ) and with OpenFMRI. 
In addition, this project involves a Genome Wide Association Study (GWAS) and will involve sharing of 
genotyping data under NIH’s GWAS policy NOT-OD-07-088 (see attachment #20 “Resource sharing 
plan”).  
 
As with other data from the PI's studies, data will be stored on restricted access servers and/or in locked 
filing cabinets in a locked room, to which only the PIs and members of the research team have access. 
These procedures will minimize the risk of personal information being divulged to non-members of the 

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research team. No identifying information will be linked to the data from the study (including saliva 
samples), except by a master list accessible only to the PI and research coordinator. Data from the REDCap 
surveys will be stored on secure servers to which only the Screening Coordinators will have password-
protected access. Respondents to the consent form may indicate their willingness to be contacted for 
other studies. Participants’ information will be retained indefinitely unless a participant explicitly asks to 
have their data removed from the database. 
 
The consent form will ask participants if they are interested in participating in future studies. These forms 
will be kept in a locked filing cabinet in a locked room, to which only the PIs and members of the research 
team have access to. These consent forms will contain identifiable data (contact information and name), 
but will only be accessed when future studies are designed using the population sample described in this 
study. At the conclusion of this study (final review by IRB) the consent forms will be retained for 5 years. 
After the allotted time has passed the consent forms will be destroyed.  
 
Similarly, saliva samples will be stored in a locked freezer until processing. Processing entails one of two 
procedures, which will be decided depending on feasibility at the time data collection starts: (1) hand-
delivery of samples to the IBG genetics core, which will conduct GWAS analysis; (2) sending the samples 
via registered mail to a commercial service, which will conduct GWAS analysis. In both cases, only 
alphanumeric codes will be provided to the off-site analysis core. The UK Biobank pipeline protocol483 has 
been established by the UK National Health Services to share this data in a de-identified form with 
investigators worldwide. The UK biobank is a separate study that is sharing de-identified data around the 
world. As part of this project we are receiving de-identified data from the UK biobank study in order to 
compare the placebo predictors in terms of genetics and identify brain structural correlates of the genetic 
patterns we identify from the GWAS and brain data. 
 
Basic identifying information (name, address, phone number/email address) is collected from every 
research participant for the purpose of research logistics (schedule visits, etc.) and mailing of the 
radiological review letter, as appropriate. These data will be stored separate from other study data 
assessments, samples, and images, along with alphanumeric codes for each participant. Only these 
alphanumeric codes will appear on both participants’ identifying information and study data, allowing us 
to link these until study completion. 
 
MRI data will be stored according to standard INC data management procedures. MRI images will be 
housed on a CU Boulder server. Metadata (name and contact information) will be entered into the 
COINS database by study personnel. Each COIN entry will receive a unique research subject identifier. 
This code will be associated with the images. A copy of the MRI images will be sent to Georgia State 
University for data sharing and storage. No identifying information is included in the images. 
 
A common tool,  Georgia State University’s (GSU) Neuroinformatics website called COINS is used to 
register and store MR data for all researchers in INC. The COINS web service employs the highest 
security for registering participants, and requires each researcher to gain permission from GSU before 
being allowed access to COINS.  Each researcher is given a portal where they may register participants 
for their own studies. Each researcher and/or their staff will be responsible for registering participants in 
COINS. Unique research subject identifiers (URSIs) are created for each participant in order to de-

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identify their imaging and research data according to good clinical practice. The URSIs are generated via 
the COINS database. Every participant is given an URSI, and all MR data are stored at INC and a secure 
server using Amazon Web Services (AWS) using the URSI. Participants are asked to register for the MRI 
with personal information including their names, birthdate, phone number and address.  This 
information is necessary in cases of incidental findings where GSU radiological review requires that the 
participant be contacted.  GSU will maintain information associated with the URSI but not the scanning 
data (e.g., participant names and contact information) in a highly secure cloud computing service via 
AWS. 
 
In order to access a study protocol in the secure, password protected COINS database investigators must 
have IRB approval. Participant names and other identifying information will be maintained in this 
restricted database, available only to authorized members of the research team for the duration of the 
study protocol. In COINS, the record linking a participant’s name and URSI will be kept indefinitely at the 
GSU in a confidential manner (in order to maintain participants radiological review information).  
 
Research staff will use secure, password-protected portals to access data stored on the COINS/AWS 
Database Repository. The Investigators will oversee all data management and give research staff 
permission to access data. Identifying information (i.e. name, address, phone number or email) will not 
be included in these secure portals.  
 
CU Boulder Research Computing, Dartmouth College Research Computing and  AWS  are currently 
involved in the storage and security of imaging data. Research staff at GSU will not be engaged in the 
research (they will not be interacting or intervening with subjects, nor will they obtain subject’s private, 
identifiable data).  Staff at GSU may be involved in helping researchers interpret the data collected 
during the protocol, but will not receive personal or identifiable data about the subjects. De-identified 
data will be managed through the COINS system, through the CU Boulder Research Computing, AWS, 
and Dartmouth College. Access to personal information is restricted to PIs and project staff for the 
specific study in which the participant is enrolled and senior GSU and INC staff (for cases of incidental 
findings where a radiological review and letter to the participant would be necessary). Other (‘third-
party’) investigators may access the de-identified data for individuals who indicated a willingness to 
share their data for a specific study if the PI who collected the initial data approves of this use of the de-
identified data. Third-party investigators will be able to access de-identified data from either the secure 
server at CU Boulder (RC Peta Library) or the secure server at Dartmouth College (RC Discovery). Any 
analysis of such data will be performed on the secure server, so that the de-identified data is not 
compromised by moving it to a different location. Only analysis derivatives (aggregate scores, group 
statistics) might be stored elsewhere if needed.  
 
 
XIII. 
WITHDRAWAL OF PARTICIPANTS 
Participants will be ruled ineligible for further participation if pain thresholds fall outside a safe range 
and/or scientifically useful range. Participants who are ruled ineligible for subsequent studies will be 

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compensated for their time at the normal rate, but will not be further contacted or invited to participate 
in subsequent study opportunities. 
 
Participants may withdraw voluntarily from a study at any time. Participants may either withdraw from a 
particular experimental session, or request that they be removed entirely from the laboratory subject 
pool. In the latter case, all of the participant’s partial or full data will be destroyed immediately. 
 
XIV. 
RISKS TO PARTICIPANTS 
1) Burn due to thermode malfunction: There is a very slight risk to the participant in case of thermode 
malfunction. Thousands of participants are tested using this equipment (Pathway system, Medoc, Inc.) 
annually throughout the U.S., usually without adverse events. However, several reported cases of 
thermode malfunction have occurred in the past 5 years (four cases, to our knowledge), which have 
resulted in minor 1st or 2nd degree burns. The manufacturer (Medoc, Inc.) has responded to these reports 
by building in enhanced hardware safety mechanisms; thus, we do not anticipate a substantial risk. The 
PI's lab has conducted experiments on approximately 400 subjects at Columbia University and over 400 
at UC-Boulder with no adverse events. Although it is not possible to precisely determine the probability 
of a burn, we estimate based on our prior experience that it is considerably below 1%. We also note that 
the vast majority of potential burns that could potentially result from equipment malfunction would 
consist of minor blistering that would heal naturally without any treatment within several days.  
 
2) Injury due to pressure pain device malfunction: There is a very slight risk to the participant in case of 
pressure pain device malfunction. We have tested the device on 12 in-lab participants (see Attachment 
14) and have done a large number of preliminary tests on study personnel without any incidents. In 
addition, we systematically tested if participants could readily remove their thumb from the device under 
high-pressure stimulation (e.g., 8 kg/cm^2) and specifically when pressure was experienced as too high to 
tolerate for an extended period.  Eleven participants finished the test, and all were able to remove their 
thumb from the device. One participant terminated the experiment after the first few trials due to 
hypersensitivity to pressure. We also asked participants whether they experienced any long-term harmful 
effects of pressure pain. The survey showed that there was no remaining mark on the thumb after 3 hours 
for all participants who finished the test, and minimal tenderness was found after three hours. In addition, 
all participants who finished the test had no remaining sensation after three hours. The participant who 
discontinued the pilot experiment early reported discomfort for several hours after the experiment, but 
it disappeared within 24 hours. Therefore, we expect that most participants experience no risk due to the 
pressure pain device, and we can exclude those who may be uncomfortable with the procedure using the 
calibration procedure described above (see “RESEARCH STUDY DESIGN”).  
 
3) Safety concerns in MR environment:  
➢ The magnetic field of the MR environment has the potential to cause burns or bodily injury if ferrous 
metal objects are implanted in the body, or if personal articles containing ferrous material are 
brought into the environment. 
➢ The risk of MRI to pregnant women and fetuses is currently unknown.   

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➢ The MRI may cause discomfort due to scanner noise.  
➢ There may be some discomfort from lying still and in one position for a long time 
➢ Peripheral nerve stimulation (PNS/tingling). At sufficient exposure levels, peripheral nerve 
stimulation is perceptible as “tingling” or “tapping” sensations. PNS symptoms will usually subside 
shortly after the scan is completed. 
➢ Participants may feel nervousness or feelings of claustrophobia. 
➢ There is a risk that the image will reveal an observation concerning an individual research 
participant that has potential clinical importance but is beyond the aims of this protocol. In the 
event of the confirmation of a significant anomaly in a participant’s brain image, this information 
will likely be distressing to the participant. 
 
4) Psychological discomfort: Studies involving administration of pain by definition require the induction 
of psychological discomfort, so this is an unavoidable risk of participation. However, as described above, 
the level of pain administered is calibrated to always be within participants’ tolerable level, and 
participants are informed prior to each session involving pain that they are free to discontinue the 
experiment (e.g., removing the thermode, withdrawal of the thumb from the pressure device) at any time 
should they wish. Participants may also experience some emotional distress due to the images in the task. 
Subjects may experience nervousness and/or claustrophobia during the MRI. While generally safe, it is 
not known whether an MRI would harm a fetus. 
 
5) Use of deception: some level of deception is inherently necessary to induce placebo analgesia.  
Participants will be informed that they will receive an application of a topical cream, but in fact be 
receiving an application of Vaseline®, an over-the-counter petroleum jelly product that has no analgesic 
properties.  We feel that this use of deception is justified for our research in this way because the risk is 
minimized by the target of the deception: we are not withholding treatment for a medical condition, 
rather using deception as a manipulation to study experimentally-induced pain.  As a result, we do not 
anticipate any significant long term effects of using deception in this study. 
 
6) Saliva Sampling: There are no known risks to donating DNA saliva samples. However, there is a risk 
associated with possible loss of confidentiality. In the Data Management section, we have described our 
procedures to maintain the confidentiality of the data, including the genomic data. 
 
7) Skin prick test: This procedure involves no more than minimal risk. Participants will be fully informed 
about all potential risks as a part of the study. Discomfort from the skin prick procedure is expected. Skin 
prick tests involving histamine are used as controls in routine allergy diagnosis tests. This test has been 
used in numerous studies to induce itching e.g., Bromm, Scharein, Darsow, & Ring, 1995; Darsow, Ring, 
Scharein, & Bromm, 1996; Pfab et al., 2006, 2010. Further, this procedure induces an itching sensation, 
but did not cause pain or any other sensations to any participants in previous studies. Participants will be 
informed about the allergy test, that we are using histamine in the allergy test and that histamine is a 
routine control in allergy tests, will be told how it will feel, and any risks in advance, and thus will not 
encounter any risks beyond deciding to take the skin prick test. A review by Liccardi, et al., 2006 found 
less than 0.02% risk of generating a systemic reaction to skin prick test.  
 

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8) Legal Risks: Participants will be asked to divulge information about illegal activities in one of the 
surveys in this study. There is a slight risk that this information could be accessed by a third party if the 
data from this study becomes compromised. We believe this risk is low due to our strict standards of 
confidentiality for information collected throughout this study (see Data Management).  
 
There are no known less risky alternatives to the use of any of the procedures proposed in these 
experiments that would provide comparable scientific information.  
XV. 
MANAGEMENT OF RISKS 
1) Burn due to thermode malfunction: Pain stimulation will always occur within well-tested and verified 
parameters by the Wager laboratory, through previous pain studies. The equipment used is widely 
available and includes several built in safety mechanisms including an auto-shutoff as well as maximum 
temperature restrictions. Additionally, participants are given an emergency shut-off button that they can 
press at any time and instantly stops heat delivery. The equipment is regularly maintained and tested by 
our trained personnel. All personnel who use the equipment are trained on equipment procedures. 
 
2) Injury due to pressure pain device malfunction: In order to minimize risks to participants, we will 
exclude participants who are hypersensitive to pressure pain or have difficulty to remove their fingers 
from the device when pressure is high using the calibration procedure (see “RESEARCH STUDY DESIGN”). 
In addition, the pressure pain software has a “Stop” button, which can be used to stop the pressure stimuli 
anytime by experimenters. The participants will be given a hand-squeezable pneumatic signaling device 
for communicating with experimenters during scanning and therefore should be able to signal intolerable 
discomfort of any kind. The device is regularly maintained and tested by our trained personnel. All 
personnel who use the equipment are trained on equipment procedures. 
 
3) Safety concerns in MR environment: This protocol will be performed using an MR scanner employing 
pulse sequences and hardware that have been approved by the FDA for human clinical use. The field 
strength is 3 Tesla and all relevant operating characteristics (RF power deposition, rate of change of the 
field gradients, coil design) fall within the limits of FDA guidelines for NMR exposure. Participants will be 
carefully screened to exclude those who may have metal in or on their bodies that cannot be removed (e 
g., bullets, metal filings, body piercings, etc.). MR Facility rules strictly forbid staff from entering the 
magnet room carrying metal objects. The risk of claustrophobia is minimized by screening subjects for 
self-reported claustrophobia and making sure the subject is lying comfortably with head and neck 
supported and providing ear protection with headphones, a mirror to see out, a button to signal distress, 
and an intercom.  Scan time will be kept to a minimum.  If they are unsure about whether or not they may 
be pregnant, female participants will be given the opportunity to complete a urine pregnancy test 
immediately before the scanning period, and those with a positive result will not be scanned. With regard 
to PNS, participants are given a squeeze ball to use in case of an emergency. They are informed that if 
they experience PNS related sensations or are otherwise uncomfortable, they can alert the MRI 
technologist via the squeeze ball and the technologist will stop the scan immediately.  
 
In the case of an anomalous finding in a brain image, the following procedure is followed: 

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1. The technologist and/or research personnel flag potential abnormalities. 
2. The MRI technologist notifies the INC Director of Operations, the GSU Director of Research and 
Clinical Operations, and the P.I.  
3. The scan gets queued to the radiologist worklist in COINS.  All cases of suspected incidental 
findings are sent for formal neuroradiologic review at GSU. 
4. The radiology review contains a written summary of the findings and classifies the referral status 
into one of these categories: 
● There is not enough information from the MRI scan to complete a full review. No obvious 
abnormalities found.       
● MRI shows nothing obvious that needs medical attention.  
● MRI shows something that may or may not be of medical concern.  Participants should 
consider discussing the enclosed report with their doctor.  
● MRI shows something that needs to be brought to the attention of your doctor. Participants 
may also be contacted by the study team and/or GSU Medical Director about this report. 
5. The PI will get an electronic copy of the radiology review (coded via URSI) as soon as the review 
is completed. If an urgent referral is recommended, the PI should discuss the review with the 
Medical Director prior to contacting the participant. 
6. If a referral is recommended, Corinne Gunn will contact the participant and explain that an 
unusual feature was observed in their scan. Corinne Gunn provides the contact information for 
the Medical Director who reviewed the image (this information is in the letter mailed to the 
participant as well). Routine referrals are handled on a case by case basis and up to the 
PI/Medical Director to determine if the participant should receive a call in advance. 
7. All cases reviewed will generate a formal radiology report, which is printed on letterhead and a 
copy of which is mailed to the participant. In the case of an urgent referral, someone from the 
study team or the Medical Director will contact the participant prior to the letter being mailed.  
 
 
4) Psychological discomfort: Participants are clearly informed of this risk prior to participation during the 
instruction period, and the ability to tolerate heat and pressure pain is explicitly listed as one of the first 
screening questions (Attachment 1). There is virtually no possibility of long-term psychological distress or 
unanticipated psychological discomfort that exceeds the proximal response to pain, as the amount of pain 
delivered is comparable to or less than that experienced in many day-to-day situations (e.g., holding a hot 
cup of coffee). However, they will be encouraged to inform the experimenter if they are uncomfortable 
with the nature of the stimuli. If participants experience any lasting negative effects related to the 
emotional content of this study, they will be encouraged to contact the Principal Investigator, Dr. Naomi 
Friedman, at Naomi.Friedman@colorado.edu. She will discuss options for counseling referrals and 
provide a referral. The cost of any follow-up counseling, should any be required, would be borne by the 
participants and/or their insurance provider. The participant will be informed that neither the study team 
nor any of its individual members will be responsible for follow-up treatment. 
 
5) Use of deception: After the experiment has completed, participants will be given written and verbal 
disclosure from the experimenter that deception was used (see attachment 12) , and given a chance to 

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ask any questions they have at that time.  In the case that no issue with the use of deception arises initially 
with the participant but does after an extended period of time, the contact information for the PI is given 
in the debriefing form so that participants may contact him directly. 
 
6) Negative reaction to skin prick test: The researcher checking vitals and conducting the allergy test will 
be trained research staff. Researchers have received training from a physician from Boulder Valley Asthma 
and Allergy on assessment of vitals, as well as administering the allergy test. The research staff is trained 
to recognize the symptoms of anaphylactic shock, as well as trained in epi pen administration (AuviQ, 
epinephrine injection, USP). We will also keep a step-by-step procedure outlining steps to take if an 
anaphylactic response occurs and will be kept with the epi-pen on site with each study session (see ‘Epi-
pen administration guide and allergy task instructions’). CINC room 174 will be outfitted with necessary 
equipment. We will observe participants for 60 minutes to ensure that no negative effects from the 
reaction occur and none are anticipated. In the very small chance that participants do react negatively, 
then epi pens will be available to inject epinephrine in the lateral thigh of 0.3mg concentration, as per 
standard allergy testing procedures (’Allergy task instructions, AUVIQ label instructions, and direct training 
from physician’). We have obtained a prescription for an Epi Pen (AuviQ, epinephrine injection, USP) from 
an allergist. 
 
7) Legal risks: In order to maintain privacy and confidentiality, the survey with the question about illegal 
activities will be administered in a private setting. All data submitted will be stored on secure, password 
protected servers and coded (see Data Management) to prevent 3rd parties from accessing potentially 
incriminating information. Participants will be aware of the question about illegal activities prior to 
completing the survey and will have an option to not disclose any information if they choose to do so. 
These measures will reduce the risk of privacy and/or confidentiality breaches.  
 
XVI. 
POTENTIAL BENEFITS  
There are no direct benefits to the subject from this study save for the knowledge that their 
participation may help scientists understand the psychological and neural mechanisms involved in pain 
processing and placebo analgesia.  
XVII. 
PROVISIONS TO MONITOR THE DATA FOR THE SAFETY OF PARTICIPANTS 
In the event that a medical professional determines that a severe burn or tissue damage occurred due to 
study procedures, the study will be put on hold until the cause of the issue is determined, the IRB will 
immediately be notified via email, and a formal report will be filed with the IRB by one of the study co-
investigators. 
 
The MRI technologists are not trained to identify potentially significant clinical anomalies in the brain 
images. Should the MRI technologist notice something he or she believes to be a potential anomaly in a 
brain image, he or she will follow the procedure noted in section XVI to ensure appropriate radiological 
review. The participant will be contacted if the radiologist recommends a scan to determine the clinical 

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significance of any anomaly. Additional action will be taken to insure the subject’s personal safety as per 
recommendations made for that specific subject. 
 
Additionally, part of our experiment involves sampling participants’ depressive thoughts via the BDI 
(Beck’s Depression Inventory), and it is possible that participants will reveal clinically sensitive 
information. An experimenter will read the participants’ responses on the BDI within a day of the 
participation.  If a participant relays thoughts of suicide during this or any other part of the experiment, 
the participant will be followed up with a clinically trained psychologist, who will provide a list of 
psychological resources, both on the college campus and within the community.  If the psychologist 
forms the impression in the conversation that the participant is experiencing elaborated thoughts of 
suicide (including suicidal plans), a licensed clinical psychologist will be contacted immediately.  If he / 
she is unavailable, the Boulder Community Hospital Suicide line will be called. Additional action will be 
taken to insure the subject’s personal safety as per recommendations made for that specific subject. 
 
XVIII. PROVISIONS TO PROTECT THE PRIVACY INTERESTS OF PARTICIPANTS  
Observations or data collection will occur only in private contexts. Therefore, there are no anticipated 
concerns with regard to privacy interests. All participants have the right to cancel at any time, as well as 
have their DNA/data destroyed.  
XIX. 
MEDICAL CARE AND COMPENSATION FOR INJURY 
In the event that a participant has experienced a burn, they will be directed to rinse the affected location 
under cool water for several minutes, and to seek independent medical attention if symptoms persist 
after a day or two. If there is any unexpected medical issue, we will call 911. No on-site medical care will 
be provided, and no compensation is available in the event of research-related injury. This is clearly 
explained on the informed consent sheet participants must agree to before they may participate in any 
study involving pain.  
 
XX. 
COST TO PARTICIPANTS 
Participants who travel to the research site with their own vehicle will be compensated for gas, and in 
some cases we will compensate out-of-state participants in the twin sample with airfare and lodging for 
2 nights for their participation. We will reimburse participants for, or cover, all other expenses associated 
with participation, such as parking.  
 
 
XXI. 
DRUG ADMINISTRATION 

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The placebo cream used in this study will be Vaseline, petroleum jelly (Unilever Co., United States), and 
stored at CINC room 171. The skin prick test will involve administration of 1mg/ml of histamine gel (1% 
histamine dihydrochloride in 2.5% methylcellulose) using the UniTest PC Individual skin test system 
(Lincoln Diagnostics; Decatur, IL).  
 
XXII. 
MULTI-SITE STUDIES 
All laboratory procedures will be performed at the Center for Innovation and Creativity (CINC) in east 
Boulder. Online behavioral assessments will be completed by the participant at home. As co-
investigator, Tor Wager will be a collaborator on the project from Dartmouth college. PI Friedman and 
Prof. Wager will meet weekly with the study team in Colorado over video-conference. During these 
meetings, the team will review all aspects of the research. In addition, they will have ad hoc meetings as 
needed, and individual meetings with graduate students and post docs working on the project (including 
Dr. Marta Ceko in Boulder, Dr. Xiaochun Han at Dartmouth College, and Bogdan Petre, a graduate 
student at Dartmouth). They also have open lines of communication on Slack, a messaging and file-
sharing service for collaboration, which allows them to post events and issues on any aspect of the 
project to the study team. Bogdan Petre will be involved in data management and analyses; he will only 
have access to de-identified data. Key personnel, Xiaochun Han, is responsible for management and 
analysis of online behavioral assessments of all participants, including MTurk participants; therefore, she 
will have access to identifiable participant information. An IRB Authorization Agreement between CU 
Boulder and Dartmouth College further explains this arrangement.  
XXIII. SHARING OF RESULTS WITH PARTICIPANTS 
There are no plans to share the results of this study with participants as a standard procedure. Participants 
or any other individuals who inquire about the results of the study at a later date will be informed of any 
publications that have resulted from the study.