fMRI Investigation of Explicit Cue and Contextual Fear
Phase: NA
Conditions: Anxiety Disorders, Fear
Sponsor: National Institute of Mental Health (NIMH)

PROTOCOL SUBMISSION TEMPLATE  
CNS IRBProtocol Template (rev.1-17-08) 
 
IRB Panel: Blue    White 
 
1. Title Page  
 
 
  
 
Protocol Title: 
fMRI Investigation of Explicit Cue and Contextual 
Fear 
 
Protocol Number: 
 
 
02-M-0321 
 
Date of This Submission/Version:   May 01, 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 
Total accrual ceiling: 1952 
(726) Patients   
  
  (1226) Healthy Volunteers   
  
 
Project Uses Ionizing Radiation:    No  
 Yes (attach RSC/RDSC documentation) 
  
 
 Medically-indicated only 
  
 
 Research-related only 
  
 
 Both 
 
IND/IDE 
 
 
 
No   
Yes (attach FDA documentation) 
  
Drug/Device/#:  
Acoustic startle  
Shock device 
7T fMRI 
  
Sponsor: _________________________ 
 
Durable Power of Attorney  
 
 No   
 Yes  
 
Multi-institutional Project 
 
No   
 Yes  
 
 
 
  
 
Data and Safety Monitoring Board  
No   
 Yes  
 
Technology Transfer Agreement  
 No   
 Yes 
 
Confidential Disclosure Agreement     
 No  
 Yes 
 
Samples are being stored  
 
 
 No   
 Yes  

 
 
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Table of Contents (number pages) 
 
Précis ......................................................................................................................... 4 
1. Introduction and Background ……………………………………………. 
 4 
2. Study Objectives................................................................................................... 15 
3. Subjects ................................................................................................................. 16 
4. Study Design and Methods .................................................................................. 19 
5. Management of Data and Samples……………………………………………   
7. Risks and Discomforts ......................................................................................... 39 
8. Subject Safety Monitoring ...................................................................................41 
9. Outcome Measures................................................................................................43 
10. Statistical Analysis……………………………………………………………...44 
11.Human Subjects Protection…………………………………………………….45 
12. Anticipated Benefits……………………………………………………………52 
13. Classification of Risk……………………………………………… …………..52 
14. Consent Documents and Process……………………………………………..  53 
15. Data and Safety Monitoring…………………………………………………... 53 
16. Quality Assurance………………………………………………………………54 
17. Reporting of Unanticipated Problems, AEs and Protocol Deviations.............54 
18. Alternatives to Participation…………………………………………………...55 
19. Privacy……….…………………………………………………………………..55 
20. Confidentiality…………………………………………………………………..55 
21. Conflict of Interest……………………………………………………………   55 
22. Technology Transfer…………………………………………………………    56 
23. Research and Travel Compensation ………………………………………… 56 
24. References ........................................................................................................... 57 
25. Attachments/ Appendices……………………………………………………... 64 
26. Consent forms…………………………………………………………………. 64 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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3. 
Précis: 
This protocol examines the neurobiology of fear and anxiety using various approaches. 
During fear conditioning in which a phasic explicit cue (e.g., a light) is repeatedly 
associated with an aversive unconditioned stimulus (e.g., a shock), the organism develops 
fear to the explicit cue as well as to the environmental context in which the experiment 
took place. Experimental evidence suggests that cued fear and contextual fear model 
different aspects of anxiety. Studies in patients indicated that contextual fear may model 
an aspect that is especially relevant to anxiety disorders (Grillon et al., 1994, 1998a,b; 
1999). However, the neural basis for the expression of contextual fear has not previously 
been elucidated in human imaging studies. 
 
One important determinant of contextual fear is predictability: contextual fear increases 
when a threat (e.g., electric shock) is unpredictable, as opposed to when the threat is 
predictable. The aim of this study is to compare the neural substrates underlying fear 
evoked by predictable versus unpredictable shocks. Animal studies have indicated that 
conditioned responses to predictably cued threat and to less explicit threat are separate 
processes mediated by distinct brain structures. Psychophysiological data suggest that the 
proposed procedure can differentiate between these two responses. Hence, we anticipate 
that this procedure will allow us to compare brain correlates of these responses in 
humans. 
 
Another objective is to study effects of threat of shock on processing and learning of 
threat cues in the amygdala, the visual and auditory systems, and motivation/reward 
systems. This will be investigated by means of event-related magneto-encephalography 
(MEG) and fMRI measurements using various paradigms. 
 
Finally, a last project will examine how pharmacologic manipulation of gamma-
aminobutyric acid (GABA) levels with the benzodiazepine alprazolam affects the 
relationship between GABA concentration (quantified with magnetic resonance 
spectroscopy, MRS), visual- and auditory-induced gamma oscillations (measured with 
MEG), and fMRI BOLD response. This project was completed in 2014.  
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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1. Introduction and Background:  
 
1.1 Fear versus anxiety: different underlying brain mechanisms? 
The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) identifies several 
anxiety disorders, suggesting heterogeneity of symptoms and etiology among them. In 
people suffering from explicit cued fear (e.g., specific phobia), fear is associated with an 
identifiable stimulus that is thought to predict the occurrence of an aversive event. By 
contrast, sustained anxiety occurs when people have learned that aversive events are both 
inevitable and unpredictable, in other words, that harm will come without warning 
(Kandel, 1983; Barlow 2000; Grillon, 2002). The purpose of the fast and intense fear-
response is to mobilize resources to enable appropriate flight/fight action. This response 
is adaptive in the face of an identifiable threat, on which the action can be based. In 
contrast, anxiety is a feeling of apprehension that is more akin to worrying. It is more 
sustained and is not generally associated with a clear event. The physiological symptoms 
of fear and anxiety are rather similar. Hence, the animal experimental literature has often 
taken these responses as similar besides their intensity and duration. For ease of design, 
up to date most human imaging studies have focused on cue-specific fear (e.g., fear 
conditioning, Labar, 1998; Büchel, 1998). The present protocol aims to dissociate brain 
mechanisms involved in fear and anxiety responses. The characteristics of fear on the one 
hand, and anxiety on the other hand, can be modeled by responses that are evoked 
experimentally by either an explicit cue or a context, respectively. Whereas 
experimentally evoked responses to specific cues capture aspects of (pathological) fear, 
responses evoked by experimental contexts are un-signaled and, thus, capture some 
essential characteristics of chronic anxiety. This protocol seeks to understand the neural 
basis of these different responses. 
 
We have reported data consistent with the hypothesis that patients with anxiety disorders 
and high trait anxious subjects are excessively sensitive to contextual fear (Grillon et al. 
1994; 1998a; 1998b; 1999). For example, veterans with posttraumatic stress disorder 
(PTSD) exhibit unconditioned enhanced startle responses in environmental contexts 
where aversive stimuli are expected, compared to experimental contexts where no such 
stimuli are expected. Moreover, differences in the means of evocation of the response 
(specific stimulus only in simple phobia) between anxiety disorders suggest differences 
in the networks involved. Moreover, sensitivity to pharmacological treatment differs 
across disorders (benzodiazepines are not really successful in the treatment of simple 
phobia). Additionally, fear-potentiated startle evoked by a discrete stimulus (such as the 
discrete cues in the proposed fMRI study) has been shown to be insensitive to 
benzodiazepine treatment in 4 different experiments across two different laboratories 
(Baas et al., 2002). 
 
Animal research has provided evidence for differentiation between two defense systems. 
A fast acting, phasic system associated with fear responds to cued threat. A more 
sustained system associated with anxiety responds to contextual threats that are not tied 
to a specific cue (Blanchard et al., 1993; Davis, 1999; Lang et al., 2000). Furthermore, 
these studies have revealed that whereas cued fear is predominantly modulated by the 

 
 
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amygdala (LeDoux, 2000; Davis, 1992), the amygdala, hippocampus (Kim and 
Fanselow, 1992; Philips and LeDoux, 1992), and bed nucleus of the stria terminalis 
(BNST, Walker and Davis, 1997) may be involved in contextual fear, though the 
hippocampus may be primarily involved in processing of contextual cues rather than in 
generating the fear response. These findings suggest that cued fear and contextual fear are 
separable processes that are mediated by distinct brain structures. Numerous human 
imaging studies have demonstrated the activation of the amygdala during cue specific 
fear responses (e.g., Labar et al., 1998; Büchel et al., 1998). In contrast, sustained states 
of anxiety in healthy humans have not been extensively explored. Therefore, the main 
objective of this protocol is to examine the brain mechanisms associated with a sustained 
state of fear or anxiety in humans. 
 
1.2. Rationale: Unpredictability, contextual fear, and sustained anxiety 
Explicit cue fear can be readily evoked using threat or classical conditioning. Contextual 
fear can be observed in experiments that elicit fear as a response to the experimental 
context (e.g., the cage, the experimental room) in which the experiment was conducted. 
Contextual fear is partly determined by the predictability of the aversive event (Seligman 
and Biknik, 1977; Odling-Smee, 1975). Contextual fear increases with increased shock 
unpredictability (Odling-Smee, 1975). Unpredictability of aversive events may cause any 
part of the context in which they may occur to be regarded as unsafe. In contrast, when a 
discrete cue predicts the occurrence of the shocks, the context becomes a safety signal, 
reducing contextual fear. In this case, the specific cue will evoke the fear response and 
the context will become inhibitory (Seligman, 1977; Ameli et al, 2001; Grillon, 2002). 
Studying the neural systems involved in modulation of defense systems during 
predictable and unpredictable shock is important for understanding the interplay between 
fear activation and fear inhibition mechanisms. This is highly relevant to understanding 
clinical anxiety and fear, since generalization of fears to situations that do not justify 
them is characteristic of most anxiety and trauma-related disorders. 
 
The present set of experiments will enable us to study the processes associated with 
phasic cue fear, sustained contextual fear, and inhibitory fear mechanisms. We have 
shown significant behavioral differentiation between contexts that induce high and low 
contextual fear by modifying shock predictability. In this procedure, subjects are 
instructed that in one condition a transient visual stimulus predicts possible shock 
administration (cue fear), while in another condition a similar visual stimulus (e.g., a blue 
square) is still presented, but is no longer predictive of the shock. Recent data from this 
procedure using the startle reflex as a measure of fear showed that giving shocks 
predictably or unpredictably increased contextual fear compared to a control condition 
(no shock administered). In addition, contextual fear was smaller in the predictable 
compared to the unpredictable condition. Furthermore, replicating our previous studies, 
startle was increased during the presence of the discrete cue in the predictable condition 
(replicating Grillon 1991, 1993, etc, see section C preliminary results). These results 
indicate that in this experimental design cued fear and sustained anxiety can be assessed 
separately. In this study we will explore to what extent the brain systems activated by 
predictable and unpredictable threat are different. 
 

 
 
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1.3. Human brain imaging studies 
Brain imaging studies of the fear or anxiety systems in healthy controls have mostly used 
conditioned responding to explicit cues (as our specific aim 1). Human studies have 
yielded ample confirmation of the involvement of the amygdala in emotion in general 
and in fear and anxiety in particular. Patients with lesions of the amygdala and 
surrounding areas show impaired fear conditioning. (LaBar et al, 1995). Fear-
conditioning (Labar et al., 1998; Büchel et al., 1998), emotional face processing (Morris 
et al., 1998), instructed threat (Phelps et al., 1999), and processing of threat-related words 
(Isenberg et al., 1999) all involve the amygdala.  
 
Compared to explicit cue fear, few studies have been conducted on contextual fear or 
anxiety in humans. In one imaging study that explicitly studied contextual fear 
conditioning (Armony and Dolan, 2001), a behavioral and a psychophysiological 
measure failed to confirm a differentiation between conditioned responding (CS+ versus 
CS-) in a safe (CS+ not reinforced) and a conditioned (CS+ reinforced) context. There 
was an increase in skin conductance responding to both the CS+ and CS- in the 
reinforced context suggesting a general increase in arousal. The results showed 
differences in activation of auditory and parietal cortices, but not in areas implicated in 
anxiety processing such as the limbic system or areas intimately connected with limbic 
structures. This pattern of findings is consistent with general arousal or attention, rather 
than with anxiety.  
 
The crucial comparison in the present design will be to compare background activity (i.e., 
activity in absence of the transient visual cues) in predictable, unpredictable, and neutral 
conditions. Our preliminary results suggest that structures involved in contextual fear will 
be more active in the unpredictable compared to the predictable condition, and in the 
predictable condition compared to the neutral condition. Hypotheses that can be derived 
from animal literature are that hippocampus, the amygdala, and related subcortical 
structures (e.g., extended amygdala, such as the BNST) will be involved in contextual 
fear. In addition, orbitofrontal areas of the prefrontal cortex (PFC), as well as parts of the 
anterior cingulate gyrus (medial PFC), are activated in states of anxiety (e.g., Benkelfat et 
al., 1995; Rauch et al., 1997), and have been implicated in the regulation of the limbic 
system (Simpson et al., 2001; Drevets, 2000; Davidson and Irwin, 1999; Hariri et al., 
2000; Garcia et al., 1999). Therefore, areas of the prefrontal cortex may be involved in 
both the generation of anxiety, as well as its inhibition. We hypothesize that areas 
involved in generation of sustained anxiety are activated in the unpredictable condition 
relative to the predictable and neutral conditions. In addition, inhibitory processes may 
mediate the decreased contextual fear when threat is predictable. Areas involved in 
inhibition will be activated during the absence of the specific cue in the predictable 
condition in comparison with the neutral and unpredictable conditions. Several areas of 
the prefrontal cortex are likely to be involved in either of these processes: the orbital, 
medial, and dorsolateral PFC, as well as the anterior insula (see Charney and Drevets, 
2002, for a review). In sustained anxiety we predict specifically that the anterior insula, 
the posterior orbital cortex, and the dorsomedial prefrontal cortex will be activated 
(specific aim 2a).  
 

 
 
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The anterior insula is activated in several kinds of anxiety-induction tasks, such as threat 
of shock as a background to a motor learning task (Chua et al., 1999; PET), and as 
common area of activation symptom provocation in three different anxiety disorders 
(Rauch et al., 1997; PET). The orbital prefrontal cortex has been associated with the 
generation of anxiety, as again in the symptom provocation study (right medial orbital 
cortex; Rauch et al., 1997), and in threat of shock (Chua et al., 1999, left orbitofrontal 
cortex; Drevets et al., 1994, posterior orbital cortex). 
 
In the medial PFC / ACC several studies indicate activation with the generation of 
anxiety. Benkelfat et al. (1995) reported that with CCK4-induced anxiety CBF in the left 
ACC is increased. Chua et al. (1999) reported activation of the left anterior cingulate 
gyrus with threat of shock as a background to a motor learning task, if state anxiety 
scores were used as a covariate (PET). On the other hand, Simpson et al. (2001) reported 
increased blood flow in two areas of the medial inferior prefrontal cortex  (subgenual 
prefrontal cortex and anterior medial prefrontal cortex) while anticipating shock relative 
to a baseline non-anxious condition, only in those subjects that reported the highest levels 
of anxiety. In contrast, subjects with lower levels of anxiety actually showed decreased 
activation in these two areas.  
 
These results suggest that activation in the medial prefrontal area can correlate with 
generated anxiety positively. However, various support for an inverse relationship 
between mPFC activation and anxiety has been reported. Lesions of the prelimbic cortex 
in rats, corresponding to the human dorsomedial PFC, lead to increased freezing to a 
conditioned tone (Morgan & LeDoux, 1995). Also, an inverse relationship between 
amygdala activation and firing rate of medial prefrontal neurons has been reported 
(Garcia et al., 1999).  
 
When human subjects anticipate aversive shocks, the dorsomedial anterior cingulate 
cortex activation is increased with respect to resting or teeth clenching control, but the 
degree of activation within each condition is inversely correlated with anxiety ratings 
(Drevets, 1994) This would suggest that this region is activated to modulate emotional 
responses (Drevets, 1999). Indeed, a recent other study showed a similar inverse 
relationship between activation of a specific region of the right lateral orbital cortex and 
the amygdala (Hariri et al., 2000). In addition, research on depression has suggested that 
in that disorder abnormalities in the ventral anterior cingulate regions (subgenual and 
pregenual anterior cingulate cortex) may result in abnormal modulation of emotional 
processing in limbic structures (for a review, see Drevets, 2000). The present protocol 
allows evaluation of inhibitory influences on the limbic structures in the absence of 
specific cues in the predictable shock condition. We intend to explore the role of (parts 
of) the medial prefrontal cortex in the inhibition of anxiety. The prediction is that parts of 
the medial PFC will be activated in the predictable condition (specific aim 2b). Given 
evidence mentioned above, parts of the dorsomedial / ACC region may (also) be expected 
to be also activated in a condition that generates anxiety. Therefore, to test specific aim 
2b we will evaluate the predictable condition with respect to the neutral baseline 
condition. In both of the predictable and neutral conditions we expect relatively low 
contextual anxiety, which in the predictable condition may be due to active inhibitory 

 
 
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processes. In addition, we will also explore the differential involvement of medial 
prefrontal cortex in the predictable and unpredictable conditions against each other.  
 
1.4. Processing of threat-related stimuli 
MEG (magnetoencephalography) monitors the activity of the brain by detecting the 
magnetic fields generated by neurons, both with a time resolution in the order of 
milliseconds. MEG is a relatively new technique. The advantage over EEG is that tracing 
back the sources of magnetic activity is much more reliable than determining the sources 
of electric activity. Moreover, Dr. Coppola and his staff are working on ways of detecting 
amygdala activity with MEG. We will conduct investigations of visual and auditory 
processing and learning of threat information using MEG and fMRI.  
 
1.5. Relationship between GABA concentration, gamma activity, and hemodynamic 
response 
Recent studies suggest that oscillations in the gamma frequency range (30–100 Hz) are 
well correlated temporally, spatially, and functionally with hemodynamic changes in 
cortex (Niessing et al., 2005). For example, a recent study (Muthukumaraswamy, Singh, 
Swettenham, & Jones, 2010)  reported that 1) endogenous resting concentration of 
GABA was positively correlated with the frequency of stimulus-induced gamma 
oscillations in the occipital cortex and 2) the magnitude of the BOLD response was 
strongly negatively correlated with both GABA concentration (R=-.64) and gamma 
oscillation frequency (R=-.88). These results indicate a close relationship between 
gamma activity and the BOLD response, and further suggest that the level of neural 
inhibition (i.e., GABA concentration) in the brain contributes to the variability of gamma 
oscillations and BOLD signal. They also provide important clues as to the role of 
inhibition/excitation on the BOLD signal and gamma oscillations, as well as possible 
mechanisms of action of classical anxiolytics such as benzodiapezines. One issue with 
the above study (Muthukumaraswamy, Edden, Jones, Swettenham, & Singh, 2009) is the 
correlational nature of the results. We propose to obtain more direct evidence of the 
influence GABA on BOLD activity and gamma oscillations by modulating 
experimentally GABA levels in healthy participants. More specifically, we will increase 
GABA activity with administration of the benzodiazepine alprazolam and we will reduce 
GABA activity with increasing subjects’ levels of anxiety using a threat of shock 
procedure. Using MRS, we recently showed 18% reduction in GABA activity in healthy 
subjects anticipating shocks (Hasler, van der Veen, Grillon, Drevets, & Shen, 2010). 
 
As an initial study, we determined the influence of the benzodiapezine alprazolam on 
gamma oscillations under threat of shock and safe conditions.  This involved using MEG 
only.  This study has been completed and results have been published (Cornwell et al., 
2017). We do not plan to pursue this study in the fMRI scanner or using MRS techniques.   
  
 
1.6. Preliminary Results 
In the proposed experiment we will use a verbal threat and conditioning through 
experience to manipulate contextual fear. The procedure was piloted using 
psychophysiological measures in 15 healthy subjects. The procedure consists of 3 

 
 
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conditions, neutral (N), predictable shocks (P), and unpredictable shocks (P), each lasting 
2 minutes. Subjects were informed of the current condition by means of a computer 
monitor that displayed the following information: “no shock” during the neutral 
condition, and “shock only during cue” during the predictable and “shock at any time” 
during the unpredictable shock conditions. During each condition a visual cue (a colored 
geometric shape) was presented twice, with 8 s duration. As instructed, these cues were 
relevant only during the predictable condition. In this condition, the cue signaled the 
possibility of receiving a shock. In the unpredictable condition, shock could be given at 
any time and in the neutral condition no shock could be administered. Acoustic startle 
stimuli were delivered in the presence and in the absence of the cues. Figure 1 presents 
the results, which show that startle was potentiated both by the explicit threat cue and by 
the threatening context. Indeed, 1) startle was significantly potentiated by the cue only in 
the predictable shock condition (FPS to specific cue) and 2) there was a significant linear 
increase in baseline startle in the absence of cues from the N to the P to the U condition 
(FPS to context). Additional pair-wise comparisons showed significant differences in no-
cue startle magnitudes between the N and P conditions and between the P and U 
conditions. The finding that P elicited contextual fear, compared to N is consistent with 
our previous studies (Grillon et al., 1993). The finding that U resulted in more contextual 
fear than P is consistent with both the safety signal hypothesis (19) and conditioning 
theories (20), which predict that the presence of a signal for shock reduces fear to the 
context.  
 
 
Figure 1.  
Preliminary startle data: Three shock conditions are no shock (neutral), predictable shock, and 
unpredictable shock. The cues predict shock administration in the predictable condition only. Error bars 
denote SEM. 
 
Although this procedure was designed to differentially affect two response systems 
assumed to be associated with cued fear ('fear') and contextual fear ('anxiety'), an 
alternative explanation of these startle results is possible. The startle results may reflect 
quantitative differences in shock expectation in the presence and absence of the threat cue 
during predictable versus unpredictable conditions. Because startle is a unidimensional 

 
 
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measure (magnitude), we cannot make a distinction between these accounts based on 
startle results alone. There are different strategies possible to approach this issue: either 
study these responses in various conditions that are thought to affect the two responses 
differently (e.g., with drugs or patient groups), or study the underlying neural 
mechanisms directly. In the present proposal the latter approach will be taken. Although 
we expect qualitative differentiation in neural structures activated by predictable and 
unpredictable shocks, it is also possible that the same structures will be activated in these 
conditions, but to a different degree. 
 
The threat of shock procedure provides a powerful tool to investigate brain correlates of 
the responses that are characteristic of fear and anxiety, because both responses can be 
measured within one experimental session using the same timing and sensory stimulation 
parameters. The instructed threat of shock procedure has been studied extensively in our 
and other laboratories. It yields highly robust fear-potentiated startle, as well as robust 
effects on autonomic and subjective measures (Grillon et al., 1991, 1993, 1998; Baas et 
al., 2002). The effect of instructed threat is highly reliable in that it is observed in 
virtually every subject tested. For these reasons, this procedure is highly suitable to study 
expression of fear and anxiety in an fMRI scanner. Unlike skin conductance and heart 
rate measurements, startle requires (auditory) probes to be evoked. This circumstance 
precludes concurrent monitoring of startle potentiation in the fMRI or MEG scanner. 
However, results from previous experiments have shown that the instructed threat 
procedure yields reliably results during a second session (Baas et al., 2002). Reliable 
correlations have been demonstrated between amygdala activation and 
psychophysiological measurements in a separate session using skin conductance as the 
dependent variable (LaBar et al., 1998). 
 
The proposed procedure was designed and tested with acoustic startle to tap into the two 
response systems proposed (i.e., fear versus anxiety). However, with startle data it is not 
possible to conclude on a qualitative distinction between brain responses associated with 
explicit cue fear and contextual fear. An alternative hypothesis is that predictable and 
unpredictable shock activate the same neural structures, but to a different degree. 
 
 
 
1.7 Interaction between cognitive performance and emotional pathology (anxiety & 
depression) 
 
High levels of anxiety and depression have been associated with poor performance and 
processing inefficiencies on cognitive (Derakshan & Eysenck, 2010; Hembree, 1988), 
and motivation/reward processes (Scott-Parker et al., 2012; Der-Avakian and Markou, 
2012; Schechner et al., 2013) suggesting that cognitive and motivational changes may 
be a central component of clinical anxiety. Yerkes and Dodson (1908) proposed that the 
detriment in performance due to high emotional arousal (e.g., anxiety) can described by 
a U-shaped function, where performance increases as arousal increases to an optimal 
level and then as arousal levels continue to increase, performance begins to decrease. In 
support of this proposal, high trait anxious individuals (i.e., those with an above-

 
 
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optimal level of emotional arousal) have been shown to experience disruption in 
executive processes (Fales, Becerril, Luking, & Barch, 2010; Eysenck, Derakshan, 
Santos, & Calvo, 2007) and in the ability to perform at work, which can lead to career 
dissatisfaction and job loss (Waghorn, Chant, White, & Whiteford, 2005). However, the 
mechanisms by which performance is affected (e.g., attentional narrowing, executive 
processing deficiencies, perceptual focus), and the degree to which performance is 
reliably hindered or facilitated by anxiety is not clear. The use of fMRI to study anxiety 
patients (target populations and procedures as described in protocol 0093) will allow us 
to discern the neural substrates of such processes and in turn provide us with evidence 
of the types of processes that support the interaction between cognitive performance 
and emotional psychopathology.  
 
Further, in addition to demonstrating that anxiety affects cognition, cognitive processes 
have been shown to effectively decrease fear in anxious individuals (e.g., Cognitive 
Behavioral Therapy [CBT]-like strategies; Kalisch et al., 2005). Identifying the neural 
correlates that support successful mitigation of anxiety (via cognitive loading) will 
provide important information for therapeutic interventions. Selectively targeting these 
cognitive mechanisms will provide an important avenue of intervention in addition to 
the ubiquitous use of intervention techniques that modulate emotional response (e.g., 
benzodiazepines). 
 
Similar to individuals with anxiety disorders, individuals with major depressive 
disorder (MDD) exhibit impaired executive functioning, and have particular trouble 
inhibiting negative information (Lissnyder, Koster, Derakshan, & Raedt, 2010). 
However, unlike anxious individuals, depressed individuals have lower tonic levels of 
emotional arousal, and therefore their task performance may be impacted by 
mechanisms different from those affected by clinical anxiety. Depressed individuals 
tend to exhibit decreased activity in dorsolateral PFC and increased activity in 
subgenual ACC and amygdala in comparison to healthy controls (Mayberg et al., 
1999), whereas anxious individuals tend to exhibit decreased activity in VmPFC and 
increased activity in amygdala and insula (Stein, Simmons, Feinstein, & Paulus, 2007). 
By investigating the differences in cognitive processing under stress between anxious 
and depressed individuals, we will be better equipped to treat their similar behavioral 
deficits via different therapeutic vehicles.  
 
The effects of anxiety on reward processing have also been documented, particularly in 
adolescents (see Schechner et al., 2013). In addition, harm avoidance and intolerance to 
uncertainty have been documented in patients with anxiety disorder as well as fearful 
temperament (e.g., Helfinstein et al., 2012; Krain et al., 2008), raising the question of 
the potentially differential effect of fear vs. anxiety on reward/motivational systems and 
decision-making.  
 
Inarguably, understanding the link between emotion and cognition in patient groups 
with emotional pathology is of particular importance to the development of successful 
therapeutic interventions. In order to target this interaction, we will induce variable 
levels of stress in subjects and monitor their brain reactivity and behavioral 

 
 
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performance on several different tasks. We will use a threat paradigm based on one that 
we used to collect pilot behavioral data from patients outside the scanner. The paradigm 
will consists of periods where subjects are under the threat of receiving a shock, and 
period where they are safe from shock. During these conditions, subjects will engage in 
a series of different cognitive tasks (e.g., 1) working memory verbal n-back: identify a 
letter that was presented one, two, or three trials ago; 2) working memory spatial n-
back: identify a location where a stimulus appeared one, two, or three trials ago; 3) 
long-term memory task where subjects will be asked to remember lists of words and 
pictures), or engage in no task at all (resting state). By parametrically modulating the 
cognitive load (i.e., difficulty) and emotional content (i.e happy or fearful) of the 
stimuli, we will be able to more precisely identify the point at which cognitive 
disruption occurs along with the corresponding neural correlates. In addition, we will 
be able to estimate the optimal point of cognitive facilitation and determine whether or 
not this differs between patient groups. We will also use reward/decision-making tasks 
to probe the interaction between fear/anxiety systems and motivation/reward systems. 
 
This approach to studying stress has been successfully established outside of the 
scanner environment, thus providing an effective tool with which to study the changes 
in brain connectivity and event-related responses associated with stress, and the 
interaction between stress and cognitive performance. We have reliably shown that 
stress (induced by a threat-of-shock paradigm) is reduced when subjects are fully 
engaged in difficult cognitive tasks, and that performance on easier, and potentially 
more susceptible cognitive tasks, decreases as stress increases (Vytal et al., in prep). 
This protocol can uniquely and discretely identify three essential transition points in the 
relationship between stress and cognition: 1) where anxiety overpowers performance, 
and 2) where the level of attentional and perceptual engagement overcomes anxiety and 
3) where anxiety facilitates certain types of cognitive processing (such as threat stimuli; 
Robinson et al, In press; Robinson et al, In submission). By studying stress across 
several different levels of task difficulty and stimuli valence (parametric modulation) 
we will have a direct window into the nuanced interactions between emotion and 
cognition in two different populations with affective disorders. Both types of mood 
disorders are marked by impaired cognitive processing and changes in emotional 
reactivity to affective stimuli(increased in patients with anxiety disorders, and 
decreased in patients with MDD e.g. Robinson et al, In submission).  This approach 
will allow us to study the changes in neural mechanisms associated with changes in 
stress and cognitive performance in such populations, thus providing important 
information about how these mechanisms differ from healthy controls. At the neural 
level, we will be able to better elucidate the differences in these complex cognition and 
emotion interactions between patients with mood pathology and healthy controls. 
Ideally, this study will increase our understanding of when and how patient populations 
with different types of emotional pathology experience emotional interference that 
negatively affects their behavioral performance. Further, we will be able to establish 
how their behavioral and neural patterns differ from individuals with a healthy response 
to stress.   
 
1.8 Rationale: Hippocampal dysfunction in posttraumatic stress disorder (PTSD) 

 
 
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Hippocampal dysfunction is implicated in the symptom expression of PTSD. We will be 
conducting a series of experiments to better understand this vulnerability: (a.) behavioral 
pattern separation (memory) task (BPS), (b.) whole brain resting state task, and (c.) the 
Morris water maze (spatial memory) task (MWM). 
 
(a.) It has long been established that individuals with PTSD tend to overgeneralize 
attributes of fearful stimuli to non-fearful stimuli.(Lissek et al., 2010) While the 
hippocampus has been implicated in this vulnerability, there is little mechanistic 
understanding of the contributing neural systems that support overgeneralization. To 
address this knowledge gap, this study aims to examine the effect of experimentally-
induced contextual anxiety on stimulus generalization using the behavioral pattern 
separation (BPS) task (Stark, Yassa, Lacy, & Stark, 2013). Ideally this study will increase 
our understanding of the contexts (safe vs. threat of unpredictable shock) for which 
stimulus generalization may occur, as well as the neural systems that may relate to 
stimulus generalization in participants with PTSD (vs. no-PTSD). 
 
(b.) Human fMRI studies have long investigated the hippocampus without differentiating 
between its subfields, even though theoretical models and rodent studies suggest that 
hippocampal subfields support different and potentially even opposite 
functions.(Leutgeb, Leutgeb, Moser, & Moser, 2007) Of focus, the dentate gyrus (DG)-
cornu ammonis 3 (CA3) subfield circuit of the hippocampus minimizes interference 
between new information and previously stored similar memories.(Besnard & Sahay, 
2016) This process is dependent on two complimentary operations, pattern separation 
(implicating the DG) and pattern completion (implicating the CA3). This study will use 
resting state scans during ultra-high field fMRI (7-Tesla) to delineate the different resting 
state networks of the hippocampal subfields. We aim to define different processes and 
functions that are presumably carried out by the DG and CA3 subfields, in particular 
regarding the seemingly opposing functions of pattern separation and pattern completion, 
as well as specific processes that are related to behavioral, functional, and structural 
alterations in participants with PTSD (vs. no-PTSD). 
 
(c.) The Morris water maze (MWM) task (Deuker, Doeller, Fell, & Axmacher, 2014) 
is one of the most widely used tasks to test hippocampal-dependent learning, including 
acquisition of spatial memory and long-term spatial memory.(Bromley-Brits, Deng, & 
Song, 2011) The task is relatively simple, which relies on distal cues to navigate from 
start locations around the perimeter of a virtual reality open swimming arena to locate a 
submerged escape platform. Spatial learning is assessed across repeated trials. The aim of 
this study is to further investigate the impact of hippocampal-dependent deficits in PTSD 
(vs. no-PTSD). 
 
1.9 Rationale: Avoidance symptoms in posttraumatic stress disorder (PTSD) 
 
Active avoidance is the commission of an overt action which functions to directly reduce 
exposure to threat. Although avoidance symptoms are relevant to understanding 
heterogeneity within PTSD diagnosis (Asmundson, Stapleton, & Taylor, 2004), no 

 
 
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research has empirically investigated whether persons with PTSD exhibit altered 
behavioral or neural responses during active avoidance paradigms. Previous research has 
demonstrated that the active avoidance of signaled threat (AAST) paradigm can be used 
to measure facilitated behavioral performance in response to threat contingencies, and 
that individual differences in how participants respond during the AAST are linked to 
stable personality traits (Gorka, LaBar, & Hariri, 2016). To address this knowledge gap, 
this study aims to investigate whether persons suffering from PTSD exhibit abnormal 
behavioral responses during performance of the AAST, and to determine how neural 
circuits underlie between-subject differences in active avoidance behavior. 
 
2. Study Objectives: 
 
The overall objective of this protocol is to investigate the brain correlates of responses to 
predictable and unpredictable shocks. Several contrasts derived from the design will 
allow this investigation. 
 
A) Explicit cue fear will be evoked by the presentation of a specific cue that is 
associated with an electric shock. In order to study explicit cue fear we will examine the 
BOLD response to the presentation of cues that predict a shock (fMRI study). We predict 
that the amygdala will be activated in this comparison, along with several cortical areas 
that are involved in explicit cue fear, in particular the anterior insula (Phelps et al., 2001) 
and the anterior cingulate (Büchel et al., 1998). In addition, sensory processing of 
auditory and visual stimuli that predict threat will be facilitated (MEG/fMRI study). This 
will be reflected in the augmentation of the activation evoked in sensory areas and the 
amygdala to those stimuli relative to non-threatening stimuli. 
 
 
B) In contrast, sustained contextual fear or anxiety will be elicited by unpredictable 
aversive events. To measure this background state, we will compare the BOLD responses 
during conditions in which subjects anticipate shocks being delivered predictably and 
unpredictably to conditions where no shocks are anticipated. In all hypotheses concerning 
the background state of anxiety, BOLD responding during the absence of the explicit 
cues will be utilized.  
1) In order to evaluate activations associated with the sustained anxiety state in 
the unpredictable condition, the contrasts of unpredictable shock versus neutral and 
versus predictable shock will be used. Regions that are hypothesized to be activated are 
the amygdala, and the anterior insula, as well as posterior orbital cortex and dorsomedial 
prefrontal cortical areas. 
2) A second hypothesis with regard to the sustained state of anxiety is that in the 
condition where shock is predictable, this sustained state is inhibited in comparison to the 
condition in which the shock is unpredictable. To evaluate this hypothesis, the contrasts 
predictable shock versus neutral and versus unpredictable shock will be used. We predict 
that in the predictable shock condition, areas of the lateral orbital cortex, and ventral 
anterior cingulate cortex are activated in comparison with the unpredictable shock 
condition, as well as the neutral condition. 
 

 
 
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C) A third hypothesis is that while anticipation of unpredictable shocks will reduce 
GABA concentration, leading to low peak frequency of stimulus-induced gamma 
oscillations in the occipital cortex and increases BOLD response, the benzodiazepine 
alprazolam will have the opposite effect (increased GABA, leading to high peak gamma 
frequency and decreased BOLD signal). 
   
D) A fourth hypothesis is that under threat of shock, prefrontal executive control regions 
will be less successfully engaged by patients with high anxiety or MDD than healthy 
controls. Further, we predict that both patient groups will recruit the cortical and 
subcortical limbic regions (e.g., amygdala, bed nucleus of stria terminalis (BNST), and 
insula) to a greater extent than healthy controls when under threat of shock. In contrast to 
anxiety patients, MDD patients will additionally recruit subgenual/rostral ACC in 
response to threat. 
 
E) In the PTSD hippocampus investigation, we hypothesize that the hippocampus and 
dorsolateral prefrontal cortex (dlPFC) will play a key role in the effect of anxiety on BPS 
Task performance and that participants with PTSD will exhibit amplified pattern 
separation deficits. We also hypothesize that pattern separation deficits will be associated 
with PTSD symptom severity in the dentate gyrus as indicated in the high-field 7-Tesla 
resting state scan. Lastly, we hypothesize that the PTSD group (vs. no-PTSD) will have 
increased hippocampal-dependent spatial memory deficits on the Morris water maze 
(MWM) task. 
 
F). We hypothesize that during the AAST paradigm, participants will exhibit enhanced 
neural responses to the avoidance cue during the threat-of-shock condition, within areas 
of the medial prefrontal cortex, amygdala, and striatum. Furthermore, we hypothesize 
that participants with PTSD will exhibit reduced medial prefrontal cortex responses 
during threat contexts, and elevated avoidance behavior. Lastly, we hypothesize that 
individual differences in avoidance behavior will be associated with the magnitude of 
task elicited neural activity within the medial prefrontal cortex, amygdala, and striatum. 
 
3. Subjects: 
 
a. Description of study populations 
 
This study will recruit 1226 healthy volunteers and 726 psychiatric patients with a current 
diagnosis of generalized anxiety disorder (GAD), panic disorder, social anxiety disorder 
(SAD), specific phobia, posttraumatic stress disorder (PTSD), or major depression. All 
participants enrolled in this study must be in good physical health and between 18-50 
years old for the duration of the study. This study is open to all persons regardless of sex, 
race, ethnicity, sexual orientation, or rank. 
 
 
b. Inclusion criteria 
All screening procedures described in this section are conducted under screening 
protocol 01-M-0254.  Subjects must meet the following inclusion criteria in order 
to participate in the study: 

 
 
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1) Male or female volunteers ages 18-50 years old.  
 
2) Judged to be in good physical health on the basis of medical history, a clinical 
MRI scan, and physical examination.  Physical exams will be conducted by a 
NIMH credentialed physician or nurse.  Clinical laboratory tests will be ordered 
based on his/her discretion.   
 
3) Healthy subjects judged to be in good psychiatric health on the basis of the 
Structured Clinical Interview for DSM-IV-TR.  The SCID will be administered by 
a credentialed NIMH clinician. The PTSD comparator group (i.e., war-zone 
exposed healthy controls), must endorse war-zone exposure on the SCID, PTSD 
module. 
 
4) Able to understand procedures and agree to participate in the study by giving               
written informed consent.  
 
5) This protocol (02-M-0321) will include patients with a primary diagnosis of 
generalized anxiety disorder, panic disorder, SAD, PTSD (from war-zone 
exposure), specific phobia, and major depression according to DSM-IV.   
 
6) Abstain from drinking caffeinated beverage including coffee, tea and 
caffeinated soft drinks and from smoking for at least 1 hour prior to testing. They 
will also be instructed not to drink alcohol on the night prior to testing and on the 
day of testing. 
 
 
8) Speaks English fluently  
 
 
 
c. Exclusion criteria 
Subjects will be excluded from the study if they meet the following exclusion 
criteria: 
 
1) Clinically significant organic disease, e.g., cardiovascular disease. 
 
2) Clinically significant abnormalities in physical examination. 
 
3) Any medical condition that increases risk for fMRI (e.g. pacemaker, metallic 
 
foreign body in eye). 
 
4) History of any disease, which in the investigators’ opinion may confound the 
results of the study, including, but not limited to, history of organic mental 
disorders, seizure, or mental retardation. 
 

 
 
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5) Have a current diagnosis of alcohol or substance abuse ACCORDING TO 
DSM IV CRITERIA 
 
6) Have a lifetime diagnosis of alcohol or substance dependence ACCORDING 
TO DSM IV CRITERIA. 
 
7) Unless subject is enrolled as a patient, subjects should not have current Axis I 
psychiatric disorders as identified with the Structured Clinical Interview for 
DSM-IV, non-patient edition (SCID/NP).  
 
8) If a healthy volunteer, past bipolar depression and any history of psychosis or 
delusional disorders. 
 
9) If a healthy volunteer, first degree relative with history of psychotic disorder 
such as schizophrenia or bipolar disorder.  
 
10) Healthy participants may not be on psychotropic medications. 
 
11) Pregnancy, i.e., a positive -HCG urine test conducted prior to each 
experiment session. 
 
12) Current or past history of cubital tunnel syndrome or carpal tunnel syndrome 
for shock studies that use the wrist for placement of electrodes. Cubital tunnel and 
carpal tunnel syndrome are exclusionary only for diagnosis on same arm as 
electrodes and are not exclusionary for studies that place shocks on ankles or feet.  
 
13) Reynauds syndrome for the cold pressor test experiment 
 
14) Color blindness (for the active avoidance task only)  
 
 
Additional exclusion criteria for patients 
 
• Patients who would be unable to comply with study procedures or 
assessments 
• Patients will be excluded if they have a current or past history of any 
psychotic disorder, bipolar disorder, delirium, dementia, amnestic 
disorder, cognitive disorder not otherwise specified, any of the pervasive 
developmental disorders, or mental retardation 
 
In terms of the rationale for the inclusion of healthy participants with past (but not 
current) mood or anxiety, there are no data, to our knowledge, suggesting that 
patients with past but not current mood or anxiety disorders are dissociable from 
healthy controls in terms of phasic reactivity to anxiogenic stimuli and thus past 
mood or past anxiety disorders are not criteria for ineligibility.  
 

 
 
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d. An eligibility checklist for this protocol is provided as an attachment. 
 
4. Study Design and Methods: 
i. 
Study overview 
This protocol consists of three scanning sessions. The first two are fMRI tasks 
with durations of 2.5 hours each (healthy participants and patients) and the third 
is an MEG task lasting 2.0 hours (healthy participants ONLY). Proceeding any 
of these testing sessions, participants will come to the NIH for a screening 
session (see Screening Methods below under Section 7.a.iii.). Qualifying 
participants will complete one of these three tasks (depending on subject 
preference and recruitment needs of each task) and will then be offered to 
participate in a second and perhaps 3rd task with the limitation that they will 
have at least a two week break between studies including electric shocks.  
 
A collaborative agreement exists with Dr. Carlos Zarate’s group to recruit, 
screen and test all the participants with Major Depressive Disorder under this 
protocol.   
 
ii. Recruitment 
English speaking healthy volunteers ages 18-50 will be recruited via 
advertisements in the local media.  Individuals with an anxiety disorder or major 
depression will be recruited with assistance from PRPL.  
 
 
Recruitment strategies will include advertisements placed through university 
newspapers, the city paper, and local gazettes, Web Links, DC and Montgomery 
buses and metrorails, and public service announcements. We also will utilize 
websites, such as college papers and local media.  The web ads will direct readers 
to the NIH Patient Info website.  
 
We also will advertise on approved listservs such as those provided by OPR, 
advocacy groups, campus groups, and Club PCR used by the research assistants at 
NIH.  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. Additional study 
information will be distributed to local chapters of the Anxiety Disorders 
Association of America, Freedom from Fear, and the National Alliance for the 
Mentally Ill.  
 
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 

 
 
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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.   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.  
 
Healthy volunteers may also be identified through the NIMH protocol #17-M-
0181 titled “Recruitment and Characterization of Research Volunteers for NIMH 
Intramural Studies. Healthy volunteers screened through protocol #17-M-0181 
may be given information and recruitment materials for this protocol. We may 
receive a list of potential participants that were identified as healthy volunteers 
under protocol #17-M-0181 and have agreed to be contacted by other NIMH 
groups.  
 
All such advertisements have been submitted to the IRB by Susanna Sung and 
recently received IRB approval. Any new advertisements or changes to existing 
advertisements will be submitted to the IRB for approval prior to publication. The 
written advertisements will be used in color as submitted, or may be printed in 
black and white.  The color of the ads may vary. Color changes will not be used to 
change the emphasis of an ad. The size of the ads may vary, but all parts of the 
ads, including fonts and pictures, will be changed proportionately to the rest of 
that ad.  Disproportionate changes in size will not be used to change the emphasis 
of an ad. 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.  
 
Recruitment efforts will be made to match the number of healthy controls to the 
number of patients in each experiment, except in studies that enroll only healthy 
controls (e.g., MEG) or in studies that include multiple healthy control groups 
(e.g., Active Avoidance task). Participant groups will include both men and 
women and recruited in a 1:1 ratio to the best of our recruitment capacity. 
Additionally, we will make all efforts to recruit healthy controls who are age, sex, 
race, and IQ matched (as assessed by the Wechsler Abbreviated Scale of 
Intelligence™ (WASI™) to our patient groups. 
 
All potential participants will undergo a telephone prescreen questionnaire either 
under this protocol (see attached pre-screen questionnaires for healthy participants 
and patient participants) or under the screening protocol 01-M-0254 if recruited 
by Dr. Zarate’s lab. Potential patient participants will only be pre-screened by 
clinician level investigators (RNs, MSWs, PAs, MDs, MSs) whereas potential 
healthy volunteer participants may be pre-screened by any investigator or research 
contact, including IRTAS.  During the pre-screening, the experiment will be 

 
 
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described. In particular, subjects will be informed that unpleasant stimuli (shocks) 
will be administered. They will also be told that the procedure involves fMRI, 
MEG, or MRS scanning.  All questions asked during the pre-screen are based on 
inclusion and exclusion criteria (e.g., "What is your age?" or “Do you wear 
braces, have a permanent retainer, or have any metal implants?”). After the initial 
telephone prescreening, individuals who appear to qualify for inclusion will be 
invited to complete a more comprehensive outpatient screening at the NIH 
Clinical Center through screening protocol (01-M-0254) 
 
1. 
A PDF of the employed IRB-approved advertisement is attached. 
 
2. 
Telephone pre-screen questions for this protocol are attached. 
 
iii. Screening methods  
The outpatient screening for participants in this protocol will take place via the 
mood and anxiety disorders screening protocol, 01-M-0254, and subjects will be 
asked to provide informed consent for 01-M-0254 on the day of screening. 
Screening procedures may consist of the following criteria: 
 
• 
Pregnancy test (urine beta HCG test) for women of child bearing age  
• 
Blood draw for genetic analysis (3 EDTA [purple top] tubes) 
• 
Subject demographic information 
• 
Vital signs (sitting blood pressure and pulse), height, weight 
• 
Medical history and physical examination 
• 
Structured Clinical Interview for DSM-IV  (patients only) 
• 
Structured Clinical Interview for DSM-IV-TR  non-patients edition (non-patients 
only) 
• 
Risk for self-harm screen 
• 
Life Events Checklist 5 – Extended Version (LEC-5)  
• 
The Clinician Administered PTSD Scale (CAPS) 
• 
Self administered Questionnaires:  These questionnaires including BDI-II, IDSR, 
HAM-D, HAM-A, MADRS PSWQ and POMS..  
 
• 
Concomitant medication and pharmacotherapy history 
• 
Relevant inclusion/exclusion criteria (see above) 
 
Some participants may have participated in the NIMH healthy volunteer protocol 
#17-M-0181.  For those participants, identifiable data may be shared between 
protocols 17-M-0181, 01-M-0254 and this protocol 02-M-0321.  The information 
from 17-M-0181 may be used for screening for this protocol as long as: it has 
been within a year for the demographic information, medical history and physical 
examination, SCID, WASI, and questionnaires. The urine pregnancy and drug 
screen will need to be within two weeks. The urine pregnancy is also repeated 
prior to any study procedures on the study visit day under this protocol. 
 

 
 
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Participants meeting medical and psychiatric eligibility criteria will be invited 
back for additional testing visits at which time they will undergo informed 
consent for this protocol (02-M-0321). 
 
 
 
iv. Study design 
Threat of Shock 
The design is similar to that employed psychophysiologically in protocols 03-M-
0093 and 01-M-0185, with some changes to adapt it for fMRI measurement. 
Figure 2 clarifies the experimental design. 
 
 
 
Design Overview 
 
 
Figure 2. 

 
 
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Experimental design: upper portion gives the design of the entire experiment. Lower part depicts 
an example of each run type (order of conditions balanced within and between subjects). Shock 
blocks can be concurrent with tasks described below. When subjects are participating in a no-
shock run, the tasks will replace the shock blocks as indicated above. Subjects do not engage in 
any specific task during resting state runs.   
 
Each condition will have an identical duration of two minutes, and be identical 
with respect to number of stimulus cues presented.  Virtual reality environments 
(e.g., restaurant scene, airport scene, pool scene) may be used as cues to allow for 
a richer and clearer differentiation of predictable, unpredictable and neutral 
conditions.  The predictable condition will be defined as the context in which 
shocks can only occur during an explicit stimulus cue. Subjects will be informed 
that they will not receive a shock during any other time during this condition. The 
stimulus cue will appear for approximately 1-8 seconds. The unpredictable 
condition will be defined as the context in which shocks can be delivered without 
warning at any time.  Furthermore, shocks will be administered to reinforce these 
instructions with the same number during predictable and unpredictable 
conditions.  Occasional reinforcement is necessary to prevent the instructions 
from losing credibility.   Finally, the neutral condition will be defined as the 
context in which participants are completely safe from receiving shocks.   
 
Study Procedures and Groups 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Motiva
tion 

 
 
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Figure 3. Healthy Controls: Full procedure diagram outlining all experimental 
procedures. Note, not all subjects will participate in all procedures. In fact, most 
subjects will participate in only one or two tasks during a scanning session. 
Healthy subjects will undergo fMRI and/or MEG (depending on whether or not 
they consent to both) and they will be scanned during runs of either shock or no 
shock. Each of the bottom level categories represents one type of experiment such 
that a single run will fit into a single experiment category. Six different 
experiments are described below (Motivation/reward, Motivation/Monetary 
Incentive Delay, Perception, Memory, Resting State,  Passive Presentation, and 
ValSal); all experiments follow the general threat procedure described above. 
 
 
 
Figure 4. Patients: Full procedure diagram outlining all experimental procedures. 
Note, not all subjects will participate in all procedures. In fact, most subjects will 
participate in only one or two tasks during a scanning session. Patients will 
undergo fMRI ONLY and they will be scanned during runs of either shock or no 

 
 
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shock. Each of the bottom level categories represents one type of experiment such 
that a single run will fit into a single experiment category. Five different 
experiments are described below (Motivation/reward, Perception, Memory, 
Resting State, Passive Viewing, and ValSal); all experiments follow the general 
threat procedure described above. 
 
 
Description of studies (subordinate categories: Perception, Memory, Resting 
State, Passive Presentation, Loss Aversion Task, ValSal Task ) 
 
Perception (Healthy N=40; Patient N=20 from each patient group) 
Subjects will be asked to identify the expression on faces (e.g. neutral, happy, 
fearful, sad) or identify the emotion of a word (e.g. ‘safe’,‘fear’ or ‘+$’).  They 
will be asked to respond during these stimuli themselves and during neutral 
pictures (i.e. boxes, stars, circles, scenes) which are paired with (i.e. appear just 
before) these stimuli. 
 
Time perception (Healthy N=50) 
Participants will be asked to judge for how long stimuli (emotional faces: fearful, 
happy, neutral, taken from a standardized battery, NimStim OR simple shapes) 
remained on the screen. They then have to make a button press denoting the 
relative duration of these stimuli, compared to durations they learned. 
 
Memory (Healthy N=80; Patient N=90 from each patient group, 20 of which will 
overlap with the patients in the Perception experiments because they will be run 
in the same session) These memory tasks will also include the below noted 
groups. 
 
Morris Water Maze task (Healthy controls without war-zone exposure N=30, 
Healthy controls with war-zone exposure N=30, PTSD from war-zone exposure 
N=30)  In one procedure, participants navigate a virtual reality water maze with a 
joystick to learn the location of an escape platform.  Participants perform a series 
of trials.  On some trials, the platform is visible to allow them to encode its 
location, and on other trials, they must retrieve its location by using spatial 
information in the virtual environment. 
 
Behavioral Pattern Separation Task (Healthy controls without war-zone exposure 
N=30, Healthy controls with war-zone exposure N=30, PTSD from war-zone 
exposure N=30) The BPS Task consists of two phases; in the first encoding 
phase, participants assign an “indoor/outdoor” verdict to pictures of neutral 
objects. Following this encoding phase, participants complete a memory test (i.e., 
the retrieval phase), where they must identify pictures of neutral objects as 
‘‘Old’’, ‘‘Similar’’, or ‘‘New’’. One-third of the objects in the retrieval phase are 
“Old” or exact repetitions of the objects presented in the encoding phase; one-
third of the objects are “New”, not presented during the encoding phase; and one-
third of the objects are perceptually “Similar” to the objects presented during the 
encoding phase, but not identical (i.e., lures). We are particularly interested in the 

 
 
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lure object responses and the rates at which participants correctly identify these as 
‘‘Similar’’, avoiding the inclination to identify these as ‘‘Old’’. Identifying the 
lure objects as ‘‘Old’’ (i.e., overgeneralization) is likely driven by pattern 
completion processes. In contrast, discriminating the lure objects from the “Old” 
objects requires a distinct representation—a hallmark of pattern separation. The 
encoding and retrieval phases will be presented under safe and threat contexts 
(i.e., threat of unpredictable shock) to investigate the role of experimental threat 
on pattern separation and pattern completion processes. 
 
In a separate procedure, 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). Subjects will also 
be asked to simply view the stimuli (without responding) as well as remember 
words or International Affective Picture System (IAPS) pictures that they were 
presented with previously after a short delay. IAPS pictures include both positive 
and negative images. Examples of pleasant pictures include baby faces, ice cream, 
puppies, fireworks, and erotic pictures. Examples of unpleasant pictures include 
car accidents, guns, surgery, snakes, tumors, and bleeding faces. Subjects will be 
told that these pictures distressing and they will be reminded of the option to 
withdraw from the experiment at any time. 
 
Resting State (Healthy N=40; Patient N=60 from each patient group, all of which 
will overlap with some of the patients in the Memory and Perception experiments 
because they will be run in the same session) This task will also include (Healthy 
controls without war-zone exposure N=30, Healthy controls with war-zone 
exposure N=30, PTSD from war-zone exposure N=30) 
Subjects will be asked to lie still with their eyes open and stare at a fixation cross 
for 6-9 minutes. They will be asked to remain as still as possible and not fall 
asleep. During a resting state run, subjects will not engage in any task. However, 
on some rest runs, they will be under the threat of a shock they have already 
experienced. On other runs, they will be told they will not receive a shock. 
 
Passive Presentation (Healthy N=60) 
These procedures involve simple, passive experience (visual, auditory, tactile) to 
measure sensory cortical responses under threat and safe conditions.  Participants 
are asked to remain attentive to whether they are at risk to receive shock or safe 
from shocks but do not need to respond to the stimuli. 
 
Loss Aversion “Motivation” Task (Loss aversion: Healthy N=30, Patients N=30; 
development of versions of the task: Healthy N=60) 
This paradigm provides a quantitative measure (lambda) of sensitivity to loss, and 
is designed after the work by Tom et al., 2007. This task requires subjects to 
decide between taking or dismissing a series of monetary gambles.  
In the clinic, versions of this task will also be developed. These modifications of 
the task have several goals. (1) Enhanced salience of stimuli: The main task is 

 
 
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fairly dry, presenting just numbers. We would like to pilot a similar task with 
more salient stimuli, such as dollar bills, or thermometer-type indicators of 
amount. (2) Manipulate probability: this task is currently only probing magnitude 
of 50% chance of winning or losing hypothetical money. We expect that 
probability (level of uncertainty in choice) plays an important part in decision-
making, next to magnitude of incentives. Therefore, we would like to modify the 
task to manipulate probability. For example, instead of a fair coin-toss scenario, 
we would present a biased coin-toss, which could favor either losses or gains. (3) 
Manipulate the self-agency: anxiety is known to enhance salience of events that 
are determined by one’s own actions. We are interested in understanding to what 
extent anxiety affects self-agency. To examine this aspect, we would pilot a task 
in which some incentives will not depend of the participants’ choice, but will be 
set by the experimenter. These various versions of the task will be piloted in the 
clinic. The most successful manipulations would then be brought to the fMRI 
setting.  
The need to use real money is based on feedback from participants which 
indicated lack of motivation in the absence of real money for this task, which can 
be boring. The use of real money will make the gains and losses more salient.  
The weaker than expected effect of loss aversion with use of token money is the 
reason to request the use of real money.  While the weak effect of hypothetical 
money is clearly seen in our pilot data, the literature supports this notion.  Most 
neuroimaging studies clearly show increased activation of reward pathways with 
increased value of monetary rewards (see review Richards et al., 2013).  
 
The determination of the amount of money disbursed will be as follows. First, to 
provide a more real-life situation and enhance motivation to gamble and 
engagement in the task, we will provide an initial endowment of $20.00 that can 
be lost during the task. This endowment will make the potential losses real. At the 
end of the task, we will randomly select three gambles from the total number of 
gambles (n=512) presented to the participants. To do so, we will ask participants 
to randomly pick 3 numbers between “001” and “512”. We will then identify the 
gambles corresponding to these 3 gambles and add the gains and losses from 
these 3 gambles. Participants can earn up to $50.00 or lose up to $20.00 (entire 
endowment). We have already used this strategy in previous protocols (e.g., 01-
M-0192).  
 
Motivation/ "Monetary Incentive Delay" Task (Healthy N=150; Patients N=30):          
 
Participants will perform one of two versions of the “Monetary Incentive Delay” 
task. During the first version of the task (N = 80 HVs on the 3T scanner and N= 
30 HVs on 7T scanner), we will examine the effect of threat of shock on goal-
driven/ motivational stimuli. We will use a paradigm during which subjects make 
speeded responses to a central target under conditions of potential monetary gain, 
loss or neutrality during threat of shock and during safety. During each trial of the 
task, participants will first see one of three shapes, either a circle, a square, or a 
triangle, then wait for a variable time interval, and finally respond to a white box 

 
 
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with a button press. Trials beginning with a circle indicate that the subject will 
win money if s/he presses the button fast enough once the white box appears; 
trials beginning with a square indicate that the subject will lose money if s/he does 
not press the button fast enough once the white box appears; trials beginning with 
the outline of a triangle indicate that the subject will neither lose nor win money, 
independent of task performance. Participants will perform this version of the 
Monetary Incentive Delay task within the MRI scanner. 
 
During the second version of the task (N = 40 HVs), we will examine the impact 
of monetary incentives and aversive stimuli on goal-driven / motivational 
processing. We will use a paradigm during which subjects make speeded 
responses to a central target during four types of trials. Accurate responses to the 
central target will determine whether participants: 1) win money (gain trials), 2) 
avoid losing money (loss trials), 3) avoid the presentation of an aversive electrical 
stimulus (aversive trials). During 4) even trials, participants will never receive 
monetary or aversive stimuli, regardless of performance. During each trial of the 
task, participants will first see a cue denoting the type of trial, then wait for a 
variable time interval, and finally respond to a white box with a button press. 
Participants will perform this version of the Monetary Incentive Delay task within 
the MEG scanner. 
 
During both versions of the modified Monetary Incentive Delay task, the shape at 
the beginning of the trial serves as a signal characterizing the trial type. 
Additionally, participants will receive feedback after each trial indicating whether 
they succeeded in responding to the target fast enough.  The duration of the white 
box is adjusted such that each participant succeeds on approximately 66% of his 
or her responses. Participants will be informed that “Your performance during 
each trial will determine whether you win money or lose money. However, the 
task may become more difficult or less difficult over time. Please try your best at 
all times.” 
 
Participants will begin with an initial endowment of $20.00 and will perform a 
task which includes both gain trials and loss trials. Successfully responding to a 
target during a gain trial will result in more money being added to the 
participant’s total. Unsuccessfully responding to a target during a loss trial will 
result in money being subtracted from the participant’s total. As such, the 
participants total winnings can increase or decrease depending on performance 
during gain trials and loss trials, respectively. $0 serves as a floor (i.e. participants 
can lose their total endowment but cannot win negative amounts of money). All 
participants will receive $10 in compensation regardless of their performance 
during the task. Although participants can lose their entire $20 endowment, 
performance during the Motivation/ "Monetary Incentive Delay" Tasks has no 
impact on compensation from participation in other parts of the study. We have 
used this strategy in this and previous protocols ("Loss Aversion" task and 01-M-
0192). The need to use real money is based on feedback from participants in the 

 
 
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"Loss Aversion" task, which indicated lack of motivation in the absence of real 
money. The use of real money makes the gains and losses more salient. 
 
ValSal Task (Healthy controls N = 46, Anxiety participants, N=46) 
The goal of this experiment is to extend the question of the mechanisms 
underlying symptoms of anxiety disorder, particularly those implicated in 
motivated behavior including aversion to uncertainty and avoidance behavior.   
The ValSal task was designed to probe two related, but distinct, processes that are 
integral to motivated behavior, i.e., valence and salience. Valence refers to the 
positive vs. negative values tagged to stimuli/situations.  For example, a gain of 
$5.00 is a positive value, and a loss of $5.00 is a negative value.  Salience refers 
to the subjective importance of stimuli/situations. For example, a gain of $5.00 
may have the same subjective importance as a loss of $5.00, both being more 
important than no loss/no gain. The ValSal task uses monetary values to assay 
these processes at the neural (fMRI) level.  
The task lasts 25 min long and comprises two runs. Each run consists of 4 blocks. 
Two blocks feature positive valence trials, and two blocks feature negative 
valence trials. The order of the blocks is counterbalanced across runs, i.e., 
positive-negative-positive-negative, or negative-positive-negative-positive.  
Each block presents 16 trials. There are 6 types of trials. The positive blocks 
present 3 types of trials: 100% probability of $5.00 gain, 50% probability of $5.00 
gain, and 0% probability of $5.00 gain.  The negative blocks present 3 parallel 
types of trials: 100% probability of $5.00 loss, 50% probability of $5.00 loss, and 
0% probability of $5.00 loss. Types of trials are randomized across each block. 
For each block, there are 4x100%trials, 4x0% trials and 8x50% trials, for a total 
of 128 trials for the whole task. 
Each trial is 8 s long and consists of 3 screens: (1) a 2 s cue signaling the type of 
trial (e.g., 50% change of gain), (2) a key-press delay screen, and (3) a 2 s 
feedback screen. An intertrial interval (ITI) of 2 s separates the trials. An 
illustration of the composition of the trials is presented in Figure below.  
 
This task can be modified in terms of the nature of the positive vs. negative 
stimuli. This could be important to distinguish among different types of anxiety 
(e.g., social anxiety vs. generalized anxiety). 
 

 
 
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Squeezer Task/ Follow-up to ValSal Task (Healthy controls = 40, Anxiety 
participants = 40) 
The valence/salience study targets the circuits associated with the coding of 
valence and salience, including medial temporal structures (amygdala and 
extended amygdala and dopaminergic pathways (e.g., ventral tegmental/substantia 
nigra).  However, preliminary analyses of the salience/valence study revealed the 
limits of the design, which, for the sake of simplicity, does not permit to assess 
instrumental responses to valence and salience stimuli. The salience/valence 
design provides essential knowledge on the circuits evaluating valence and 
salience, but not the responses to such stimuli that motivate behavior. This 
complementary study fills this gap by querying motivation operationalized as the 
amount of motoric effort extended to approach or avoid incentive stimuli (see 
example in Pessiglione et al. 2008). We will use the grip force measure (described 
on page 37 of this protocol) to compare motivation coding between adults with 
anxiety and healthy adults to complete our initial valence/salience study. 
 
It has been shown that effort is modulated by incentive magnitude (e. g. effort 
towards $10 > effort towards $1) and that this modulation most likely originates 
from structures of the basal ganglia. Reports on how anxiety modulates effort, 
particularly in function of incentives, are few, and inconsistent. To address this 
gap in knowledge, we propose a new task that uses grip force as a proxy to 
motivation, based on (Pessiglione et al. 2008). The task timeline is depicted in 
Fig.1: while in the MRI scanner, the participant will hold a hand dynamometer 
(“squeezer”). The task lasts 20-30 minutes. After device calibration, baseline 
recordings and instructions, the participant will work on 5 runs of two blocks of 6 
trials with a 5s break in between, making up a total of 72 trials. Next, they will 
complete 19 questions detailing their experience. This debriefing questionnaire is 
uploaded into iRiS. The task ends with a message telling the participant that they 
won a total amount (will be made fixed for all participants, e.g., $25). 
 
There will be two types of trials in which the participant will squeeze to win 
(positive valence trials) or to avoid losing (negative valence) a monetary amount. 
See Fig. 2 for an example. The valences and amounts are pseudo-randomized a 
priori, so all participants go over the same experiment timeline. The trial timeline 
is: first, participants see a fixation cue for 1s. Next, they are cued with the valence 
(“Avoid lose” or “Try to win”) together with the amount at play ($1, $5 or $10) 
for a random duration of 1.75, 2, or 2.25s. Next, participants respond by 
squeezing and holding for as much as they want, up until the dash reaches the 
maximum level or the screen times out, whichever happens first. The farther up 
they can bring the dash, the more money they are able to win/avoid lose. The trial 
ends with a feedback screen that displays how much money they won/lost that 
lasts 1.5s. 
 

 
 
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Figure 1. Task design. 
 
 
Figure 2. $1 trial timelines. Top: negative valence. Bottom: positive valence. 
 
 
 
Active Avoidance of Signaled Threat Paradigm (Healthy controls without war-
zone exposure N=90, Healthy controls with war-zone exposure N=30, PTSD from 
war-zone exposure N=30). 
 
Participants will perform a modified version of the active avoidance of signaled 
threat paradigm (Gorka, LaBar, & Hariri, 2016), during which, they will hold 
down a button and view sequentially presented pairs of colored squares. The 
squares will differ based on the color’s hue (low, medium, and high). Participants 
will be instructed to lift their finger when they see the medium-medium color 
combination (i.e. “go” trials), and to refrain from lifting their finger during all 
other combinations (i.e. “stop” trials). Participants will perform alternating blocks 
in safe and threat conditions, and each block will consist of a series of “go” and 
“stop” trials.  
 
We will use independent stair-case procedures to titrate task difficulty so that all 
participants achieve a ~75% accuracy during go trials for each condition. During 
the threat condition, participants will receive an electrical shock whenever they 
make a mistake. Participants will be informed that “Your performance during 
each trial will determine whether or not you receive an electrical shock. However, 
the task may become more difficult or less difficult over time. Please try your best 
at all times.” During the safe condition, participants will never receive an 
electrical shock. The duration of each electrical shock will not last longer than 

 
 
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100 milliseconds. The frequency of delivery for electrical shocks will be based on 
performance, but a minimum of 10 seconds will separate each electrical shock. 
No participant will receive more than 10 electrical shocks in this substudy 
regardless of their performance. The amplitude of the electrical shock will be 
selected by the participant, through the shock workup protocol (see section 8, 
Electric shocks: work-up), to correspond to a level which the participant perceives 
to be tolerable, yet aversive. The shock intensity will not go above the 100 mA 
maximum shock intensity outlined in the protocol (see section 4iv., Electric 
shocks as unconditioned stimulus). These parameters have been well tolerated by 
participants in our previous studies. 
 
Healthy controls without war-zone exposure will perform the task in the MEG or 
MRI scanners. Healthy controls with war-zone exposure and participants with 
PTSD from war-zone exposure will perform the task in the MRI scanner. 
Differences in accuracy and reaction times between the safe and threat condition 
will serves as measures of active avoidance, and will be used to determine 
whether persons with PTSD exhibit impaired or facilitated behavioral responses 
to threat. Indices of neural circuit function will be used to determine whether 
persons with PTSD exhibit altered neural responses during active avoidance, and 
whether changes in neural circuit function are associated with individual 
differences in active avoidance behavior (i.e. accuracy and reaction time). 
 
Additional Procedural Description 
 
fMRI and MEG investigations of threat  
These experiments will have a similar design for both the event-related fMRI and 
event-related MEG. Separate groups of subjects will be run in the fMRI 
experiment (consisting of just one session in the MRI scanner) and in the MEG 
experiment (including the acquisition of a structural MRI scan). The fMRI 
experiment may be performed in a 3-Tesla or 7-Tesla fMRI machine depending 
on the availability of the 7T scanner.  
 
The primary focus of the study will be the selective facilitation of processing and 
learning of threat-related information (Baas et al., 2002; cf. Weinberger et al., 
1995; Thiel et al. 2002). A visual or auditory stimulus that indicates whether or 
not an electric shock and/or loud sound stimulus may be anticipated will be 
presented in a repetitive manner to allow averaging over enough trials to achieve 
acceptable signal-to-noise ratio.  
 
The first run will consist of a baseline measurement to determine processing of 
the visual or auditory cues before these are associated with a threat manipulation. 
Subsequently, the subjects will receive an instruction that links one of the stimuli 
to the possibility of shock and/or loud sound stimulus reinforcement. The second 
run will be again a series of presentation of these stimuli, but with occasional 
reinforcement of the instruction by presenting a shock and/or loud sound stimulus 
during the presentation of the threat cue. During both runs participants may be 

 
 
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asked to use an MRI compatible fiber optics response box to rate their level of 
anxiety at multiple time points. Additional runs that include the procedures listed 
above (Perception, Memory, Resting State, and Passive Presentation) will be 
applied such that participants will be tested during 2-6 runs, depending on the 
number of tasks. Experimental procedures will not exceed 6 functional imaging 
runs in a single session. 
 
Previous experiments (Baas et al., 2002) proved that reinforcement of 1 in 16 
threat cues was sufficient to maintain expectation of shock throughout the 
experiment. Previous experiments have also shown that sufficient signal to noise 
ratio was achieved with event-related potential measures with about 200 
presentations of each stimulus condition. This amounts to administration of about 
12 to 15 electrical shocks during the experiment. This is the same duration of the 
experiment and total number of shocks as proposed as the experimental procedure 
proposed in the amendment to protocol 01-M-0185. The discomfort to the patient 
will not be increased by adding MEG measurements. MEG measurements are non 
invasive, painless, and are used routinely in clinical practice, including in children 
and infants. 
 
MEG assessment of the cold pressor test 
This MEG experiment will involve exposure to an acute stressor, i.e., immersion 
of hand into ice-cold water for 1min, to increase baseline anxiety.  The procedure 
consists of a 5 min interval, during which the subject has his hand immersed up to 
the wrist level in water at room temperature (70F). After this interval the subject 
is asked to immerse the same hand up to the wrist level in ice water for 60 
seconds. Ice water with temperature of 33-36F is prepared by mixing 2 liters of 
crushed ice and 2 liters of tap water in a plastic foam bucket shortly before the 
procedure takes place. After 60 seconds the hand is taken out of the ice water and 
dried with paper towels.  Subsequently, we will measure autonomic responses and 
neural responses with MEG during resting periods to assess the effects of stress 
exposure.   
 
 
Alprazolam/threat study 
 
This will be a double-blind cross-over design; each subject will receive each drug 
treatment (placebo, 1 mg alprazolam). Subjects (healthy only) will be tested on 2 
occasions on separate days as follows: 
 
1) fMRI/MRS/MEG/placebo 
2) fMRI/MRS/MEG/Alprazolam 
 
For the initial study involving only MEG scanning after alprazolam and placebo 
administration, subjects will be tested during periods of threat, when they 
anticipate unpredictable shocks, and during safe periods.  Tasks during MEG 
scanning include passive presentation of visual (3 cycles per degree, square-wave 

 
 
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gratings) and auditory stimuli (pure tones) and memory tasks (n-back, virtual 
reality) to evoke gamma responses.    Following this initial study, a new sample of 
subjects will be tested in similar way during fMRI, MEG, and MRS scanning.  
 
Recruitment, exclusion/inclusion, and safety precaution for this study are the 
same as in protocol 10-M-0049, which examines the effects of various 
compounds, including Alprazolam, on psychophysiological responses during 
anticipation of shock.  Administration of the drug/placebo will take place in an 
outpatient clinic or a Day Hospital of the Clinical Center by a certified healthcare 
provider (nurse, nurse practitioner, or physician) and under the supervision of the 
medical staff of the Clinical Center.  Participants will remain in the clinic or Day 
Hospital of the Clincal Center for 2 hours to allow the drug to take effect, and 
will return to that site following the testing procedures, which take place in the 
NMR center.  A certified healthcare provider will be present for testing in the 
NMR center, and participants will be monitored (but not recorded) via a video 
camera. There will be two-way communication between the testing/patient room 
and the experimenter room. This study has been completed.  
 
 
v. Study procedures 
This protocol includes an fMRI study of cognition and emotion without threat of 
shock (2.5 hours; 90 minutes in the scanner), an fMRI threat of shock study (2.5 
hours; 90 minutes in the scanner), an MEG cold pressor study (1.5 hours), and an 
MEG threat study (2.5 hours), an alprazolam/threat MEG study (5-6 hours, 2 
sessions), and an alprazolam/threat fMRI/MEG/MRS study. An additional 
telehealth consenting visit may be added to each during the pandemic.  The 
alprazolam studies have been completed. Participants will not necessarily 
complete all these components of the study but will initially be recruited for one 
component and then invited to participate in additional components as needed. 
MEG and alprazolam studies will only recruit healthy subjects. Healthy 
volunteers and patients may undergo the wrist shock task in 7T or 3T fMRI 
machine. 
 
After initial telephone screening, individuals who appear to qualify for inclusion 
will be invited for an in-person screening at the NIH.. On the screening day, 
subjects will be asked to sign an informed consent for screening protocol 01-M-
0254. A medical and psychiatric history will be taken and a physical exam will be 
conducted. Subjects will also be asked to fill out various questionnaires of mood 
and anxiety. Subjects will complete informed consent for protocol 02-M-0321 
prior to study procedures. Questionnaires may be administered on the day of the 
study procedures through the Clinical Trial Survey System (CTSS)/ Clinical 
Trials Database (CTDB). 
 
We estimate that 1186 healthy subjects and 686 patients will be needed: about 30 
subjects per experiment (in at least 6 studies), so that high-quality data is acquired 
from at least 15-20 subjects per experiment, with enough subjects to account for 

 
 
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data lost to artifact or subject withdrawal. Subjects will be told that the study 
examines their responses to anticipation of shock. In addition, to pilot an 
adaptation of this procedure to make it suitable for children, adolescents and 
patients, we will test 12 more subjects (healthy adults) using a mildly aversive air 
blast to the neck instead of aversive shock. Air blasts have been used as 
unconditioned stimuli successfully to examine explicit cue fear but not contextual 
anxiety (Grillon et al., 1999; Pine et al., 2001). The original procedure will 
employ threat of shock to maximize the chance of finding significant effects. 
Shocks are inherently more anxiogenic than air blasts. The air blast manipulation 
will be piloted in the psychophysiological laboratory. 
 
Anxiety, mood & subjective measures 
We will use Mood and Anxiety Symptom Questionnaire-Short Form (MASQ-SF), 
Spielberger State/Trait Anxiety Inventory (STAI, Spielberger, 1983), Penn State 
Worry Questionnaire (PSWQ, Meyer et al., 1990), the Beck Anxiety Inventory 
(BAI, Beck et al., 1988), and the Profile Of Mood States questionnaire (POMS, 
McNair et al., 1971). It will be noted whether subjects have had previous 
experience with fMRI, because previous experience may lead to a lower baseline 
anxiety. To evaluate the effect of several experimental parameters, analogue 
scales will be employed. Visual analogue scales will be used for measurements 
outside of the scanner (a 10 cm long, indicating at the left-hand side ‘none’, right 
‘extremely high’; the middle (at 5 cm) will be scored as neutral). First, the 
subjects’ level of discomfort induced by the shock will be evaluated after shock 
sample administration. On the test day, subjects’ anticipation of the aversiveness 
of the shocks will be measured. Non-analogue measures may be completed by the 
participant on the Clinical Trial Survey System (CTSS) online system. AIs may 
then collect data from the Clinical Trials Database (CTDB). Participants may 
enter their responses while at NIH using a wireless-device interface to access the 
NIH-intranet secure CTDB. CTSS will not require real-time monitoring.  
 
Subjective measures in the scanner 
After each run subjects will be asked to retrospectively indicate their level of fear 
on a numeric scale from 1-100 during the absence of the visual cue in all three 
conditions (neutral, predictable, unpredictable), and in the presence of the cues in 
the predictable condition. The instruction texts that indicate these conditions will 
be projected on the screen one by one and the subject will be asked to give their 
rating orally through the intercom or by pressing response buttons. 
Function Magnetic Resonance Imaging 
The parameters for acquisition of fMRI data that we will start out with in the 
current study will be as follows. First, a sagittal localizer scan will be acquired to 
orient subsequent scans. Second, the functional scans will be acquired. Although 
BOLD signal drop-out during EPI sequences is more severe at higher field, 
preliminary analysis of 7T data confirms that the signal obtained is adequate to 
image our primary regions of interest, the BNST and dorsal nuclei of the 

 
 
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amygdala. These scans will include series of contiguous 4 mm axial slices 
extending covering the entire brain, parallel to the AC-PC. These scans will use a 
64 x 64 matrix with echoplanar single shot gradient echo T2* weighting 
(TR=2000 ms; TE=40 ms; FOV=200 mm; 64 x 64 matrix, 3.75 mm voxels). This 
slice thickness and voxel size has been optimized on the NIH scanner for 
obtaining changes in BOLD measures in the human amygdala (Dr. Pessoa, 
personal communication). These parameters have also yielded good results for the 
amygdala, the lower orbital cortex, and the anterior cingulate cortex, in terms of 
minimizing signal susceptibility artifacts. However, if we fail to see reliable 
activations using these parameters in the pilot subjects, we may need to acquire a 
more limited number of slices focused on the amygdala and prefrontal regions 
(axial oblique angle). We are aware that on restricting the field of view in this 
manner, the analyses will have to be restricted to ROI analyses.  Finally, a high-
resolution T1-weighted anatomical scan of the whole brain will be acquired using 
a magnetization prepared gradient echo sequence (MPRAGE). This yields 0.9 x 
0.9 x 1.2 mm3 voxels optimized for gray-white-CSF contrasts. Finally, a set of 
standard perfusion scans will be acquired to evaluate cerebral vasculature 
structure and function. Participants may be scanned in the 3-Tesla or 7-Tesla 
fMRI scanner.  
 
GABA MR Spectroscopy 
The procedure will be similar to that implemented in 05-M-0006. Subjects will be 
scanned on a 3 Tesla GE whole body scanner using a GE resonator head coil that 
provides a homogenous radiofrequency field and capability of obtaining 
spectroscopic measurement from cortical regions. The voxel will be located on 
the basis of a MPRAGE structural MRI acquired in the same scan session. GABA 
will be measured using an interleaved PRESS-based J editing method. Individual 
peak areas will be fitted using MRUI (http://carbon.uab.es/mruiwww), which 
performs time domain spectral. Concentrations of GABA, choline, N-acetyl 
aspartate (NAA), and co-edited Glx (i.e., glutamate and glutamine) will be 
expressed in mmol/liter (mM) referenced to concentration of creatine.  This 
creatine referencing method has been used in the field for over a decade and has 
been validated by a number of research groups. The spectroscopy data will be 
processed in two steps. First the unedited spectra will be fitted for the amplitudes 
of choline, creatine, and NAA. Secondly the GABA at 3.0 ppm and co-edited 
Glx-2 at around 3.8 ppm will be extracted from the edited spectra and fitted 
accordingly. The GABA signal will be corrected for macromolecule 
contaminations (Shen, Rothman, & Brown, 2002). At experimental conditions 
optimized for GABA editing, a small fraction of Glx-2 at 3.8 ppm and Glx-4 at 
2.4 ppm will be co-edited because of their J couplings to the Glx-3 signal at 2.1 
ppm. The clean co-edited Glx-2 signal will be used for measurement of Glx as its 
intensity is proportional to the total concentration of Glx. The GABA signal 
closest to the Glx-2 peaks resonates at 3.0 ppm. 
 
Visual and auditory threat 

 
 
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For the MEG recordings, the participant will either sit or lie supine in the shielded 
recording room with her head in the helmet. Brain magnetic fields will be 
recorded with the 275-channel OMEGA system. The 275 SQUID sensors are 
uniformly distributed, in a grid, over the inner surface of the helmet that covers 
the entire head with provisions for the eyes and ears. Visual and two-way audio 
communication with the participant will be maintained throughout the session. 
Head position within the magnetometer will be determined continuously during 
the scan by digitizing the position of three indicator coils that are attached to the 
preauricular and the nasion fiducial points. The positions define the coordinate 
system for the signals and allow for detection of head movement artifacts. Digital 
photographs of the fiducial points will also be taken to localize the same points on 
the participant's anatomical MRI scan. Participants in the MEG experiment will 
be invited back for a short MRI session, in which a structural MRI scan will be 
acquired, to be used in the source localization of the MEG data. 
 
Eyetracking in MEG 
Subjects are situated in the seat of the MEG, and the camera of the eye-tracking 
unit (SR Research Ltd. Ottawa, Canada) is moved into place. Afterward, the 
subject undergoes a calibration procedure where they are instructed to fixate on 
repeated presentations of a randomly positioned fixation cross. Afterward, the 
eye-tracking camera passively monitors the position of the subject’s gaze, and the 
diameter of the subject’s pupil. The unit also creates events at the onset and offset 
of each blink or saccade. 
 
 
Scanning: On the day of the scanning session, subjects will again fill out state 
questionnaires, and visual analogue scales to assess anticipation of the 
experiment. Electrodes for shock administration and for skin conductance 
monitoring will be attached. Subjects will then enter the scanner. They will be 
informed of all conditions via a display monitor in the scanner. The conditions are 
as follows: neutral (no shocks), predictable (shocks only during display of 
stimulus cue) and unpredictable (shocks can be administered at any time). During 
these three conditions, scans will be made in the presence and the absence of the 
stimulus cues. Shock reinforcements will occur during as few runs as possible.  
 
Psychophysiological testing: Following scanning, the experiment may be repeated  
in a psychophysiological laboratory, where we will assess their startle, skin 
conductance, and heart rate responses during the different threat conditions. The 
psychophysiology will likely take place on a different day. 
 
 
Electric shocks as unconditioned stimulus (US): Electric shocks will be used as 
the US. Electric shocks are among the most efficient ways to induce anxiety in the 
laboratory. The shocks will be delivered through two disk electrodes placed on 
one of the forearms, feet or ankles. Given high heterogeneity of skin resistance 
and pain threshold between participants, each shock will have intensity up to 100 

 
 
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mA and will be delivered as trains of 1-2 ms pulses. The total shock duration will 
not exceed 500ms. For each subject a level of shock that is tolerable, but 
uncomfortable will be selected (see shock workup below, section 8, Electric 
shocks: work-up). The shock is generally described by participants as rather 
anxiogenic and unpleasant, but tolerable. We have been using shocks in various 
protocols at the NIMH since 2000 without major complains. Participants will 
have the opportunity to withdraw from the study if they wish at any moment, and 
are informed explicitly about this option in the informed consent. Our experience 
is that over 95% of subjects who received the shock chose to participate in the 
experiment.  
 
Startle Reflex: The acoustic startle stimuli will be a 40-ms burst of white noise 
(103 dB) with instantaneous rise time. Auditory stimuli will be delivered 
binaurally via headphones. The eyeblink component of the startle response will be 
measured by recording electromyographic (EMG) activity of the left orbicularis 
oculi muscle as we have done in all of our previous psychopharmacology studies.  
 
 
Motion induced by the shock reinforcements: To reduce the impact of the motion 
artifacts on the data caused by the reaction to a shock (typically a startle response, 
i.e., a jerk-like motion), as few shocks as possible will be given during the 
experiment. . The normal procedures for movement artifact detection and 
correction will be employed (AFNI/ SPM). However, due to movement induced 
by the shocks there may be a substantial change in position of the head during the 
shocks, which would change the head position in the scanner. The alignment 
procedure of functional images with respect to each other work well with motion 
up to 1 or 2 voxel sizes (see http://afni.nimh.nih.gov/afni/edu/README.registration, and Cox 
and Jesmanowicz, 1999).  
 
The runs in which the shocks are delivered will be closely evaluated with respect 
to the occurrence of motion artifacts, by examining the data visually, and by 
relying on the parameters provided in the dedicated software (AFNI/ SPM) that 
yield measures of amount of movement. TRs in which excessive movement 
occurs will be discarded. If either based on the visual inspection or based on the 
movement parameters movement appears excessive, the data from that run will be 
discarded. The AFNI software can successfully correct movement up to the order 
of about a voxel size (Cox and Jesmanowicz, 1999). 
 
An alternative strategy is to prevent head movement in the scanner by the use of a 
bite-bar.  Our experience suggests that movement associated with shock 
reinforcement is marginally higher than the levels of movement observed across 
functional imaging studies, in general.  This leads to a slightly higher than 
average amount of data rejection in our studies involving shock reinforcement.  
Nevertheless, the use of a bite-bar does not appear warranted because of the 
additional baseline anxiety and muscle stress caused by restraining the subjects in 
this way.     

 
 
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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. 
 
 
v.i.  End of participation:  Participation in this study will end after the completion 
of each visit, following an assessment for adverse reactions to experimental 
procedures and a psychiatric interview when clinically indicated. Participants will 
remain under the care of their medical provider. Additionally, depressed patients will 
receive care as outlined under the direction of Dr. Zarate and the mood disorders 
program staff. Medical care will not be offered at the completion of study 
procedures. Participants will be informed of any findings that require further 
evaluation. Clinical MRI brain scans are completed one time per year, and sent to 
radiology for assessment. In rare cases when an abnormality is discovered, 
participants are offered further assessment at the NIH via a neurology consult. Minor 
abnormalities with no clinical significance to participants may not be shared. 
 
b. Clearly identify which procedures are research and which are clinical care 
  
 
N/A 
 
c. Identify any medications/ devices requiring IND/IDE 
 
NSR devices: 
- 
Acoustic Startle 
- 
Shock Device  
- 
7T fMRI 
 
d. Identify if radiation is medically-indicated or for research only 
 
N/A 
 
e. 
Identify relationship of this study to other protocols (include if subjects are required  
 
to participate in other protocol) 
 
N/A 
 
5. Management of Data and Samples: 
 
Storage 
Any saliva samples collected will be stored in Building 10, Room 3D55. These samples 
will be assayed for salivary cortisol levels as well as for alpha-amylase activity. 

 
 
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Information is stored using a confidential case number (subject research code number), 
and no identifiers (name, address, phone number, etc.) are placed on data that could allow 
direct linking of database information to individual subjects.  Additionally, data will be 
kept in password-protected computers.  Samples are kept in locked storage. Only study 
investigators have access to the samples and data. Any loss or destruction of samples will 
be reported to the IRB. Samples will be destroyed at the end of the study.   
 
Data and sample sharing plan 
This protocol is not subject to the Genomic Data Sharing (GDS) Policy. Data and 
samples may also 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 and/or samples from this 
protocol may be open-access or restricted access.  
 
Samples and 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 and samples 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.    
 
6. Additional Considerations: 
 
a. Research with investigational drugs or devices 
Acoustic startle, shock device, and 7T fMRI  used in this protocol are considered non-
significant risk (NSR) devices and will only be used within published guidelines.  
   
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.  
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  

 
 
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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.  
 
The 7T fMRI 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 7T fMRI is not an implantable device. 

 
 
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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 7T fMRI 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 7T fMRI, 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 7T fMRI 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 7T fMRI studies on an NSR device basis. Hence, the CNS IRB, like most 
US IRBs, has accepted NSR designation for 7T fMRI within these limitations.  
 
 
 
b. Gene therapy 
N/A 
 
7.  Risks and Discomforts: 
 
fMRI 
MRI is widely regarded as a safe, noninvasive procedure for visualization of brain tissue. 
The risks involved with fMRI are the same as those involved in standard anatomic MRI, 
since these three procedures rely on the same physical properties of brain tissue. This 
study will be performed on an FDA approved 3T scanner at the NIMH.  
 
MRI at 3 Tesla is a routine clinical procedure, and issues regarding radio frequency 
deposition, time varying magnetic fields, and the static field at 3 Tesla do not require 
detailed discussion.  
 
MRI at 7 Tesla (ultra high field strength) is less commonplace but becoming more so, as 
there currently are approximately 60 high field scanners in global operation. fMRI at 7T 
poses the minimal risk of vertigo-like sensations during movement in and out of the bore, 
but subjects in one study rated their discomfort of such sensations well below the length 
of scan session, which is the same for 7T as for 3T (Theysohn, et al. 2008). Furthermore, 
tissue heating from radiofrequency deposition is a minimal concern at small flip angles 
for fMRI at high fields (Gonzales-Castillo, et al 2011). Essentially, the risks of shocks in 
the scanner environment, with 7T as well as 3T, include heating of the electrodes with 
discomfort or burns. These risks, however, are actually less likely in the 7T scanner than 

 
 
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in 3T MRI scanner because in the 7T scanner the RF field does not extend to the whole 
body, as it does with the 3T. 
 
MRS 
There is no known hazard to the exposure of magnetic waves during MR imaging. MRS 
does not introduce additional risks compared to fMRI (the gradient noise is much milder 
with MRS than MRI).  
 
Electric shocks: work-up 
Electric shock poses minimal risk to participants.  The shocks will be delivered through 
two disk electrodes located on one of the subjects’ ankles or feet or wrist. Prior to testing, 
a shock work up procedure will be used to allow us to control for heterogeneity of skin 
resistance and pain threshold between participants and to administer a range of mildly 
painful stimuli in an ethical manner during the task itself. Shocks with step increases in 
amplitude will be administered up to a level tolerable to each participant, while 
participants will provide subjective ratings of shock intensity. The initial shocks are of 
mild intensity while participants familiarize with the setup. Up to three series of shocks 
will be administered (up to 20 shocks in total), as repeated escalation allows participants to 
adapt to initial anxiety about the shocks, since thereafter ratings are more consistent 
(Seymour et al., 2004, 2005). Participants are explicitly informed of their right to 
discontinue the research without consequence at any point during the consent process and 
then again following the sample shock procedure. Our experience is that over 95% of 
participants choose to participate in the experiment following the sample shock workup. 
Anticipation of the shock increases subjective anxiety and enhances physiological arousal.  
 
Electric shock within the MRI scanner 
Introduction of electrical wires that are connected to the subject in the strong magnetic 
field of the scanner may constitute an additional risk. The main risk associated with 
administering shocks is the introduction of electrode wires directly in the RF- (radio 
frequency) field of the magnet. Wires will not be exposed to the radio frequencies 
induced in the RF head coil. The shock stimulator equipment will be outside the magnetic 
area, and a plastic and copper cable is taken through the wall for connection to the subject 
electrodes. To eliminate the possibility that electrode wires will enter the RF-field from 
subject movements, shock electrodes will be attached to either the foot or ankle region. 
Similarly, electrodes for skin conductance recording in the scanner will be attached to the 
sole of the other foot. Area between the wires will be minimized by twisting the wires 
together, and coiling of the wires will be prevented by inspection prior to each session 
(i.e., wires run without loops). Studies employing wires for electrical stimulation (e.g., 
Disbrow et al., 1998) or for psychophysiological measurement (skin conductance; Büchel 
et al, 1998; LaBar et al., 1998 etc) have been reported in the literature.  
 
MEG 
MEG is a noninvasive procedure to measure brain responses by recording magnetic fields 
and electrical potentials outside the head with sensors,.  No known risks is associated 
with this procedure. 
 

 
 
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Eyetracking in MEG 
The unit is a passive monitoring system, and nothing is attached to the subject. There are 
minimal risks associated with this procedure. 
 
Startle stimulus 
The auditory stimuli that will be used in the startle studies are 40-ms duration 103 dB 
white noise. Auditory startling sounds of much higher intensities are frequently used in 
startle studies. Sounds of higher intensities and longer duration are also widely used in 
aversive conditioning in human subjects, where they serve as unconditioned stimuli. The 
short duration (40 ms) of these sounds minimizes risk of hearing loss (Blumenthal et al., 
2005). In addition, a white noise is safer than a pure tone. The PI has been involved in 
similar studies and collaborations involving over 1000 of subjects with no adverse 
reactions. The auditory stimulus may trigger a migraine. 
 
Cold Pressor test 
The cold pressor test is a simple, safe and reliable procedure, widely used to determine 
cardiovascular reactivity, pain perception and stress response in healthy subjects, as well 
as in clinical conditions such as hypertension or depression (Velasco et al. 1997, Kelly et 
al. 1998).  There are no known risks associated with the cold pressor procedure.   
 
Psychophysiological recording 
The psychophysiological measures that will be obtained are non-invasive, requiring the 
administration of no 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 since the salt concentration of the paste is 
similar to that of human sweat. The risk is equivalent to that of an EEG recording. 
 
Alprazolam 
 Alprazolam (Xanax™) is a benzodiazepine widely used in the treatment of anxiety 
disorders or for the short-term relief of anxiety symptoms. For chronic treatment of 
anxiety disorders, the PDR (2000) recommends a daily dose up to 4 mg, given in divided 
doses. Plasma levels are proportionate to the dose given. Peak concentration in the 
plasma occurs in one to two hours following administration. For dose 0.5-3.0 mg, peak 
levels of 8.0 to 37 ng/mL are observed. Mean plasma elimination half-life is 11.2 hours. 
Contraindications include sensitivity to benzodiazepines. Side effects in patients using 
chronic alprazolam include insomnia, drowsiness, fatigue, headache, involuntary 
movement, nausea, and memory difficulties. Besides its effect on anxiety in clinical 
populations, alprazolam has also been frequently used to test the effects of 
benzodiazepines on experimental anxiety at doses of 0.5 and 1 mg. This study has been 
completed.  
 
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, & Vaitl, 1988). Because the intensity of these pictures might make some people 
uncomfortable, their contents will be fully described to the subjects before participation 

 
 
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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 of psychological supports by 
a clinician.  A clinician will then talk to them and will follow up with a call. 
 
Delayed Treatment (patients only)  
Although participants will not be taken off any medication for the purposes of this study, 
we will only include those patients who are not currently taking any psychotropic 
medications, with the exception of PTSD participants only. Thus patients included in this 
study may be at risk of increased symptoms because of the absence of 
psychopharmacologic treatment. During the initial psychiatric assessment, clinical staff 
will inform patients that delaying psychopharmacological treatment may increase their 
risk of increased symptoms and that such treatment is readily available at mental health 
care providers outside of the NIH. PTSD participants may continue on antidepressants 
and benzodiazepines. 
 
Storage/sharing of data and samples:  There is minimal risk that data or samples could be 
identified.  To minimize this risk, data and samples that are shared will be de-identified.  
Data and samples will be sent with a code.  This linking code will be kept at the NIH.  
 
Grip force measures: This measure is not expected to increase risks in human subjects. 
Gripping hard may cause muscular fatigue or soreness but is not expected to increase risk 
of injury or harm to the subject. The fatigue or soreness is expected to be mild and self 
resolve without treatment. 
 
 
8. Subject Safety Monitoring: 
a. Parameters to be monitored 
 
General Subject Monitoring 
 
Over the course of the outpatient visits to the NIH, Clinical Center, the Principal 
Investigator, Associate Investigators, Independent Study Monitor, Research Nurse 
Coordinator, and attending clinical staff will monitor the participants for distress, 
discomfort, and desire to discontinue the study. Participants will be provided with the 
contact information of the Principal Investigator should concerns arise in between visits. 
 
Alprazolam Subject Monitoring 
 
For the alprazolam experiment, a certified healthcare provider (nurse, nurse practitioner, 
or physician) will be on hand throughout the procedure and will be responsible for 
participants’ discharge. A sedation assessment, the Observer’s Assessment of 
Alertness/Sedation Scale (Chernik 1990), will be administered by the certified healthcare 
provider prior to and following the procedure before discharge.  The certified healthcare 
provider will also contact the participant the following day after each testing session to 
ensure that there are no adverse reactions to the drug or experimental procedures. This 
study has been completed.  

 
 
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Increased symptom severity 
If, during the course of assessing a participant, a study staff member identifies a 
significant worsening of symptoms, a mental health provider currently treating the 
participant will be contacted, and the necessary treatment steps (e.g., 
hospitalization, referral to a care provider, etc.) will be offered.  
 
Risk for self-harm:  Participants will be monitored for risk for self-harm via a history 
and physical exam and clinical interview on Visit 1, and subsequently during all study 
visits. If a participant is considered a positive screen for self-harm, the participant will not 
be able to leave the premises until evaluated for safety and study procedures will be 
terminated for that day. If following the safety/mental health evaluation it is determined 
that a participant is at risk for self-harm, the participant will be excluded from the study. 
Participant transfers to alternative medical facilities will be conducted according to NIH, 
Clinical Center Guidelines. For off-hour psychiatric emergencies, the study investigator’s 
voice message instructs participants to call 911 or visit the nearest Emergency 
Department.  If a participant leaves a voicemail in distress, the clinical team will attempt 
to make contact with the participant within one business day.  If contact by phone is 
successful, the participant will be triaged by phone and offered a list of mental health 
resources. If the participant does not respond to calls, the clinical team will try calling an 
alternative number if available. 
 
b. Toxicity tables/ criteria to be used 
 
N/A 
 
c. Criteria for individual subject withdrawal 
Participants will be withdrawn from the study if the Principal Investigator determines a 
serious adverse event is related to the study procedures or if the severity of an adverse 
event warrants withdrawal. Participants will also be withdrawn if they develop high 
levels of anxiety, fear, or panic during/from any shock experiments. Participants will also 
be excluded from the study, if they become at risk for self-harm. Additional criteria 
include withdrawal of consent and/or participant decision, non-compliance with protocol 
procedures, and lab results that preclude participation (i.e., positive drug and/or 
pregnancy result). This study nor the NIH will pay for outside treatment. 
  
 
9.  Outcome Measures: 
 
a. Primary 
 
Functional magnetic resonance imaging (fMRI), measuring the event-related 
haemodynamic response related to neural activity in the brain.  
 
Magnetoencephalography (MEG) a neuroimaging technique employed in the 
current protocol to measure the event-related magnetic fields produced by 

 
 
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electrical activity in the brain via extremely sensitive devices such as 
superconducting quantum interference devices (SQUIDs). 
 
b. Secondary 
 
Psychophysiological measures of anxious arousal including the skin conductance 
response (SCR), heart rate, respiration, and EMG measures of the fear-
potentiation of the startle reflex. 
 
Salivary cortisol assays to measure the stress response to the cold pressor, saliva 
will be collected at several intervals throughout the session with plain cotton swab 
Salivettes. All samples will be frozen and stored at -70ºC until assayed in one of 
the freezers located in bldg 10, 2D corridor. On the day of assay, the Salivette 
tubes will be thawed at room temperature and centrifuged at 3000g for 10 
minutes. Cortisol concentration in saliva will be measured by enzyme 
immunoassay (Salimetrics LLC, State College, PA).  Management and analysis of 
saliva samples will be done by Roman Duncko, MD, in the Section on Genetic 
Developmental Epidemiology.  Samples will be number coded, stored for 
approximately a year, and then properly disposed in medical waste. 
 
Self-reported measures of anxiety, level of risk, and CS-US contingency 
awareness. 
 
10. Statistical Analysis: 
 
a. Analysis of data/ study outcomes 
Analysis of functional imaging data involves a series of initial steps including 
realignment (i.e., correction for subtle head movement). Hypotheses regarding task-
associated changes in the BOLD signal will initially be tested by analyzing task-related 
hemodynamic responses in four regions-of-interest (ROI) selected based on hypothesized 
areas of activation: the amygdala, the dorsal anterior hippocampus (Williams et al., 
2001), the anterior insula, and the anterior cingulate. These ROI will be defined using 
AFNI (Analysis of Functional NeuroImages; http://afni.nimh.nih.gov/afni/). The ROIs 
will be defined on each subjects’ anatomical MRI image, and the corresponding changes 
in BOLD data will be extracted from fMRI images that have been coregistered to the 
anatomical MRI. Correction to control for inflation of α (Type I error) will be applied to 
the tests performed on these 8 regions (4 regions in each hemisphere). 
 
In addition, a voxel-wise analysis will be performed to investigate activations outside of 
these regions using SPM 8 (Statistical Parametric Mapping; 
http://www.fil.ion.ucl.ac.uk/spm/). Spatial transformation and smoothing will be 
performed prior to the voxel-wise analysis. This analysis will assess activations in areas 
outside the ROIs defined a priori, as well as to localize the voxel-coordinates for the peak 
difference between conditions within the primary ROIs. We will model the effects of 
presenting the cues depending on the background, and of presenting the background itself 
on BOLD activity.  

 
 
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The factor context (neutral, predictable, unpredictable) will be entered as a blocked factor 
to model the background state during these 2-min conditions. In addition, the transient 
response to the cues will be modeled with respect to the onset of the cue. The hypothesis 
is that in the areas critical for cue fear there will be a transient response to the cue in the 
predictable relative to the neutral and unpredictable condition. This model of analysis 
constitutes a mixed block/event-related design.  
 
All experiments except for those that include patient groups will use within-subject 
designs, such that each participant will experience each comparison condition of interest 
(note: this does not imply that ALL subjects will participate in ALL procedures, just that 
the procedures in which they elect to participate will include the conditions necessary to 
perform the intended analyses). Experiments that include patients will operate both 
within-subject and between-subject designs; the within-subject factors being the 
experimental conditions, and the between-subject factor being subject group (patient or 
healthy control). 
 
b. Criteria for significance  
 
For voxel-wise analyses on normalized whole brain data, we will control false positive 
rates per map at α=.05, using random-effects models (see e.g., Zarahn, 2000; Zarahn et 
al., 2000; Pine et al., 2001; 2002). 
 
c. Power analysis  
Sample size of 15 subjects with good quality data (no major artifacts or other problems) 
is usual for previous published studies on activation of limbic structures, such as Phelps 
et al. (1999; N=12), and Whalen et al. (1998; N=10). Although power computations are 
possible for ROI analyses using conventional statistical tests in which average BOLD 
signal intensities are treated as dependent variables, because the current study constitutes 
a pilot experiment that has not been previously performed, the mean and standard 
deviation of the changes in BOLD responses that will be obtained using the proposed 
methods are not known. In general, with sample sizes as small as n=10 and for alpha = 
.05 (two-tailed), power exceeds .80 to detect effect sizes of 1.2 in one pre-specified brain 
region. Fortunately, most fMRI studies of neurobiologically meaningful task-associated 
changes in BOLD signal document larger effects than this. For patient subtypes, (i.e., 
GAD, SAD, panic, phobia, PTSD, and MDD) an N of 30 will be targeted for each study. 
This larger sample size will provide us with a greater range of symptom severity in order 
to conduct correlation analyses with regional brain activity and it will buffer against 
potentially higher rates of attrition (excessive motion, symptom-related dropout) in our 
psychiatric samples.  
For the Time Perception, cognitive task, a power analysis was conducted to determine the 
number of participants that will be used for that sub-study. In a previous fMRI 
experiment, our largest effect size (η2=0.24) was for brain activation in the insula under 
our anxiety manipulation. Assuming a somewhat smaller effect size (η2=0.20) due to 
regression to the mean, if we set power at .90 and experiment-wise, two-tailed alpha at 
0.05. Based on these parameters, we will need 46 subjects per experiment. Assuming 4 

 
 
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subjects to initially adjust the experimental procedure, a N of 50 subjects will be the 
target for the experiment.  
 
For the first modified version of the Motivation/ "Monetary Incentive Delay" Task, we 
propose to collect 80 participants on the 3T fMRI scanner in order to assess the role of 
between subject factors such as gender and trait anxiety on neural responses to incentives. 
Assuming a medium effect size (R2=0.15), a power of 0.8, and an alpha of 0.05, power 
analyses suggest that we would require a sample of 75 participants to detect an 
association employing two-tailed multiple regression analyses. Assuming that five 
participants are lost due to head motion, we will need to collect 80 participants to detect a 
medium sized effect of gender or trait anxiety on these neural responses.  
 
For the first modified version of the Motivation/ "Monetary Incentive Delay" Task, we 
propose to collect 30 participants on the 7T fMRI scanner in order to assess the role of 
small midbrain structures in reward/threat processing. Assuming a slightly larger effect 
size due to the improved signal to noise ratio of 7T imaging (Cohen’s dz=0.6), a power of 
0.8, and an alpha of 0.05, power analyses suggest that we would require a sample of 24 
participants to detect a difference between conditions employing two-tailed paired T 
tests. Assuming five participants are lost due to head motion. we will need to collect 30 
participants to detect a medium sized effect of trial type on neural responses to 
incentives. 
 
For the second modified version of the Motivation/ "Monetary Incentive Delay" Task, we 
propose to collect 40 participants on the MEG scanner to assess anticipatory responses to 
aversive and monetary motivational stimuli. Assuming a medium effect size (Cohen’s 
dz=0.5), a power of 0.8, and an alpha of 0.05, power analyses suggest that we would 
require a sample of 34 participants to detect a difference between conditions employing 
two-tailed paired T tests. Assuming five participants are lost due to head motion, we will 
need to collect 40 participants to detect a medium sized effect of trial type on anticipatory 
neural responses. 
 
Collectively, the three research projects listed above (80+30+40=150) will increase our 
accrual from 30 to 150 participants for the Motivation/ "Monetary Incentive Delay" Task. 
 
For the first modified version of the Active Avoidance of Signaled Threat Paradigm, we 
propose to collect 45 healthy participants without war-zone exposure on the fMRI 
scanner. Assuming a medium effect size (Cohen’s dz=0.5), a power of 0.8, and an alpha 
of 0.05, power analyses suggest that we would require a sample of 34 participants to 
detect a difference between conditions employing two-tailed paired T tests. Assuming ten 
participants are lost due to head motion and poor performance, we will need to collect 45 
participants to detect a medium sized effect of trial type on neural responses during 
avoidance. 
 
For the second modified version of the Active Avoidance of Signaled Threat Paradigm, 
we propose to collect 45 healthy participants without war-zone exposure on the MEG 
scanner. Assuming a medium effect size (Cohen’s dz=0.5), a power of 0.8, and an alpha 

 
 
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of 0.05, power analyses suggest that we would require a sample of 34 participants to 
detect a difference between conditions employing two-tailed paired T tests. Assuming ten 
participants are lost due to head motion and poor performance, we will need to collect 45 
participants to detect a medium sized effect of trial type on neural responses during 
avoidance. 
 
Collectively, the two research projects listed above (45+45=90) will increase our accrual 
from 30 to 90 healthy participants without war-zone exposure for the Active Avoidance of 
Signaled Threat Paradigm. 
 
d. Accrual number  
Our ceiling is set at 1952 participants.  This will account for the addition of patients 
(726) as well as a separate group of 1226 healthy matched (age, sex, IQ) controls. 
 
11. Human Subjects Protection: 
 
a. Subject selection 
 
i. 
Statement of equitability 
This study provides equal opportunity to participate for all eligible persons and prohibits 
discrimination on the basis of race, ethnicity, color, religion, creed, sex, sexual 
orientation, gender identity, national origin, ancestry, disability (unrelated to exclusion 
criteria), genetic information, or handedness.  Participants above the age of 50 will be 
excluded to achieve some homogeneity in our sample and thereby reduce the variability 
of the neurobiological signal across subjects. An age limit of 50 years provides for the 
inclusion of the majority of participants we recruit, without compromising the results by 
including older adults who have been shown to exhibit functional reorganization in the 
brain (Reuter-Lorenz, & Lustig, 2005). The initial age limit was set at 40 years old 
because it resulted in a relatively homogenous sample, without unnecessarily excluding 
subjects. This limit has since become too stringent, and based on similar studies (e.g., 
Blair et al. 2008; Blair et al., 2006; Hsu et al., 2010), we have increased our age limit to 
50 years old. 
 
ii. Rationale for selection if not equitable 
We plan to enroll men and women in 1:1 ratio. 
 
The race distribution of the samples studied in this project will be similar to that of the 
greater Washington DC metropolitan area, and will include representation from all 
minorities except American Indians, who are less prevalent. Since the NIMH is located in 
a suburban area in which African-Americans may be under-represented, if at any point 
the race composition of subjects recruited during the proposed imaging study fails to 
reflect the race distribution of the larger geographic area, we will intentionally emphasize 
recruitment of subjects from more urban areas through collaborations with Howard 
University. While we have not found significant race effects on the clinical or 
psychobiological parameters assessed, we will continue to perform secondary analyses to 
explore potential effects of race on these domains.  

 
 
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Participants  who do not speak English fluently will be excluded because our primary 
instruments are not validated in languages other than English. 
 
b. Justification for inclusion/ exclusion of children 
 
We will not enter subjects under age 18 because of ethical concerns about exposing them 
to threat of shock. Though shock stressor is well tolerated by adults (and used frequently 
in the literature to assess stress reactions in healthy and disordered individuals), such 
methods may be inappropriate for younger children. Moreover, concerns regarding the 
legal inability to provide informed consent before age 18 (and the consequent 
dependence on parental decision) preclude inclusion of subjects under age 18. 
 
c. Justification for inclusion/exclusion of other vulnerable subjects, e.g. cognitively 
impaired, pregnant, mentally ill  
 
Pregnant females will be excluded due to the risk of electric shock and MRI during 
pregnancy. 
 
We have specific hypotheses regarding the effects of stress on different psychiatric 
populations, and the data that we gather will have implications for clinical intervention 
as well as basic understanding of behavioral and neural differences associated with the 
different subtypes of mood and anxiety disorders. Patients will undergo clinical 
assessment and care as outlined above, and will be consented, enrolled, and retained as 
participants only if they a) fully understand the procedures and risks involved, b) are 
willing to participate, c) do not display any contraindication to participating (see 
exclusion criteria above), and d) do not display significant psychiatric symptom increase 
during periodic clinical assessment throughout the study. 
 
d. Justification of sensitive procedures (use of placebo, medication withdrawal, 
provocative testing) 
 
Participants will be asked to abstain from smoking and from consuming caffeinated 
beverages including coffee, tea, and soft drinks for at least 1 hour prior to testing. They 
will also be instructed not to drink alcohol on the night prior to testing and on the day of 
testing. To reduce any associated withdrawal symptoms, participants will not be asked to 
completely stop smoking or consuming caffeine for the entire duration of this study. 
 
Participants who are currently on certain types of psychotropic medications will be 
excluded from study participation (exceptions made for PTSD participants only). To 
reduce any associated withdrawal symptoms, patients will not be taken off medications 
for the purpose of the study 
 
Electric shocks are used as the stressor in this study. Our use of electric shocks stems 
from our experience that electric shocks are among the most efficient ways to induce 
anxiety in the laboratory setting. Because of this advantage along with the fact that such 

 
 
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shocks have been very well tolerated by 100’s of past participants in our 
psychophysiology experiments, we decided to again use electric shocks in our 
neuroimaging investigations described herein. 
 
 
e. Safeguards for vulnerable populations e.g. DPA, pregnancy testing, contraception 
use, ethics consult, HSPU involvement 
 
We assess pregnancy via urine test within 24 hours of any MRI session and exclude all 
participants who are pregnant. 
 
f. Informed consent procedure 
Informed consent for the study will be obtained in-person at the initial study visit or using 
NIH-approved Telehealth platforms prior to any diagnostic or medical procedure.  AIs 
listed on the KSP form are qualified to obtain consent for the studies.. 
 
All consent forms will be approved by the Institutional Review Boards (IRB) at the 
NIMH.  
 
 
Subjects can be invited back to participate in future experiments. If a subject is not 
contraindicated in any way (e.g., contraindication cases include: a patient who enters 
into treatment and thus is not eligible, or a subject who does not wish to participate in 
other aspects of the study), he or she will be invited back for additional testing. Subjects 
will undergo informed consent each time they participate in a different experiment in 
this study, and will be explicitly reminded that they are not required to participate in 
additional procedures. Further, all participants will be re-consented, even for repeat 
procedures, when more than two months have passed since they were last consented. 
Full SCIDs will be performed for all subjects annually; if a SCID was done within the 
year and the subject returns to participate in another study, then a credentialed staff 
clinician will spend time with them to do a brief assessment of current symptoms and 
functioning.  
 
The consent forms include a checklist which includes all procedures outlined in the 
protocol. Prior to consenting, all procedures subjects will experience will be marked so 
that the participant understands exactly what they will be doing. If a subject returns to do 
a different task within the same protocol, they will be re-consented with appropriate 
procedures marked 
 
 
 
 
 
14.  Consent Documents and Process: 
a. Who will obtain consent? 
 

 
 
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Study investigators designated as able to obtain consent are listed on the study 
personnel sheet..  All study investigators obtaining informed consent have completed 
the National Institute of Mental Health, Human Subjects Protection Unit "Elements 
of Successful Informed Consent” training. 
 
b. How will consent will be obtained if special procedure is needed? 
 
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 
before signing.  
 
The informed consent document will be provided as a physical or electronic 
document to the participant or consent designee as applicable for review prior to 
consenting. A designated study investigator will carefully explain the procedures and 
tests involved in this study, and the associated risks, discomfort and benefits. In order 
to minimize potential coercion, as much time as is needed to review the document 
will be given, including an opportunity to discuss it with friends, family members 
and/or other advisors, and to ask questions of any designated study investigator. A 
signed informed consent document will be obtained prior to any research activities 
taking place. 
 
The initial consent process as well as re-consent, when required, may take place in 
person or remotely (e.g., via telephone or other NIH approved remote platforms used 
in compliance with policy, including HRPP Policy 303) per discretion of the 
designated study investigator and with the agreement of the participant/consent 
designee(s). Whether in person or remote, the privacy of the subject will be 
maintained. Consenting investigators (and participant when in person) will be 
located in a private area (e.g., clinic consult room). When consent is conducted 
remotely, the participant will be informed of the private nature of the discussion and 
will be encouraged to relocate to a more private setting if needed. If the consent 
process is occurring remotely, participants and investigators will view individual 
copies of the approved consent document on screens at their respective locations; the 
same screen may be used when both the investigator and the participant are co-
located but this is not required.  
 
Note: When required, the witness signature will be obtained similarly as described 
for the investigator and participant below. 
 
Consent will be documented with required signatures on the physical document 
(which includes the printout of an electronic document sent to the participant) or on 
the electronic document. The process for documenting signatures on an electronic 
document is described below. 
 

 
 
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When a hand signature on an electronic document is used for the documentation of 
consent, this study will use the following electronic platform to obtain the required 
signatures: 
• 
iMedConsent platform (which is 21 CFR Part 11 compliant) 
 
Both the investigator and the participant will sign the electronic document using a finger, 
stylus or mouse. Electronic signatures (i.e., the “signature” and a timestamp are digitally 
generated) will not be used. 
 
 
c. If special documents are needed (minor assent, Braille, another language, etc). 
 
N/A 
 
15. Data and Safety Monitoring: 
a. Monitoring plan for the study as a whole 
 
Data and Safety Monitoring Plan – Independent Monitor 
This protocol will have an independent safety monitor, Dr. Joyce Chung.  She has no 
involvement in protocol implementation or investment in the outcome of the study.  The 
PI will provide, every four months, study data including enrollment, study progress, and 
outcome data to the independent safety monitor. The PI will report serious, unexpected 
adverse events and deaths related to the protocol's experimental procedures to the 
independent study monitor at the same time as they are reported to the IRB, the IC 
Clinical Director and other NIH officials. 
  
The independent monitor will provide the PI with a summary report of findings, 
including recommendations for continuation or topping of the study.  The PI submits this 
report to the Office of the Clinical Director and the IRB after each review occurs.   
 
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. Data and safety issues are reviewed at biweekly laboratory 
meetings with all research and clinical staff. 
 
c. 
Criteria for stopping the study or suspending enrollment or procedures:  
Study enrollment will be stopped or suspended for any potentially related serious adverse 
event, until the Principal Investigator, Clinical Director, the Independent Study Monitor, 
and the IRB determine there is no risk to continue. 
 
16. Quality Assurance: 
Quality assurance monitor 

 
 
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1.Quality assurance will be monitored by the PI and research team and the NIMH 
Office of Regulatory Compliance (ORO).  
 
 
Quality assurance plan 
2. 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. 
 
17.  Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations: 
a. Reporting/ non reporting of expected AEs 
 
Reportable events for this protocol will be tracked and reported in compliance with 
Policy 801. 
18. Alternatives to Participation: 
a. Treatment/ therapeutic alternatives should be listed. If none, so state. 
 
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: 
 
Information will be stored using a confidential case number (subject research code 
number), and no identifiers (name, address, phone number, etc.) will be placed on 
data that could allow direct linking of database information to individual subjects.  
Additionally, data will be kept in password-protected computers.  Samples will be 
kept in locked storage. Only study investigators will have access to the samples and 
data. 
 
Special precautions:  Confidentiality will be protected to the extent possible under 
existing regulations and laws, but cannot be guaranteed. De-identified results from 
this study will be posted on cctrials.gov. 
 
 
21. Conflict of Interest: 
 
a.  Distribution of NIH guidelines:  The NIH conflict of interest guidelines were 
distributed to all investigators. 
 

 
 
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b.  Conflict of interest:  There are no conflicts of interest to report for NIH investigators.  
Non-NIH investigators will abide by the conflict of interest policies of their own 
institutions. 
 
c.  Role of a commercial company:  There is no commercial company or sponsor for the 
study. 
 
22. Technology Transfer: 
 
A tech transfer agreement (2018-0101) with SmartMedTek LLC is in place between Dr. 
Grillon and Dr. Zeffiro.  
 
23. Research and Travel Compensation: 
 
In compliance with federal regulations, active duty service members or other federal 
employees who participate in this study while on duty may only be compensated for 
blood sample collection. They will be compensated at a rate of $50 for the single sample 
collection. Active duty participants will travel on invitational orders. Participants who are 
non-active duty/non-federal employees (or active duty/federal employees on leave with 
command approval) will be compensated for research-related discomforts and 
inconveniences in accordance with NIH guidelines. 
 
b. Amount of compensation (hourly rate, inconvenience units and maximum for 
study) 
 
Subjects will be given compensation for their participation in the study based on NIH 
standards for time devoted to research projects based on the following schedule. 
 
VOLUNTEER PAYMENT SCHEDULE PER TESTING SESSION 
 
 
Duration 
Inconveni- 
Total 
Total 
Procedure 
 
ence units 
units 
pay 
 
Testing (fMRI)  
OUTPATIENT Visit 
Time 
1.5-2 h 
1 
4 
$40 
FMRI scanning incl. shocks 90 min 
6 
6 
$130 
TOTAL 
 
 
 
$170 
 
Testing (MEG) 
OUTPATIENT Visit 
Time 
1-1.5 h 
1 
4 
$40 
MEG scanning incl. shocks 
1-1.5 h 
6 
6 
$60 
MRI scan 
                30-45 min 
 
 
$70 
TOTAL 
 
 
 
$170 
 

 
 
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Testing (Alprazolam) – Completed  
OUTPATIENT Visit 
Time 
5-6   h 
1 
20 
$200 
MEG scanning incl. shocks 
1-1.5 h 
6 
6 
$60 
MRI scan 
                30-45 min 
 
 
$70 
TOTAL (x2 MEG sessions) 
 
 
 
$590 
 
Additional compensation may be provided during the “Loss Aversion” testing session. 
This can amount up to an additional $50.00. Subjects may also be compensated an 
additional $20 if they complete an extra visit for Telehealth consent instead of in-
person consenting.  
 
c. Travel compensation (append travel form as attachment) 
  
No travel compensation will be given to participants in this protocol.  
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25. Attachments/ Appendices: 
 
An eligibility checklist, recruiting advertisement and screening questionnaire for Patient 
Recruitment Office has been attached to this protocol via PTMS. 
 
26. Consent Forms: 
 
This protocol includes three Healthy adult volunteer consents (one for fMRI studies and 
one for MEG studies,) and one Adult Patient consent form. Changes to these consents 
have been requested and subsequently addressed.