Effects of Arousal and Stress on Classical Conditioning
Phase: NA
Conditions: Healthy Volunteers
Sponsor: National Institute of Mental Health (NIMH)

 
 
CNS IRBProtocol Template (rev. 3-1-07) 
 
IRB Panel: Blue    White 
Title Page 
 
Protocol Title: Effects of arousal and stress on classical conditioning 
Protocol Number 
 
01-M-0185 
Date of This Submission 
April 25, 2023 
 
Principal Investigator 
Maryland Pao, 
MD  
Branch/Institute 
OCD, NIMH 
Bldg/Rm 10/6-
5340 
Phone 
301-435-5770 
E-mail 
paom@mail.nih.gov 
 
Total requested accrual 
  0 Patients 
  
  1575 Volunteers  
 
  
 
Project Uses Ionizing Radiation:   x No  
 Yes (attach RSC/RDSC documentation) 
  
 
 Medically-indicated only 
  
 
 Research-related only 
  
 
 Both 
 
IND/IDE 
 
 
 
 No   
XYes (attach FDA documentation) 
  
Drug/Device/#:  
Startle Device 
Acoustic Startle 
  
Sponsor: N/A 
 
Durable Power of Attorney  
 
x No  
 
 Yes  
 
Multi-institutional Project 
x No  
 
 Yes  
 
 
 
 
 
Institution_________________    FWA #_________  
Date of IRB approval_________ (attach IRB documentation) 
 
Data and Safety Monitoring Board  
x No  
 
 Yes  
 
Technology Transfer Agreement  
x No  
 
 Yes  
Agreement type and number __________________Expiration Date_____________ 
 
Confidential Disclosure Agreement     
x No  
 Yes 
 
Samples are being stored  
 
 
x No   
 Yes  
 
Flesch-Kincaid reading level of consent forms: 
  
 
Shock Stress 
 
 
7.8 
Sural Nerve Stimulation  
8.3 
 
 
 
 

 
 
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PRECIS 
 
Objective: Fear and anxiety are adaptive responses to different types of threats. Fear is a 
short-duration response evoked by explicit threat cues and anxiety a more sustained state 
of apprehension evoked by unpredictable threat. This protocol studied fear using 
Pavlovian fear conditioning in two studies, Studies 1 and 3. Study 2 focused on anxiety.  
Studies 1 and 3 will be discontinued to focus uniquely on the study of anxiety.  
Specifically, we will examine the interactions between anxiety induced experimentally 
using verbal threat and cognitive processes. We will seek to 1) characterize the effect of 
anxiety on key cognitive processes including working memory and attention control and 
2) examine the extent to which performance of cognitive tasks distract from anxiety. 
 
Study population: This more than minimal-risk protocol will test medically and 
psychiatrically healthy volunteers aged 18-50. Pregnant or nursing women will be 
excluded.  
 
Method: Fear and anxiety will be measured using the startle reflex to brief and loud 
sounds. Fear conditioning will be assessed using shock as unconditioned stimulus. 
Cognitive performance will be examined during periods of unpredictable shock 
anticipation.  
 
Outcome measures: The study will include cognitive performance and measure of 
aversive states, primarily the startle reflex. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Contents 
PRECIS ............................................................................................................................... 2 
1. 
INTRODUCTION ....................................................................................................... 4 
2. 
STUDY OBJECTIVES ............................................................................................... 7 
3. 
SUBJECTS .................................................................................................................. 9 
4. 
STUDY DESIGN AND METHODS .......................................................................... 9 
5. 
MANAGEMENT OF DATA AND SAMPLES ....................................................... 17 
6. 
ADDITIONAL CONSIDERATIONS ....................................................................... 18 
7. 
RISKS AND DISCOMFORTS ................................................................................. 19 
8. 
SUBJECT SAFETY MONITORING ....................................................................... 21 
9. 
OUTCOME MEASURES ......................................................................................... 21 
10. 
STATISTICAL ANALYSIS ................................................................................. 21 
11. 
HUMAN SUBJECTS PROTECTION .................................................................. 22 
12. 
ANTICIPATED BENEFIT ..................................... Error! Bookmark not defined. 
13. 
CLASSIFICATION OF RISK ................................ Error! Bookmark not defined. 
15. 
DATA AND SAFETY MONITORING ................................................................ 24 
16. 
QUALITY ASSURANCE ..................................................................................... 24 
17. 
ADVERSE EVENT AND UNANTICIPATED PROBLEM REPORTING ......... 26 
18. 
ALTERNATIVES TO PARTICIPATION ............................................................ 28 
19. 
PRIVACY .............................................................................................................. 28 
20. 
CONFIDENTIALITY............................................................................................ 28 
21. 
CONFLICT OF INTEREST/ TECHNOLOGY TRANSFER ............................... 29 
22. 
TECHNOLOGY TRANSFER ............................................................................... 29 
23. 
RESEARCH AND TRAVEL COMPENSATION ................................................ 29 
 
 
Abbreviations 
CS 
 
conditioned stimuli 
US 
 
unconditioned stimuli 
CR 
 
conditioned response 
CTXacq     
acquired contextual stimuli 
CTXext      
extinction contextual stimuli 
CTXnov     
novel contextual stimuli 
WM 
       
working memory 
SART   
Sustained attention to response task 
 
 

 
 
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1. INTRODUCTION 
 
The overall objective of this protocol since its inception has been to enhance our 
understanding of fear and anxiety, and associative learning using experimental 
approaches from two perspectives: fear conditioning and unpredictability. Specifically, 
we have argued that fear and anxiety can be differentiated by contrasting defensive 
responses to short-duration conditioned stimuli (CS) that predict unconditioned aversive 
stimuli (US, e.g., shock) and long-duration context associated with unpredictable US, 
respectively. The specific objectives and summary of findings of studies that have been 
completed are presented in the Appendix. Tasks that were planned but never 
implemented are also described in the Appendix.  We will no longer study fear and will 
now focus our research on anxiety by examining its interaction with cognition.  
 
Despite increasing knowledge of affective components as well as neural and 
psychopharmacological mechanisms of anxiety, very little is known about the role of 
cognitive processes and how the affective and cognitive components of anxiety interact. 
Cognitive processes could be involved in the etiology and/or maintenance of anxiety. It is 
clear that the maintenance of anxiety depends on cognitive factors such as attention, 
attention control, orienting, and working memory (Clark and Squire 1999; Carrillo, 
Gabrieli et al. 2000; Sarter, Bruno et al. 2003).  In addition, prominent symptoms of 
anxiety are cognitive. Anxious people complain of difficulty concentrating because they 
are highly distractible. It is therefore possible that poor cognitive functioning contributes 
to interference from intrusive anxious thoughts (Eysenck, Derakshan et al. 2007). 
Context, such as physical exertion can influence anxiety and its effects on cognitive 
performance (Chang, et al., 2012).The present protocol builds on our past results to study 
the impact of induced anxiety (e.g., during anticipation of unpredictable shock) on 
physiological and subjective measures of anxiety and task performance in healthy 
volunteers.  
 
This protocol was initially separated into three objectives: 1) Fear conditioning, negative 
valence, and executive functions (Study 1), 2) Interaction between cognition and anxiety 
(Study 2), and 3) mechanisms of extinction and retrieval of fear memories (Study 3). The 
protocol studied fear using Pavlovian fear conditioning in two studies, Studies 1 and 3.  
These two studies will be discontinued to focus uniquely on the study of anxiety (Study 
2). With the removal of Study 1 and Study 3 leaving only Study 2, we have dropped the 
label of Study 2. The protocol will only contain information on the current study 
(previously labeled Study 2). The completed or canceled studies are described below, 
including Study 2 sub-studies, along with a brief summary of outcomes.  
 
Study 1 
Study 1 is completed. The goals were reached and the study has been stopped. 
 
Brief Description: Study 1examined affective and cognitive processes that affect fear 
conditioning. Study 1 included three separate sessions, each lasting approximately three 
hours.  All three sessions were to be completed within a period of 6 months.  

 
 
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Outcome: Panic disorder is characterized by sudden, repeated, and unexpected attacks of 
intense fear and overwhelming anxiety about when another attack may strike. We have 
reported that patients with panic disorder and healthy individuals with a history of panic 
attacks show a hypersensitivity to unpredictable threats, suggesting a possible link 
between panic and sustained anxiety. The main objective of this study was to examine the 
degree to which CO2-induced symptoms of panic related to fear evoked by predictable 
shock or anxiety evoked by unpredictable shock, as well as activity in the neural systems 
that mediate and regulate these affective states. Psychological and physiological 
symptoms of panic were assessed during an 8-min 7.5% CO2 challenge task. 
Psychological, physiological, and neural symptoms of fear and anxiety were measured 
during 2 sessions (1 psychophysiology and 1 fMRI) where subjects experienced several 
blocks of no threat (N), predictable shock (P), and unpredictable shock (U) (NPU threat 
task) (Balderson et al, 2017). We used a principle component analysis to characterize 
panic susceptibility (PS), and found that PS significantly predicted dlPFC activity to the 
unpredictable cue during the NPU threat task. When examining the weighted beta 
coefficients from this analysis, we observed that self-reported fear/anxiety during the 
CO2 challenge negatively loaded onto dlPFC activity during the NPU task. Consistent 
with this observation, dlPFC activity during the unpredictable cue was also negatively 
correlated with anxiety during the NPU sessions. Together, these results suggest that 
panic symptoms and anxiety are regulated by the same prefrontal cognitive control 
system. 
 
Resultant Publications 
Balderston NL, Liu L, Roberson-Nay R, Ernst M, Grillon C. 2017. The relationship 
between dlPFC activity during unpredictable threat and CO2-induced panic symptoms. 
Transl Psychiatry 7: 1266 
Study 2b: Effect of anxiety on response inhibition.  
Study 2b. has been completed and the goals were reached.  
Brief Description: This study examined the effect of threat of shock on response 
inhibition in a stop signal task. During stop-signal tasks, the go signal was presented on 
all trials; on a minority of trials after the go signal and just as the subject is about to 
respond, a stop signal appeared (usually a sound), indicating that one should not press the 
button on that trial. 
Outcome: Inhibition is a key theme in psychiatric research, including anxiety disorders. 
However, inhibition encompasses many processes. Studies show that anxiety impairs 
cognitive inhibition, the inhibition of thoughts, emotions and perception.  In a first study, 
we explored the effect of anxiety on behavioral inhibition, operationally defined as the 
inhibition of pre-potent motor responses.  We hypothesized that anxiety promote a 
cautious behavioral stance. As a result, anxiety should improve response inhibition.  This 
hypothesis was confirmed in several studies that examine the effect of anxiety induced by 
the threat of shock on response inhibition in a go/nogo experiment with frequent go 
(91%) and infrequent nogo (9%).  We showed that induced anxiety increased the rate of 
correct nogo responses without affecting  go responses (Grillon et al, 2013, 2017; 
Robinson et al 2013). These study reveal that while anxiety affects negatively many 
cognitive and behavioral processes, it also has some beneficial effects. We also examined 
individual differences in the effect of anxiety on response inhibition (Grillon et al, 2016). 

 
 
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We showed that subjects who most benefited from anxiety were characterized by low 
attention control. We thus confirmed that anxiety increases inhibitory control of prepotent 
responses—a mechanism which is adaptive under threat—and show that this effect is 
greater in those who rely more upon such prepotent responding, i.e., those with low 
attentional control. 
 
Resultant Publications 
Grillon C, Robinson O, Krimsky M, O"Connell, Alvarez G, Ernst M. 2017. Anxiety-
mediated facilitation of behavioral inhibition: threat processing and defensive reactivity 
during a go/nogo task. Emotion 17: 259-66 
Grillon C, Robinson O, Marthur A, Ernst M. 2016. Effect of attention control on 
sustained attention during induced-anxiety. Cognition & Emotion 30: 700-12 
Robinson OJ, Krimsky M, Grillon C. 2013. The impact of induced anxiety on response 
inhibition. Front Hum Neurosci 7:69 
 
Study 2g: Exercise, anxiety and cognition: 
Study 2g. has been completed and the goals were obtained.  
Brief Description: This study aimed to understand the influence of acute physical 
exercise on the interaction between anxiety and cognition. The study had a within-
subjects design and took place over three outpatient study visits, one exercise screening 
visit and two study visits, each separated by five to eight days. During one visit, the 
participant will pedal lightly on the stationary bicycle for 30 minutes. During the other 
visit, the participant will pedal on the exercise bicycle at a moderate intensity, ensured by 
levels of resistance and defined by target heart rate of resting heart rate plus 60-70% of 
her heart rate reserve. Then, after completion of the exercise, participants will perform an 
N-back task under threat of shock and safe conditions to measure working memory 
(Vytal, Cornwell, Letkiewicz, Arkin, & Grillon, 2013). After 15 minutes (10 min break, 5 
min startle habituation), participants will undergo the NPU task under threat of shock and 
safe conditions to measure physiological response to fear and anxiety (Schmitz & 
Grillon, 2012). 
Outcome: Exercise has been proposed as a first line of treatment for anxiety disorders. 
We examined potential mechanisms that may be responsible for the anxiolytic effect of 
anxiety.  A first study investigated whether exercise affected fear and anxiety 
differentially.  Results showed that biking at 60-70% of heart rate reserve for 30 min 
reduced anxiety evoked by unpredictable shock but not fear evoked by predictable shock 
(Lago et al, in press). These results suggest that exercise therapeutic effect may involve 
specific modulation of the anxiety and not the fear system. A second study examined 
whether exercise anxiolytic effect was mediated by improved cognitive control. Two 
observations motivated this study. First, engagement of attention control during increased 
working memory (WM) load can decrease anxiety. Second, exercise can improve 
attention control.  Therefore, we hypothesized that exercise would boost the anxiolytic 
effects of increased WM load via its strengthening of attention control. Anxiety was 
induced by threat of shock and was quantified with anxiety-potentiated startle (APS) 
(Lago et al, in press). Subjects were tested after two types of activity, exercise (biking at 
60-70% of heart rate reserve) and control-activity (biking at 10-20% of heart rate 
reserve).  After each activity, participants completed a WM task (n-back) at low- and 

 
 
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high-load during safe and threat.  Results were not consistent with the hypothesis: 
exercise vs. control-activity increased APS in high-load (p=.03). However, this increased 
APS was not accompanied with threat-induced impairment in WM performance (p=.37).  
Facilitation of both task-relevant stimulus processing and task-irrelevant threat 
processing, concurrent with prevention of threat interference on cognition, suggests that 
exercise increases cognitive ability.  
 
Resultatnt Publications 
Lago T, Hsiung A, Leitner B, Duckworth C, Chen K, et al. in press. Acute exercise 
decreases anxiety to unpredictable threat but not fear to predictable threat in humans. 
Depress Anxiety  
Lago TR, Hsiung A, Leutner BL, Duckworth CJ, Balderston NL, et al. in press. Exercise 
modulates the interaction between cognition and anxiety in humans. Cognition & 
Emotion  
 
Study 2h. Stop Signal Task: 
Study 2h. has been stopped and will not be continued. 
Brief Description: Participants performed paradigms which tested whether threat impacts 
the initiation and the inhibition of behavioral responses. Participants were presented with 
stimuli, and asked to either initiate a response (i.e. “go”) or inhibit their response (i.e. 
“stop”), based on what stimuli is presented.  
Outcome: This study was an extension of our response inhibition study with go/nogo task 
(Study 2b).  Results did not show clear outcome.  
 
Study 3: 
Study 3 has been stopped and will not be continued. 
Brief Description: This study examined the effects of memory retrieval on extinction. It 
examined whether exposure to contextual stimuli present during acquisition or extinction 
can serve as implicit reminders of acquisition or extinction, respectively, and can 
reactivate the memory of the CS+-shock or CS+-no shock association. This study 
required 4 sessions –two at the Clinical Center and two sessions through an on-line 
program off campus.  
Outcome: In the lab, postdocs are given the freedom to develop their own research in 
their way for independence.  After the postdoc who proposed the study left, we did not 
pursue the substudy because 1) the substudy objectives were not a high enough priority 
compared to other competing studies and 2) other postdocs were not interested in 
working of this project. 
The choice is to place this study on hold until a new postdoc implements it or to remove 
it from the protocol. I selected to remove the study. It will not be continued.  
 
 
2. STUDY OBJECTIVES 
 
 Interaction between cognition and anxiety 

 
 
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It is well-established that anxiety interferes with behavior because it distracts from 
ongoing goals (Vytal, Cornwell et al. 2012). In fact, one of the main complaints of 
anxious individuals is their inability to remain focused due to distracting anxious 
thoughts. However, some individuals (e.g., soldiers) can perform very well under stress, 
down-regulating their fear and anxiety.  In addition, most people can also be distracted 
temporarily from their anxiety while being involved in a task (e.g., watching TV or 
working). Objective 2 will examine the interactions between experimentally-induced 
anxiety and performance on cognitive tasks. 
A leading theoretical account of emotion/anxiety interaction assumes that 1) worry is the 
component of state anxiety responsible for the effect of anxiety on performance on 
cognitive tasks and 2) that cognitive interference occurs especially at the level of working 
memory (WM) (Eysenck, Derakshan et al. 2007). The interaction between threat of shock 
and WM tasks depends on WM load. We have demonstrated that 1) threat of shock 
interferes to a greater extent with low load WM tasks than high load WM tasks (see Fig 2 
below) and 2) anxiety is reduced under high WM load compared to low WM load (see 
Fig 4 below) (Vytal, Cornwell et al. 2012). These results suggest that when WM is fully 
occupied by a task, there is little WM left to allocate to anxious thoughts. However, it is 
unclear whether these effects are due specifically to WM per se or to more general 
attention control mechanisms as we have also evidence of performance impairment 
during shock threat in tasks that requires little WM.  
Executive functioning is involved in goal directed behaviors. It is also probably involved 
in implicit and explicit emotion regulation, when emotion (e.g., anxiety) competes with 
our goals. There are different components of executive functioning including working 
memory, inhibition, and conflict.  How each of these components interacts with anxiety 
will be investigated in this study. 
This objective examines the role of components of executive functioning and context in 
the interaction between cognition and anxiety. The two key questions are 1) to which 
degrees various cognitive processes (e.g., 
working memory, inhibition) are affected 
(impaired/facilitated) by anxiety induced by 
shock anticipation, and 2) to which degree 
various cognitive tasks down-regulate 
experimental anxiety. 
 
 

 
 
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3. SUBJECTS 
 
a. Description of study populations 
The participants will be medically and psychiatrically healthy volunteers (males and 
females) ages 18-50.   The accrual ceiling is 1575 subjects. NIH, but not NIMH, 
employees may participate. NIH employee participation is guided by intramural institute 
policy. 
 
b. Inclusion criteria 
• Males and females 
• Age 18 – 50 years 
 
c. Exclusion criteria 
• Non-English speaking 
• Pregnancy 
• Any current ongoing medical illness 
• Current Axis I disorders  
• Past significant psychiatric disorders (e.g., psychotic disorders) according to 
DSM-IV 
• Current alcohol or substance abuse according to DSM-IV criteria 
• History of alcohol or substance dependence based on DSM-IV criteria within 6 
months prior to screening 
• Current psychotropic medication use 
• Current or past organic central nervous system disorders, including but not limited 
to seizure disorder or neurological symptoms of the wrist and arms (e.g., carpal 
tunnel syndrome).  The latter exclusion is for shock studies only. 
• Positive urine toxicology screen 
• Employees of NIMH or an immediate family member of a NIMH employee. 
 
4. STUDY DESIGN AND METHODS 
 
a) Study overview 
 
This protocol examines the interaction between cognition and induced anxiety.  This 
study consists of the following sub-studies.  Subjects can enroll in one or more sub-
studies. They will sign a consent form for each sub-study.  These sub-studies require 1 
outpatient visit to the Clinical Center for up to 4 hours or up to two visits if it includes a 
telehealth consenting visit.  
Study A: Working memory and anxiety 
Study C: Effect of anxiety on sustained attention to response task (SART) 
Study D: Effect of anxiety on Stroop task (closed for accrual; data analysis only) 
Study E: Pilot studies 
 
Study F: Pilot study of sural nerve stimulation 
 
  
 
b) Recruitment 

 
 
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Subjects will be recruited via advertisements in the local media (such as the free 
Express or Examiner; newsmagazines such as Washington Post Local Living 
sections and the like); postings on college campuses and on NIH campus; in 
coffee houses and fast food places with permission of managers; local libraries; as 
well as in Montgomery County and DC metro and buses.  We also will utilize 
websites, such as college papers (e.g. Maryland Diamondback, American 
University Student Media online publications, etc) and local media (e.g. the 
Gazette and Washington City paper).  The web ads will direct readers to the NIH 
Patient Info website.  We also will advertise on approved listservs as provided by 
Public Relations and Public Liaison office (e.g. NIMH Outreach Partnership 
Updates) or local groups (e.g. Community Service Announcements – City of 
Gaithersburg, Montgomery County Providers, Howard County Providers, 
Frederick Providers Council, etc) . IRB-approved ads will be posted on these 
listservs with the permission of the moderator and IRB-required statement on how 
the receiver was identified.    One such site will be the Club PCR site used by the 
research assistants at NIH. The ads will direct the participant to PRPL whose staff 
will conduct initial screening and pass along potentially appropriate callers to the 
PI or his staff. We may also post the advertisements on college recruiting websites 
for research studies, such as SONA. SONA is a software platform to post research 
studies. We will use IRB approved language for the posts and request that 
participants reach out to our group to express interest. No PII will be shared on 
the platform. Advertisements may also be placed on coffee sleeves and handed 
out with coffee orders at coffee shops. These advertisements will be IRB-
approved prior to use. IRB-approved text ads will be sent from Instagram, Twitter 
and Facebook Accounts such as NIH/CC and NIMH-Extramural. Twitter and 
other web language will be sent to local publications and groups, such as Family 
magazine, in hopes they will send it out to use on their own page. Notecards 
and/or flyers may be posted in places such as grocery stores, coffee shops, 
community centers, and bookstores, or placed in advocacy group offices, in 
doctor’s office waiting rooms, libraries, and retail establishments with approval of 
the venue or in accord with their policy.  They may be made available at outreach 
exhibits, speaking engagements, and professional meetings with approval of the 
venue or in accord with their policy.  They may be given directly to those 
requesting study information. Postcards may be sent using commercially-
available mailing lists via direct mail. The postcards will identify the source of the 
mailing list. ResearchMatch may be used to recruit participants for this protocol.   
We may also use the Survey tool in REDCap (Research Electronic Data Capture) 
to assist with recruitment, pre-screen potential participants, and evaluate our 
recruitment strategies. REDCap is a secure web application for building and 
managing online surveys and databases. REDCap Survey will not contain PHI 
and contains workflows that support the collection of de-identified data. 
Interested persons may be directed to REDCap from a link in our IRB approved 
advertisements. In our secure confirmation email to potential healthy participants, 
we would like to have the option to include the link to offer to allow participants 
to pass along as a WOM recruitment strategy with the disclaimer that they should 

 
 
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not forward the confirmation email. Potential participants will be given an 
automated survey when they click on the link. The survey would ask the potential 
participant our non-PII prescreening questions that we currently ask via a phone 
prescreen. The subject will be provided with a numeric code automatically by the 
database and will also be provided the phone number for our study staff and will 
be asked to call our research team to continue screening. The potential participant 
will be instructed to provide their unique numeric code during this phone screen. 
The unique code will allow the staff to connect the person to the non-PII 
information that has already been entered by the potential subject.  This allows us 
to speed up the prescreening process as we will have access to their answers. We 
will be the only ones with access to the responses. We will also use REDCap to 
develop an anonymous survey to evaluate our screening efforts. 
 
 Ads may be placed on the CC Twitter, Facebook page, and newsletters. IRB 
approved ads may be place on website such as advocacy groups, university 
student sites, and newspaper sites.  In addition ads will be place on Craigslist 
under the “Volunteer” category.  The email address will be hidden from public 
view to prevent spam.  IRB-approved Tabletop tent cards will be displayed on 
tables in public areas such as the NIH cafeterias, with the approval of the venue 
owner/manager. 
 
We may use paid advertising on social media sites, such as Facebook, Instagram, 
and Twitter, to recruit potential volunteers whom are within our age range and 
located near the NIH (within 50 miles). We will not use behavioral targeting for 
any advertising campaign. We will only use advertisements and videos that have 
been previously IRB approved. Accounts used for Twitter, Facebook, and 
Instagram are the NIH sponsored accounts. 
 
We may also identify potential healthy volunteers through the NIMH protocol 
titled “Recruitment and Characterization of Research Volunteers for NIMH 
Intramural Studies.” There is no direct solicitation of employees/staff by 
supervisors nor co-workers.   
 
Audio advertisements will be played during radio or podcasts, Potential radio 
stations may be public radio (i.e., WAMU) or other mainstream radio stations 
(i.e., 97.1 Fresh FM). Potential podcasts may be ones like Ted Radio Hour, Moth, 
Criminal, Diane Rehm, etc. An animated short video advertisement may also be 
posted on the NIMH YouTube channel. This video may also be posted on official 
NIH, NIMH, and NIH Clinical Center social media accounts such as Facebook, 
Instagram, and Twitter  It may be sent via sent via electronic listservs such as NIH 
postbac listserv, community listservs, advocacy and provider listservs, and 
running/ health listservs. 
 
Email addresses provided on the advertisements may be changed to the NIH email 
of other staff on this protocol following any staff changes or changes in the 
individual responsible for referrals. 

 
 
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During an initial telephone interview, the nature and duration of the various studies will 
be explained. Potential participants will then be screened for inclusion with screening 
protocol 01-M-0254, and subjects who qualify will be invited to participate in the study. 
A copy of the study consent form will be mailed to the subjects. This will give them an 
opportunity to review the consent form and ask questions. 
 
c) Screening  
 
Consent procedures are conducted prior to any research activities.  The screening for all 
three studies is done under the screening protocol 01-M-0254.  Screening will include a 
medical and a psychiatric history will be taken and a medical exam. In addition, the 
following laboratory tests will be obtained: 
• Pregnancy test for females of child bearing potential 
• Urine toxicology screen 
• Subject demographic information 
• In addition, a CBC, hepatic panel, electrolytes, creatinine, BUN, thyroid panel; 
and ECG may be done at the discretion of the examining physician or nurse 
practitioner.   
 
Results of the screening are recorded in CRIS. Subjects are contacted if abnormal results 
are revealed during screening. A test may be repeated to confirm a result if needed. If the 
abnormal result is confirmed, a clinician will contact the subject to advise him/her on 
treatment options outside NIH. 
 
• In addition to the medical and psychiatric assessment under protocol the 
screening protocol 01-M-0254,  participants may be asked to fill out the 
following questionnaires: 
 
o State-Trait Anxiety Inventory (STAI) (Spielberger 1983)  
o NEO-Personality Inventory (Costa and McCrae 1992) 
o Anxiety Sensitivity Index (Peterson and Reiss 1992)  
o Beck Anxiety Inventory (Beck and Steer 1987) 
o Beck Depression Inventory (BDI) (Beck and Steer 1987) 
o Fear Questionnaire (Marks and Mathews 1979) 
o Penn State Worry Questionnaire (Meyer, Miller et al. 1990) 
o Positive and Negative Affectivity Scale PANAS (Watson, Clark et al. 
1988) 
o Multidimensional Personality Questionnaire Harm Avoidance (MPQ-
HA) (Tellegen 2002) 
o Mood and Anxiety Symptoms Questionnaire  (MASQ) short-Form 
(Keogh and Reidy 2000) 

 
 
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o Adverse Life Event Checklist (Gray, Litz et al. 2004) 
o Attention Control Scale (Derryberry and Reed 2002)  
 
The non-analogue measures from these reports may be completed by the participant on 
the Clinical Trial Survey System (CTSS) online system. Subjects will have signed the 01-
M-0254 screening consent prior to filling out these measures on CTSS. AIs may then 
collect data from the Clinical Trials Database (CTDB) for the purposes of this study.  
Participants may enter their responses while at NIH using a wireless-device interface to 
access the NIH-intranet secure CTDB. Two items will require real time monitoring. Item 
#9 on the Beck Depression Inventory (BDI) and item #61 on the Mood and Anxiety 
Symptom Questionnaire-Short Form (MASQ-SF) inquire about suicidality. An associate 
investigator must check both items before the participant leaves the Clinical Center.  If 
the participant indicates anything higher than a “0” (“I don’t have thoughts of killing 
myself”) on the BDI item #9 or a “1” (“not at all”) on the MASQ-SF item #61, the 
associate investigator must notify a staff clinician. The staff clinician must assess for 
immediate safety and refer the participant to appropriate resources. If the screening 
protocol (01-M-0254) finds them to be eligible, they will undergo the informed consent 
process for 01-M-0185.   
 
d) Study procedures 
 
Procedures for the separate studies are outlined below. 
 
 
 
Anxiety will be induced with the threat of shock. 
• The threat of shock experiment requires a single session. Performance of 
cognitive tasks will be conducted during alternating periods of shock threat and 
safety.  
 
Sub-study A: Working memory and anxiety 
This study will examine whether greater WM capacity or practice is associated with 
increased or decreased anxiety during performance of a WM task during threat of shock. 
On the one hand, increase in WM capacity could be associated with more ability to 
allocate resources to anxious thoughts, therefore increasing anxiety. On the other hand, 
better WM reflects better ability to screen out intrusive stimuli and thoughts, thereby 
decreasing anxiety. As for task practice, we hypothesize that practice will make more 
resources available to allocate to anxious thoughts.   
 
Subjects will be asked to remember verbal and nonverbal stimuli. These stimuli consist of 
words, pictures, letters or spatial locations in series of stimuli. Participants will be 
instructed to remember one, two, or three stimuli back from the current stimulus on the 
screen (n-back task). There will be 2 groups. One group will practice the N-back task 

 
 
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minimally (e.g., 5 min) and the other group will have more extensive training (e.g., 20 
min).  
 
 
Sub-study C: Effect of anxiety on sustained attention to response task (SART) 
SART are sustained attention tasks during which a button-press response is made to 
frequent targets and responses are withheld to rare non targets. SART have been used 
extensively to study inattention, “off-task thinking”, task-unrelated thoughts, and ‘mind 
wandering” (Robertson, Manly et al. 1997). An advantage of this task is that it enables us 
to identify periods when the mind wanders. When the mind wanders, subjects make 
errors of commitment (they button-press to non-targets).  We will use SART to attempt to 
identify periods of anxious thoughts.  
 
Subjects will have to respond to frequent targets (e.g., digits 1, 2, 3…) and to withhold 
response to rare non targets (e.g., 3). We will examine the association between response 
speed and anxiety as assessed with startle. Mind wandering in SART is associated with 
faster RT. We will examine whether faster RT is associated with larger potentiated 
startle. We will also probe subjects’ thoughts (on-task, off-task, anxious thoughts) after 
each startle stimulus to assess whether anxious thoughts predict startle potentiation.    
 
Sub-study D: Effect of anxiety on Stroop task 
We recently wrote a review on the effect of threat of shock on cognitive performance. 
Results suggest that threat of shock impaired performance on the emotional Stroop but 
tended to facilitate performance on the classic Stroop. However, results of the classic 
Stroop studies were not straightforward. This study will examine the effect of the threat 
of shock on classic and emotional Stroop in a within-subject design. 
 
Stroop tests are used to examine conflict of emotional distraction. In the classic Stroop 
test, the name of a color is printed in a color that conflicts or does not conflict with the 
word (e.g., the word red printed in blue).  In the emotional Stroop, the words are not 
colored words but emotional words (e.g., the word death printed in blue). The subject’s 
task is to name the color of the word. 
 
Sub-study E: Pilot studies 
We will pilot experiments that examine the effect of threat of shock on various cognitive 
processes. The objectives are 1) to assess the feasibility of implementing cognitive tasks 
conducted under threat of shock while startle stimuli are occasionally administered, 2) 
improve upon study design, and 3) attempt to predict appropriate sample sizes for 
neuroimaging, psychopharmacological, and clinical studies carried out in our other 
protocols (03-M-0093, 02-M-0321).   
 
The design of the experiment will be similar to the threat of shock experiments in other 
Study 2 studies. Subjects will perform cognitive tasks during alternating safe and threat 
periods. Occasional startle stimuli will be delivered to assess anxiety. We will focus on 4 
cognitive domains: 1) sensory/perceptual processes, 2) attention/control, 3) working 

 
 
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memory, and 4) complex executive functions (e.g., planning, decision making). The 
following tasks will be piloted: 
 
• Sensory/Perceptual processing: We will examine the effect of threat of shock on 
the ability to remain focused on goal-relevant stimuli in the presence of 
potentially interfering distractors. We will use a response-competition paradigm 
during which subjects make speeded responses indicating whether a central target 
is one of two pre-specified targets while attempting to ignore peripheral 
distractors (Lavie 2005). Slower responses in the presence of an incongruent 
distractor will be compared with a congruent distractor. Perceptual load is 
manipulated by varying the number of items that are similar to the target. 
Conflict tasks: Tasks such as the Flanker task will be used. This task requires 
subjects to attend to a centrally presented stimulus and ignore the flanking stimuli 
surrounding it. When the flanking stimuli are mapped to the opposite response 
from the center stimulus (incompatible trials), subjects respond more slowly 
because of the need to exercise top-down control. We are interested in this task 
because it examines top-down control without relying on working memory. 
Executive function/cognitive flexibility: We will examine threat of shock on 
complex executive functions testing including decision making (e.g., Iowa 
Gambling task), planning ability (e.g., Tower of London), set-shifting (e.g., 
Wisconsin Card Sorting test), and solving tasks (e.g., anagrams).  
Working memory: We have found that threat of shock reduced performance on 
n-back WM tasks.  Because of the nature of N-back tasks, it is unclear whether 
reduced performance is due to anxiety interference with memory or other 
processes such as perception, encoding, or retrieval. We will investigate whether 
we can dissociate the effect of anxiety on these processes using Sternberg WM 
tasks.  
 
Sub-study F: Pilot studies of sural nerve stimulation 
In Protocol 01-M-0185, the shocks are delivered via electrodes located on the forearm or 
fingers. In order to obtain an objective measure of unpleasantness, we will pilot a new 
technique using up to 30 subjects in a session lasting up to 2 hours.  
 
This pilot experiment will be conducted under the supervision of the PI, who used this 
technique when he was a graduate student. 
 
The objective of this pilot study is to learn to implement this new technique. The shock 
will be administered on the sural nerve in the retromalleolar space and recording of the 
response from the biceps femoris muscle (see Electric shock: sural nerve below). We will 
learn how to locate the best placement of the stimulating electrodes to stimulate the sural 
nerve and of the recording electrodes to record the R3 reflex. We will also need to 
determine the best parameters of stimulation.  Finally, we will examine how to organize 
the equipment in experiments that investigate the interaction between anxiety and 
cognition. Hence, in a subgroup of subjects, we will pilot delivering sural nerve 
stimulation while they perform a working memory or vigilance task. 
 

 
 
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Procedures common to all studies 
 
Startle response  
The startle reflex will be elicited with a 102 dB white noise (40-ms duration) delivered 
binaurally via headphones. The eyeblink component of the startle reflex will be recorded 
binaurally with two electrodes placed under each eye. Eyeblink responses will be scored 
in the 20-100 ms window following the onset of the startle stimulus. 
 
Autonomic measures 
Heart rate, skin conductance activity (event-related responses and spontaneous 
fluctuations), and finger pulse volume (FPV) will be recorded during testing to evaluate 
changes in autonomic arousal. The heart rate will be monitored with two disposable 
electrodes, one on each wrist. A computer algorithm will detect the R-wave in each 
cardiac cycle and calculate the number of whole and fractional heart beats for 500 ms 
periods in each condition. The skin conductance will be measured using two Ag-AgCl 
electrodes filled with a .05M NaCl electrolyte. Electrodes will be placed on the distal 
phalanges of the index and second fingers of the left hand.  FPV will be measured with a 
finger plesthymograph. 
 
Grip force measures 
The strength of hand compression (i.e. grip force) will be recorded during testing to 
evaluate behavioral indices of motivation and performance. Participants will hold a hand 
clench dynamometer which will assess hand compression in units of kilogram-force 
(kgf). The magnitude of grip force will serve as a within-subject measure of motivation. 
Additionally, patterns of dynamic grip force behavior will serve as measures of fine 
motor skill.  
Procedures to induce anxiety 
 
Electric shock: forearm or fingers 
Electric shocks are one of the most efficient ways to induce anxiety in the laboratory. The 
shocks will be delivered through two disk electrodes located on the forearm or on two 
fingers.   
Test shocks 
At the beginning of the study a shock workup procedure is conducted to determine the 
setting for the overall procedure for each subject. The level of shock is initially set at 3.5 
μAmp (the low range).  A shock is administered and the subject is asked to rate it on a 
scale from 1 (not at all painful) to 5 (extremely painful). We then increase the level of 
shock slightly until the subject identifies the sensation at a rating level of 4.  No more 
than three test shocks are given and the level is selected based on the subjective ratings 
provided by the participant.  No subject will ever receive a shock rated more than 4 for 
the duration of their study.  In addition, subjects are reminded that they have the 
opportunity to withdraw from the study at anytime if they wish.  
 
Participant rating of the experience:  

 
 
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At the end of the study subjects are asked to retrospectively rate the shock experiment on 
a scale from 1 (not at all painful) to 10 (extremely painful). This provides us with an 
assessment of the overall unpleasantness of the experience during the entire shock 
experiment.  
 
Electric shock: sural nerve 
The shock will be administered on the sural nerve in the retromalleolar space and 
recording of the response from the biceps femoris muscle. The aim is to have an objective 
measure of unpleasantness. The R3 reflex will be evoked using a method similar to that 
described in Rhudy et al (2005). Briefly, electric stimulation of the sural nerve will be 
four to six rectangular wave pulses of 1- to 2-ms duration every 2 to 5 ms. A trial will 
begin with 0 mA and increased in 1.0-1.5-mA steps until a R3 reflex is detected. Stimulus 
intensity will then be decreased in 0.75 mA steps until a reflex is no longer detected. This 
up–down staircase procedure will be repeated two more times, but with 0.5-mA steps 
until the R3 threshold is determined. Once the R3 threshold is determined, the intensity of 
stimulation during experiments in which we used shock to evoke anxiety will be set at 1.2 
times the threshold intensity.  At this intensity, subjects usually report a slightly painful 
pin-prick sensation (Rhudy et al 2005). 
 
e. End of Participation 
 
The participant is discharged following each experiment.  Subjects will be terminated 
from the protocol if they request to discontinue participation. 
 
5. MANAGEMENT OF DATA AND SAMPLES 
 
a. Storage 
 
No samples will be collected or stored. The samples that were previously stored at the 
Neuroendocrine Core Facility (NCF) have been discarded.  
 
Questionnaires are labeled with a subject number.  The master list and computerized data 
are maintained in password protected computers. Only study personnel will be given a 
password and have access to the data. Data will be stored without identifiers, and only 
study personnel with have access to the code key.  
 
 
Additional information regarding the web site: The servers are located on the main 
NIH campus, building 10, room 4A11A.  The room is locked and accessible only by 
selected NIMH IRP Computer Support staff.  NIMH IRP computer support will collect 
userID, day/time website is accessed, and the subjects’ responses to the questions. The 
subjects’ names will not be used. Two servers will be utilized:  one server will be used to 
host the web application; the second server will be used to store the data.  The web 
application will require userID and password for access. The site itself will be SSL 
encrypted (https).  The MS SQL database that contains the data will be on a separate 

 
 
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server, isolating the data should the web server become compromised. The data stored on 
the MS SQL database will be encrypted and protected.  Before deployment to the general 
public, the ISSO and IRT will perform vulnerabilities scan on the site.  All security 
vulnerabilities will be corrected before the site goes ‘live’.  
 
b. Data and sample sharing plan 
 
This protocol is not subject to the Genomic Data Sharing (GDS) Policy. Samples are not 
collected or stored. Data may be shared with collaborating laboratories at NIH or outside 
of NIH and/or submitted to NIH-designated repositories and databases if consent for 
sharing was obtained.  Repositories receiving data from this protocol may be open-access 
or restricted access.  
 
Data will be stripped of identifiers and may be coded (“de-identified”) or unlinked from 
an identifying code (“anonymized”). When coded data is shared, the key to the code will 
not be provided to collaborators, but will remain at NIH.  Data may be shared with 
investigators and institutions with an FWA or operating under the Declaration of Helsinki 
(DoH) and reported at the time of continuing review.  Sharing with investigators without 
an FWA or not operating under the DoH will be submitted for prospective IRB approval.  
Submissions to NIH-sponsored or supported databases and repositories will be reported 
at the time of Continuing Review.  Submission to non-NIH sponsored or supported 
databases and repositories will be submitted for prospective IRB approval. 
 
Required approvals from the collaborating institution will be obtained and materials will 
be shipped in accordance with NIH and federal regulations. 
 
Data collected prior to Amendment TT will not be shared.  
 
6. ADDITIONAL CONSIDERATIONS 
 
a. Research with investigational drugs or devices 
Acoustic startle and shock device used in this protocol are considered non-significant risk 
(NSR) devices and will only be used within published guidelines. The auditory startle 
device is manufactured by Biopac and the shock device is manufactured by Digitimer. 
   
Auditory startle does not meet criteria for a Significant Risk device as outlined Under 21 
CFR 812.3(m), as an investigational device that:  
1. Is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject  
Response: Auditory startle is not an implantable device. 
2. Is purported or represented to be for a use in supporting or sustaining human life 
and presents a potential for serious risk to the health, safety, or welfare of a subject  
Response: Auditory startle is not for use in supporting or sustaining human life. It does 
not present a potential for serious risk to the health, safety, or welfare of participants 
when used as described in this protocol.  

 
 
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3. Is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential for 
serious risk to the health, safety, or welfare of a subject  
Response: Auditory startle, as used under this protocol is not of substantial importance in 
diagnosing, curing, mitigating, or treating disease, or otherwise preventing impairment 
of human health and does not present a potential for serious risk to the health, safety or 
welfare of a subject.  
4. Otherwise presents a potential for serious risk to the health, safety or welfare of a 
subject  
Response: Auditory startle has been in use numerous for decades and have been cleared 
by the FDA. Safety guidelines have been developed and updated allowing its 
dissemination to a wide range of clinical and non-clinical settings. The FDA has 
generally waived pre-IDE inquiries for auditory startle studies on an NSR device basis. 
Hence, the CNS IRB, like most US IRBs, has accepted NSR designation for auditory 
startle within these limitations.  
 
The shock device (electrical stimulator) does not meet criteria for a Significant Risk 
device as outlined Under 21 CFR 812.3(m), as an investigational device that:  
 
 
1. Is intended as an implant and presents a potential for serious risk to the health, 
safety, or welfare of a subject  
Response: The shock device is not an implantable device. 
2. Is purported or represented to be for a use in supporting or sustaining human 
life and presents a potential for serious risk to the health, safety, or welfare of a subject  
Response: The shock device is not for use in supporting or sustaining human life. It does 
not present a potential for serious risk to the health, safety, or welfare of participants 
when used as described in this protocol.  
3. Is for a use of substantial importance in diagnosing, curing, mitigating, or 
treating disease, or otherwise preventing impairment of human health and presents a 
potential for serious risk to the health, safety, or welfare of a subject  
Response: The shock device, as used under this protocol is not of substantial importance 
in diagnosing, curing, mitigating, or treating disease, or otherwise preventing 
impairment of human health and does not present a potential for serious risk to the 
health, safety or welfare of a subject.  
4. Otherwise presents a potential for serious risk to the health, safety or welfare of 
a subject  
Response: The shock device has been in use numerous for decades and have been cleared 
by the FDA. Safety guidelines have been developed and updated allowing its 
dissemination to a wide range of clinical and non-clinical settings. The FDA has 
generally waived pre-IDE inquiries for shock studies on an NSR device basis. Hence, the 
CNS IRB, like most US IRBs, has accepted NSR designation for shock device within these 
limitations.  
 
 
7. RISKS AND DISCOMFORTS 
 
The following studies are considered minimal risk studies:  

 
 
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a. Psychophysiological recording: The psychophysiological measures that will be 
obtained are non-invasive, do not involve the administration of needles, drug, or dyes. 
Little discomfort is expected. During electrode placement, the possibility of skin irritation 
from contact with the saline electrode paste exists. However, this is unlikely as the salt 
concentration of the paste is similar to that of human sweat. The risk is equivalent to that 
of an EEG recording. 
 
b. Auditory startle stimulus: The auditory stimuli that will be used in the startle studies 
are white noise with a duration of 40 ms and an intensity of 102 dB. Auditory startling 
sounds of much higher intensities are frequently used in startle studies (Hawk and Cook 
2000). Sounds of higher intensities and longer durations are also widely used in aversive 
conditioning in human subjects, where they serve as US (Hodes, Cook et al. 1985; Sandin 
and Chorot 1989). The short duration (40 ms) of these sounds makes them harmless (i.e., 
there is no danger of hearing impairment). In addition, white noises are safer than pure 
tones. The PI has been involved in various studies and collaborations involving over 1000 
of subjects with no adverse reactions. 
 
c. Electric shock: In the main studies, the shocks will be delivered through two disk 
electrodes located on the forearm or on two fingers. The shock is generally described by 
subjects as anxiogenic and moderately painful.  At the end of testing the mean participant 
rating of aversiveness on a scale of 1 (not at all painful) to 10 (extremely painful) is about 
5. Over 95% of subjects who experienced the shock chose to participate in the 
experiment.   
 
In very rare occasions, subjects have experienced symptoms that may be related to the 
shock. For instance, a participant with a condition called “cubital tunnel syndrome”, a 
repetitive motion injury similar to carpal tunnel syndrome, indicated worsening of his 
syndrome over the months subsequent to his participation. Another participant reported 
pain in her arms for several hours after testing. The pain was no longer present the next 
day. It is unclear whether these symptoms were due to the shocks. Nevertheless, subjects 
with neurological symptoms of the wrist and arms will be excluded from the study.  
 
Study 2f pilots a new method of administering the shock.  The shocks are anxiogenic and 
are usually described as inducing a slightly painful pin-prick sensation (Rhudy et al 
2005).  Typical experiments include over 20 such stimuli (Rhudy et al 2005). Our shock 
studies usually include less than 10 shocks and most frequently about 5/6 shocks. 
Subjects with “cubital tunnel syndrome” do not need to be excluded given than the 
shocks are not delivered on the arm or wrist. 
 
d. Emotional pictures: The emotional pictures will be selected from a set of 
standardized stimuli that are frequently used in psychophysiological and brain imaging 
studies of emotions (Lang, Ohman et al. 1988). Because the intensity of these pictures 
might make some people uncomfortable, their contents will be fully described to the 
subjects before participation in the study. In addition, a debriefing will be conducted to 
assess whether subjects are distressed or negatively affected by the pictures and in need 

 
 
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of psychological supports by a clinician. A clinician will then talk to them and will follow 
up with a call to see if further supportive contact is needed. If so, then a study clinician 
will meet with the participant and assess for necessary intervention. 
 
7.2 Assessment of Potential Risks and Benefits 
This is a more than minimal risk protocol in healthy volunteers.  These experiments may 
enhance our understanding of how stress and anxiety affect associative learning, that is, 
the way we learn about our environment. We have minimized risks by delivering electric 
shosks at a level that is judged by the participant as uncomfortable and tolerable. Subjects 
may stop the experiment at any time if they find the discomfort to be too great. The risks 
are reasonable in relation to the anticipated benefit.  
 
 
 
8. SUBJECT SAFETY MONITORING 
 
An Associate Investigator is in an adjacent room to the participant during all phases of 
the procedures. There is a non-recording camera that allows AI to monitor the subject.  
The subject is able to wave if there is a problem or simply call out to the AI as the rooms 
are connected with intercom. Participants are told they may end any procedure at any 
time by waving to AI or calling out.  The AI can end experiments if the subject is unable 
to follow instructions. The medically responsible physician or members of his/her staff 
are available by phone or pager for any medical problems that arise.  
 
9. OUTCOME MEASURES 
 
Primary outcome measures: The primary outcome measures will be the startle 
reflex, and performance on cognitive tasks.  
Secondary outcome measures: These measures include psychophysiological 
measures other than startle (e.g., skin conductance), psychological questionnaires 
(Spielberger state and trait anxiety), and performance on standard cognitive tests 
(e.g., working memory capacity).  
 
 
10. STATISTICAL ANALYSIS 
 
The accrual ceiling for this protocol is 1575 healthy controls. The overall current target N 
for the protocol is 148 healthy controls.  
Sub-study A (previously Study 2a) 
36 subjects 
Sub-study C (previously Study 2c)   34 subjects 
Sub-study E (previously Study 2e) 
48 subjects 
Sub-study F (previously Study 2f) 
30 subjects 
 
Study 2a: Working memory and anxiety 

 
 
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Analysis of the data: Startle and performance data will be analyzed with ANOVAs with 
repeated measures using condition (threat, safe) x Load (low, high). 
Power analysis: Based on pilot and published works in our lab, we expect an 
small/moderate  effect size for startle potentiation (Cohen’s d=0.3). A priori calculations 
indicate that the testing of 30 participants at an alpha of 0.05 would provide power of 
0.80 to detect a significant 2 (Condition) x 2 (Load) interaction on startle 
response.  Assuming a 20% attrition rate, we will collect (about) 36 subjects. 
 
Study 2c: Effect of anxiety on sustained attention to response task (SART) 
Analysis of the data: The main analysis is to compare startle magnitude when subjects 
focus on the task and when their mind wander with ANOVAs with repeated measures 
using condition (threat, safe) x thought (on task, off task). 
Power analysis: Based on pilot works in our lab, we expect an small/moderate  effect 
size for startle potentiation (Cohen’s d=0.4). A priori calculations indicate that the testing 
of 28 participants at an alpha of 0.05 would provide power of 0.80 to detect a significant 
2 (Condition) x 2 (Load) interaction on startle response.  Assuming a 20% attrition rate, 
we will collect (about) 34 subjects. 
  
 Study 2d: Effect of anxiety on Stroop task 
Study is 2d is is closed for accrual. We are currently analyzing the data.  
 
Study 2e: Pilot studies 
These pilot experiments are designed to determine the parameter of stimulation of study 
that will be used in our neuroimaging, psychopharmacological, and clinical studies 
conducted in our other protocols (03-M-0093, 02-M-0321, 02-M-0263). We use tasks in 
4 domains, 1) sensory processing, 2) conflict, 3) executive function, and 4) working 
memory. We will use 12 subjects per domain for a total of 48 subjects.  
 
Study 2f: Pilot study of sural nerve stimulation 
This pilot study investigates the location of the stimulating and recording electrodes and 
different types of stimulus delivery. We have requested 30 subjects. 
 
11. HUMAN SUBJECTS PROTECTION 
 
a. Subject selection 
i. Both genders will be equally represented. Ethnic representation will reflect the 
population from which the sample is recruited.  
 
ii. As people get older the probability that they startle decreases. Because startle is 
our main independent measure it is important to test subjects who are likely to startle in 
order to collect analyzable data.  Based on our experience, we made the decision that 50 
would be the upper age limit to ensure usable data and thus justify the minimal risk.     
 
b. Justification for inclusion/ exclusion of children 
Children will be excluded because of the aversive nature of the stimuli used in this 
protocol. 

 
 
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c. Justification for inclusion of other vulnerable subjects, e.g. cognitively impaired, 
pregnant, mentally ill 
Protections for employees and staff participating in this study include 1) assuring that the 
participation or refusal to participate will have no effect, either beneficial or adverse, on 
the subject’s employment or position at the NIH, 2) giving employees and staff who are 
interested in participating the “NIH Information Sheet on Employee Research 
Participation” prior to obtaining consent, and 3) assuring that there will be no direct 
solicitation of employees or staff. This study collects sensitive information (e.g. drug and 
alcohol use, specific medical diagnoses). The PI will train study staff regarding obtaining 
and handling potentially sensitive and private information about co-workers through staff 
discussions and written branch/section procedures.  Information about sensitive 
information (e.g. drug and alcohol use, specific medical diagnoses) will be in the 
participant’s NIH medical record. 
 
d. Justification for exclusion of decisionally impaired adults: 
All subjects must be able to provide their own consent. We do not want to enroll 
participants who do not understand the risk/benefit ratio of the study, particularly when 
there is no benefit to the participants.  
 
e. Justification of sensitive procedures (use of placebo, medication withdrawal, 
provocative testing) 
Not applicable. 
 
f. Safeguards for vulnerable populations 
Female participants will undergo urine pregnancy tests prior to experiments. Neither 
participation nor refusal to participate as a subject will have an effect, either beneficial or 
adverse, on the participant’s employment or position at NIH. The NIH Information Sheet 
on Employee Research Participation will be available to employees considering 
enrollment. 
 
 
 
 
 
12. CONSENT DOCUMENTS AND PROCESS 
 
a. Designation of those obtaining consent:  Study investigators or Associate 
Investigators identified in the KSP form will obtain informed consent.  NIH 
employees will not be consented by a coworker.  
 
b. Consent procedures: All participants will receive a verbal explanation in terms 
suited to their comprehension of the purposes, procedures and potential risks of 
the study and of their rights as research participants.  Participants will have the 
opportunity to carefully review the written consent form and ask questions 
regarding this study prior to signing.   

 
 
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Telehealth Consent Procedures: Under certain circumstances, there may be a need 
to obtain telephone or telehealth consent for this study using NIH-approved 
platforms.  Under these circumstances, the individual will be sent a copy of the 
consent form. After the document is reviewed, an AI authorized to obtain consent 
will contact the individual and review the procedures and risks.  The participant 
and the investigator will sign their respective copies, and the former will return 
their copy to the investigator.  One copy each of the signed consent form will be 
placed in the Medical Record, retained by the investigator, and returned to the 
participant or parent/guardian. Proper documentation will be made in CRIS. 
 
 
c. Consent documents 
There are two consent forms for this protocol, each containing all required elements, and 
designated as follows: 
Shock 
Sural nerve stimulation 
 
13. DATA AND SAFETY MONITORING 
 
a. Data and Safety monitor: Data and safety monitoring will be conducted by the P.I. 
who will review the results for each subject.  
 
b. Data and Safety monitoring plan: All participants are monitored while they are 
onsite.  Paper data are stored in locked cabinets within locked closets/rooms. 
Electronic data are encrypted and cannot be accessed without obtaining a 
password. All data are reviewed as they are obtained. The PI will review data and 
safety parameters at least annually. The PI will document the data and safety 
review in the research records and at the time of continuing review. 
 
 
14.   STUDY INTERVENTION AND PARTICIPANT DISCONTINUATION/ 
WITHDRAWAL 
 
14.1 Discontinuation of Study Intervention  
  
Discontinuation from the task will mean discontinuation from the study, and 
remaining study procedures will be stopped.  Only clinical evaluations of AEs and 
follow up calls may occur following discontinuation. Any new clinically relevant 
finding will be reported as an adverse event (AE). 
 
  
Participants are free to withdraw from participation in the study at any time upon 
request. 
  
An investigator may discontinue or withdraw a participant from the study for the 
following reasons: 

 
 
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• Completion of study intervention 
• If any significant worsening of symptoms or active suicidal ideation, 
clinical adverse event (AE), laboratory abnormality, or other medical 
condition or situation occurs such that continued participation in the study 
would not be in the best interest of the participant 
• Concomitant medication use  
• Investigator discretion 
• Positive pregnancy test 
 
14.2 Participant Discontinuation/Withdrawal from the Study 
Participants are free to withdraw from participation in the study at any time upon 
request.  
 
An investigator may discontinue or withdraw a participant from the study for the 
following reasons: 
• Significant study intervention non-compliance  
• If the participant meets an exclusion criterion (either newly developed or 
not previously recognized) that precludes further study participation 
• Subject has completed the study  
• Death 
• Screen Failure 
  
The reason for participant discontinuation or withdrawal from the study will be 
recorded in CRIS.  
 
14.3 Lost to Follow-up 
Not applicable. 
 
15. QUALITY ASSURANCE 
 
a. Monitor 
Quality assurance will be monitored by the PI and research team and the NIMH Office of 
Regulatory Oversight (ORO). 
 
 
b. Plan 
ORO monitors intramural research studies to ensure compliance with GCP, 
organizational policies and regulations.  Audit frequency is determined by the ORO SOP 
based on the study level of risk.  Results of ORO audits are provided to the PI, The 
Clinical Director and the CNS IRB.  This study will undergo audits at least once every 
three years and for cause. 
 

 
 
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16. ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS  
Definition of Adverse Event 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 
(a)). 
Definition of Serious Adverse Events (SAE) 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the 
view of either the investigator or sponsor, it results in any of the following outcomes: 
death, a life-threatening adverse event, inpatient hospitalization or prolongation of 
existing hospitalization, a persistent or significant incapacity or substantial disruption of 
the ability to conduct normal life functions, or a congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse. 
Classification of an Adverse Event 
Severity of Event 
For adverse events (AEs) not included in the protocol defined grading system, the 
following guidelines will be used to describe severity.  
 
• Mild – Events require minimal or no treatment and do not interfere with the 
participant’s daily activities.  
• Moderate – Events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with 
functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually potentially 
life-threatening or incapacitating.  Of note, the term “severe” does not necessarily 
equate to “serious”.] 
 
Relationship to Study Intervention 
All adverse events (AEs) must have their relationship to study intervention assessed by 
the investigator who examines and evaluates the participant based on temporal 
relationship and his/her clinical judgment. The degree of certainty about causality will be 
graded using the categories below. In a clinical trial, the study product must always be 
suspect.  
 

 
 
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• Related – The AE is known to occur with the study intervention, there is a 
reasonable possibility that the study intervention caused the AE, or there is a 
temporal relationship between the study intervention and event. Reasonable 
possibility means that there is evidence to suggest a causal relationship between 
the study intervention and the AE. 
• Not Related – There is not a reasonable possibility that the administration of the 
study intervention caused the event, there is no temporal relationship between the 
study intervention and event onset, or an alternate etiology has been established. 
 
 
Expectedness  
The LIP and/or the Medical Associate Investigator will be responsible for determining 
whether an adverse event (AE) is expected or unexpected.  An AE will be considered 
unexpected if the nature, severity, or frequency of the event is not consistent with the risk 
information previously described for the study intervention. 
Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to 
the attention of study personnel during study visits and interviews of a study participant 
presenting for medical care, or upon review by a study monitor. 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be 
captured on the appropriate case report form (CRF). Information to be collected includes 
event description, time of onset, clinician’s assessment of severity, relationship to study 
product (assessed only by those with the training and authority to make a diagnosis), and 
time of resolution/stabilization of the event. All AEs occurring while on study must be 
documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s 
condition deteriorates at any time during the study, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the 
duration of the event at each level of severity to be performed. AEs characterized as 
intermittent require documentation of onset and duration of each episode. 
At each study visit, the investigator will inquire about the occurrence of AE/SAEs since 
the last visit.  Events will be followed for outcome information until resolution or 
stabilization. 
Adverse Event Reporting  
Reportable events for this protocol will be tracked and reported in compliance with 
Policy 801. 
Serious Adverse Event Reporting  
Reportable events for this protocol will be tracked and reported in compliance with 
Policy 801. 

 
 
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Events of Special Interest  
Not applicable.  
Reporting of Pregnancy  
Not applicable.  
 
 
17. PROTOCOL DEVIATIONS 
It is the responsibility of the investigator to use continuous vigilance to identify 
and report deviations to the NIH Institutional Review Board as per Policy 801.  
All deviations must be addressed in study source documents, reported to National 
Institutes of Mental Health Clinical Director.  The investigator is responsible for 
knowing and adhering to the reviewing IRB requirements. 
17.1 
NIH Definition of Protocol Deviation 
A protocol deviation is any changed, divergence, or departure from the IRB-
approved research protocol.  
• Major deviations: Deviations from the IRB approved protocol that have, 
or may have the potential to, negatively impact the rights, welfare or 
safety of the subject, or to substantially negatively impact the scientific 
integrity or validity of the study. 
• Minor deviations: Deviations that do not have the potential to negatively 
impact the rights, safety or welfare of subjects or others, or the scientific 
integrity or validity of the study. 
 
18. ALTERNATIVES TO PARTICIPATION 
 
Subjects do not receive any treatment in this study or forego any treatment in order to 
participate in this study.  The alternative, therefore, is not to participate.   
 
19. PRIVACY 
 
All research activities will be conducted in as private a setting as possible.  
 
20. CONFIDENTIALITY 
 
a. For research data and investigator medical records 
 
We will actively protect confidentiality of the subjects and the data in each step. 
Information will be stored using a confidential case number, and no identifiers (name, 
address, …) will be used that could allow direct linking of database information to 
individual subjects. The master code list will be stored separately from the data.  
Electronic data are stored on password protected computers.  Research staff will be 
trained to respect the privacy and confidentiality of NIH employees/staff, especially for 
sensitive, private information (e.g. illegal drug or alcohol use) may be obtained during 
participation.  
 

 
 
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b. For previously stored samples 
 
Specimens are no longer being stored. We have properly destroyed previously stored 
samples.  
 
c. Special precautions 
 
Hard copy data are stored with a double lock method (in a locked cabinet in a locked 
room).  Only study investigators have access to the data.  
 
21. CONFLICT OF INTEREST/ TECHNOLOGY TRANSFER 
 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons 
who have a role in the design, conduct, analysis, publication, or any aspect of this trial 
will be disclosed and managed. Furthermore, persons who have a perceived conflict of 
interest will be required to have such conflicts managed in a way that is appropriate to 
their participation in the design and conduct of this trial.  The study leadership in 
conjunction with the National Institutes of Mental Health has established policies and 
procedures for all study group members to disclose all conflicts of interest and will 
establish a mechanism for the management of all reported dualities of interest. 
 
22. TECHNOLOGY TRANSFER 
 
No technology transfer agreement is in place for this protocol. 
 
23. RESEARCH AND TRAVEL COMPENSATION 
 
Volunteers will be compensated for time and research-related inconveniences as shown 
in table below. If subjects do not complete the study they will be paid half of the 
compensation. Travel is not compensated.  Payments are mailed after each NIH study 
session.  NIH employees or staff who participate during work hours must have 
permission from their supervisor. NIH employees or staff must either participate outside 
of work hours or take leave in order to receive compensation. 
 
 
 
 
VOLUNTEER PAYMENT SCHEDULE 
Protocol: Effects of arousal and stress on classical conditioning 
 
 
 
 
 
Procedure (shock threat – 1 day) 
Duration    
Amount  
Psychophysiology / shock 
3-4 hrs 
$120 

 
 
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Additional optional telehealth consenting visit 
30 minutes 
$20 
TOTAL 
4 hrs 
$120 or $140 
 
 
 
 
 
 
 
 
 
 

 
 
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