Cognitive vs. Emotional Psycho-Pharmacological Manipulations of Fear vs. Anxiety
Phase: PHASE4
Conditions: Anxiety Disorder
Sponsor: National Institute of Mental Health (NIMH)

 
 
CNS IRBProtocol Template (rev.1-17-08)  THIS IS THE WRONG VERSION 
 
IRB Panel: Blue    White 
Title page 
Cognitive vs. emotional psychopharmacological manipulations of fear 
vs. anxiety  
Protocol Number  
 
 
14-M-0114 
Date of This Submission/Version 
9/23/19/v.1 
 
Principal Investigator 
Ernst, M, MD 
PhD 
NIMH 
15k/118 
301 402-9355 
ernstm@mail.nih,gov 
 
Total requested accrual 
  0 Patients (separately describe patient groups)              
  
  300 Volunteers  
 
  
Project Uses Ionizing Radiation:   x No  
 Yes (attach RSC/RDSC documentation) 
  
 
 Medically-indicated only 
  
 
 Research-related only 
  
 
 Both 
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Drug/Device/#_____________________ 
  
Sponsor: _________________________ 
 
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Multi-institutional Project 
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 Yes  
 
 
 
 
 
Institution_________________    FWA #_________  
Date of IRB approval_________ (attach IRB documentation) 
 
Data and Safety Monitoring Board  
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 Yes  
 
Technology Transfer Agreement  
No   
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Agreement type and number: Human DTA 2019-0418  
Expiration Date: 09/2022 
 
Confidential Disclosure Agreement     
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Samples are being stored  
 
 
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Flesch-Kincaid reading level of consent form: 
 Startle test consent 
8.9 
 Study consent  
8.9 
fMRI study consent 
8.6 
 

 
 
 
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• Précis  
 
Objective:  The overall aim of this protocol is to examine the effect of pharmacological 
manipulations of affective and cognitive processes on anxiety and task performance. 
Ultimately, the goal is 1) to provide insight into the relative influence of cognitive and 
affective states on anxiety, 2) generate theoretical models that can be applied to a better 
understanding of the interaction between cognition and emotion, 3) develop a better 
screening approach to candidate anxiolytics, and 4) help formulate novel therapeutic 
interventions for clinical anxiety.  
 
Excessive or inappropriately sustained anxiety and fear lead to the most common 
group of psychiatric disorders. A number of theoretical models have been proposed to 
understand the mechanisms engaged in these maladaptive behaviors. Most recent emphasis 
has focused on the synergistic contribution of cognitive and emotional processes. Our 
laboratory has been instrumental in delineating aspects of behavioral and neural processes 
that are associated with fear and anxiety, using psychophysiological and neuroimaging 
measures of fear and anxiety. Evidence shows that levels of anxiety modulate cognitive 
performance, such as working memory or perceptual discrimination, and that, conversely, 
cognitive engagement influences severity of experimentally induced anxiety. The exact 
contribution of emotional processes vs. cognitive processes to the experience of anxiety is 
not clear, similarly to the neural mechanisms underlying these interactions.  
 
In this protocol, we propose to manipulate pharmacologically separately cognitive 
and emotional processes to dissociate their contribution to fear/anxiety, while using state-of-
the-art measures of anxiety derived from translational work. Indeed, we already developed 
integrative experimental models of fear and anxiety via the manipulation of predictable and 
unpredictable shock, respectively. We already employed successfully these models to 
measure anxiolytic and anxiogenic effects of various compounds such as alprazolam, 
citalopram, hydrocortisone, and oxytocin in healthy participants.  
 
In the initial version of this Protocol, we proposed in a first step (step-1) to start with 
a simple proof-of-concept study, using two pharmacological compounds in a double-blind 
randomized parallel design, each preferentially acting respectively on the cognitive (20 mg, 
methylphenidate) or affective (propranolol) domain, and using a single cognitive process 
(working memory). In a second step (step-2), we proposed to extend this work to the fMRI to 
examine the cognitive correlates of the effects seen in the step-1 behavioral study, 
specifically with 20 mg methylphenidate. Whereas the comparison among three drugs was 
planned for the electrophysiology study, we planned to study only the drug that improves 
cognition in the fMRI. The reason we focused on methylphenidate in step 2 was that our 
overall goal was to study the effect of improving cognitive functions on anxiety using 
neuroimaging. To reach this goal, we planned to use different approaches to boost cognitive 
functions in the coming years, including psychopharmacology, direct current stimulation, 
mindfulness. Methylphenidate was our first psychopharmacological study towards this 
objective. Future work will also expand to other compounds and cognitive processes, as well 
as vary the strategy to induce anxiety. In the initial study, anxiety was induced using the 
threat of shock, while participants performed the task. We examined in step-1 whether 1) the 

 
 
 
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reduction of induced-anxiety with propranolol improved cognitive performance, and 2) the 
facilitation of cognitive performance with methylphenidate reduced induced-anxiety. In step-
2, we proposed to identify the neural mechanisms underlying the effects of methylphenidate, 
the drug having beneficial effects on cognitive function. 
 
The results of the step-1 and step-2 studies were in the expected direction but did not 
reach statistical significance. We therefore decided to conduct similar studies but with a 
higher dose of methylphenidate, 60 mg instead of 20 mg. We initially submitted an 
amendment (AMD 10) that was accepted by IRB to use 60 mg MPH only in the fMRI study 
(Step-2). However, we now have decided to also use 60 mg MPH in the behavioral study 
(Step-1).  The initial version of the behavioral study used 3 treatments, placebo, MPH, and 
propranolol. We will drop the propranolol treatment because we did not find any significant 
effect of this treatment on anxiety, cognition, or the interaction of cognition and anxiety. The 
objective of the study remains the same. 
 
 
Study population: Medically and psychiatrically healthy adult males and females, aged 
18 to 50 years. 
 
Design: The study is a double-blind design. For each of the two studies (i.e., step-1/ 
behavioral and step-2/fMRI), two groups of healthy participants will come for one 
experimental session, following a screening session. During this session, they will be 
asked to perform a working memory task under the threat of shock, i.e., while 
anticipating unpleasant electric shocks. Each group will receive one drug challenge, 
either placebo or methylphenidate (60 mg).  
 
Outcome measures: In step-1, the primary outcome measures are the startle reflex and 
performance on the working memory task. In step-2, the primary outcome measures are 
the startle reflex and the cerebral fMRI blood-oxygen-level dependent (BOLD) 
responses. For both step-1 and step-2, secondary measures include skin conductance, 
heart rate, and subjective measures of anxiety. 
. 
 
 
 
 
 
 
 

 
 
 
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Table of Contents  
Title page .................................................................................................................... 1 
1. 
Introduction ........................................................................................................ 5 
a. 
Interaction between Anxiety and Cognition ................................................................5 
b. 
Effects of Anxiety on cognitive performance ...............................................................6 
c. 
Effects of Cognition on Anxiety (Step-1 and Step-2).....................................................6 
d. 
Previous work: Effect of induced-anxiety on n-back WM tasks ....................................7 
2. 
Study objectives ................................................................................................ 14 
3. 
Subjects ............................................................................................................ 15 
4. 
Study Design and Methods ................................................................................ 16 
6. 
Additional Considerations ................................................................................. 24 
7. 
Risks/ discomforts ............................................................................................. 25 
8. 
Subject safety monitoring .................................................................................. 27 
9. 
Outcome measures ........................................................................................... 28 
10. 
Statistical Analysis ......................................................................................... 28 
11. 
Human Subjects Protection ............................................................................ 29 
12. 
Consent documents and process .................................................................... 30 
13. 
Data and Safety monitoring ........................................................................... 31 
14. 
Quality Assurance .......................................................................................... 32 
15. 
Adverse event and unanticipated problem reporting...................................... 32 
16. 
Alternatives to participation .......................................................................... 32 
17. 
Privacy ........................................................................................................... 32 
20. Confidentiality .................................................................................................... 32 
21. Conflict of Interest .............................................................................................. 32 
22. Technology Transfer ............................................................................................ 33 
23. Research and Travel Compensation ..................................................................... 33 
24. References .......................................................................................................... 34 
 
List of Abbreviations: 
dB 
Decibel 
DSM Diagnostic and Statistical Manual of Mental Disorders  
ECG Electrocardiogram 
MPH Methylphenidate 
PLA Placebo 
PRO Propranolol  

 
 
 
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SBP 
Systolic blood pressure 
WM 
Working memory 
 
1. Introduction  
 
a. Interaction between Anxiety and Cognition 
 
A large literature documents the influence of anxiety on cognition (Eysenck et al., 2007), 
and conversely the influence of cognition on anxiety (McRae et al., 2009; Van Dillen and 
Koole, 2007), the latter being exploited for the treatment of anxiety disorders (Clark and 
Beck, 2010). This interaction suggests that anxiety disorders include pathology in 
functional networks that mediate emotion and cognition (Sylvester et al., 2012). Indeed, 
some of the prominent cognitive problems of anxiety are mediated by impaired cognitive 
function, independent from disruption in affective processes. Anxious individuals 
complain of being easily distracted and of having difficulty concentrating, while 
population-based studies report impairments in executive functioning and episodic 
memory across anxiety disorders (Airaksinen et al., 2005). Consequently, pathological 
anxiety may not only reflect primary overactive emotional reactivity but also independent 
cognitive impairment. For example, attentional bias for threat and increased distractibility 
associated with anxiety could reflect weak top-down attentional control rather than 
increased bottom-up influence.  
 
It is well-established that anxiety can impair cognition, and that cognitive strategies can 
be used to reduce anxiety.  For example, one effective way to down-regulate anxiety is to 
immerse oneself in a cognitive task (Vytal et al., 2012). Tasks that engage working 
memory appear to be especially efficient at reducing anxiety, probably because of 
competition for attentional resources (Vytal et al., 2012). However, the underlying 
mechanisms that mediate the interaction between anxiety and cognition are unclear. This 
protocol aimed at clarifying this question using a pharmacological approach.  
  
As a first step, we will use a threat induction method combined with a working memory 
(WM) task to test the interaction of anxiety state with WM in healthy adults. Specifically, 
we propose to employ our well-established, translational, within-subjects state anxiety 
induction method (induced-anxiety) to collect a physiological measure of anxiety 
intensity (Davis et al., 2010; Grillon, 2008). This method consists of the induction of 
anxiety through the application of unpredictable electrical shocks, and the quantification 
of anxiety level by assessing the startle blink reflex measured using EMG methodology. 
We also select a WM task, which we have shown to be reliably impaired by induced-
anxiety (Vytal et al., 2012; Vytal et al., 2013).  
 
To identify factors that influence the relation between anxiety and cognition, we will 
attempt to separately manipulate anxiety and cognition using pharmacological agents that 
modulate preferentially cognitive or affective processes. We will use methylphenidate 
(MPH) for modulating cognition, and propranolol (PRO) for modulating physiological 
arousal. This is a proof-of-concept study that tests two hypotheses: 
1) A treatment, which improves cognitive functioning, can also reduce anxiety. 

 
 
 
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2) A treatment, which reduces anxiety symptoms, can also improve cognition. 
b. Effects of Anxiety on cognitive performance 
 
Several studies have shown that anxiety impairs performance on tasks of executive 
function and working memory (Vytal et al., 2012; Vytal et al., 2013). A general account 
of the detrimental effect of anxiety on cognitive performance is competition for resources 
(Eysenck et al., 2007). Specifically, the performance on a cognitive task, together with 
the experience of being anxious, is equivalent to being engaged in a dual task, i.e., 
anxiety-related processes act as a secondary task that take up resources away from the 
primary cognitive task. This protocol seeks to better understand the nature of the anxiety-
related processes responsible for interfering with cognitive performance.  
 
It is well-established that anxiety is not a homogeneous process. At least two components 
of anxiety have been traditionally recognized one is cognitive and the other is 
physiological. The cognitive component, also termed anxious apprehension, involves 
worry and anxiety-related thoughts. The physiological component, also termed anxious 
arousal, includes interoceptive feeling state, tension, and nervousness. So far, the 
cognitive component has been assumed to play a key role because worries and anxious 
thoughts are believed to take up working memory resources (Eysenck et al., 2007). 
However, it is also likely that cognitive performance is also affected by the distracting 
influence of anxious arousal. Indeed, drugs that reduce anxious arousal, such as the beta-
blocker PRO, are used to treat performance or test anxiety (i.e., public speaking, stage 
fright) (see below).  
 
According to this bimodal model, the manipulation of either the cognitive component or 
the physiological component of anxiety could reduce interference of anxiety on ongoing 
activity (i.e., by reducing anxiety or improving attention). In this protocol we will 
examine whether psychopharmacologically reducing the physiological component of 
anxiety improves cognitive performance. We hypothesize that pharmacological blockade 
of physiological signs of anxiety with the beta-blocker PRO will reduce the detrimental 
effect of anxiety on WM performance. Since it is not possible to rule out potential direct 
cognitive effects of PRO, we adopt a design that will permit to measure such potential 
effects and control for them statistically using ANOVAs. Because of the specific effect of 
PRO on test-anxiety, and the unclear mode of action of this drug, the Step-2 will not test 
this drug in the fMRI. 
 
c. Effects of Cognition on Anxiety (Step-1 and Step-2) 
 
The effects of cognition on anxiety generate substantial interest because of their potential 
therapeutic benefits (Clark and Beck, 2010; Robinson et al., 2013). Several studies have 
shown that anxiety and threat-related physiological effects can be down-regulated by the 
use of various cognitive strategies such as self-distraction, reappraisal, or WM load 
(Dvorak-Bertsch et al., 2007; Kalisch et al., 2006; King and Schaefer, 2011). 
Specifically, this effect appears to have the greatest impact when the task involves high 
cognitive (King and Schaefer, 2011) or perceptual load (Doallo et al., 2006). Moreover, 
therapeutic techniques based on this assumption (e.g., cognitive behavioral therapy, CBT) 

 
 
 
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are highly effective in reducing anxiety in patient populations (Chambless and Gillis, 
1993). These findings suggest that when attentional demands are high, task performance 
takes precedence over anxiety-related processes (e.g., worry, interference from 
physiological arousal). Therefore, engaging in a WM task can successfully relieve 
anxiety. However, there is large individual variability in the ability to engage task-related 
WM processes when anxious. Specifically, attention control mechanisms play a key role 
in the ability to stay on task and screen out anxiety-related processes. In fact, poor 
attention control has been proposed to be a vulnerability factor for anxiety disorders 
(Bishop, 2009). This raises the possibility that improving attention-control 
pharmacologically could not only minimize the detrimental effect of anxiety on 
performance, but could also minimize attention to anxiety-related processes. This leads to 
the hypothesis that MPH may help alleviate anxiety during task performance under 
threat. Since it is not possible to rule out potential direct anxiolytic effects of MPH (see 
section Page 10, MPH and anxiety), we adopt a design that will permit to measure such 
potential effects and control for them statistically using ANOVAs. Because of the clear 
effects of MPH on cognition in general, and the well-known mechanism of action of this 
drug, we plan to test it in the fMRI in our Step-2 study. 
 
 
d. Previous work: Effect of induced-anxiety on n-back WM tasks 
 
Working memory refers to a temporary storage system that can be used to encode, 
rehearse and manipulate information in mind (Postle, 2006). One of the most commonly 
used WM tasks is the n-back paradigm (where subjects respond to successive stimuli 
based on whether they match the stimulus 1, 2, or 3 trials back etc.), because cognitive 
load or task difficulty can be parametrically modulated. Findings regarding the effects of 
anxiety on WM consistently report performance impairment (Eysenck et al., 2007). 
However, little is known about the effect of performing a WM task on anxiety. 
 
We have examined the effect of induced-anxiety evoked by threat of shock on n-back 
WM in several studies (Vytal et al., 2012; Vytal et al., 2013). Anxiety was probed by 
examining startle reactivity to brief and loud sounds. Startle is a well-established 
translational measure of anxiety and is reliably increased (fear-potentiated startle) by 
anticipation of aversive stimuli such as shock (Davis et al., 2010).  
 
We found that induced-anxiety impaired performance in the low load WM tasks (1- and 
2-back) but not in the high load WM task (3-back) (Fig. 1). In addition, as expected, 
induced-anxiety increased the magnitude of startle, a phenomenon termed fear-
potentiated startle. Most notably, the fear-potentiated startle was reduced linearly as WM 
load increased (Fig. 2). In other words, as task difficulty increased, anxiety decreased.  
These findings are consistent with the hypothesis that under low load, task performance 
does not take up all processing resources. Remaining resources are then available to 
process threat-related stimuli, including worry and physiological arousal. However, when 
these resources are depleted by high-load working memory tasks, anxious apprehension 
and anxious arousal has no workspace with which to operate, and their effects on 
performance are eliminated. 

 
 
 
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These results illustrate the interaction between anxiety and cognition and provide support 
for the two following predictions regarding the effect of PRO and MPH.  
 
• First, one would expect that reducing anxious arousal with PRO should reduce 
fear-potentiated startle during induced-anxiety regardless of whether subjects are 
involved in a task or not. Importantly, reducing fear-potentiated startle (i.e., 
reducing anxiety) during induced-anxiety should also improve WM performance.   
• Second, improving attention control with MPH during induced-anxiety should 
help subjects focus on the task and ignore threat-related processes.  This should 
reduce anxiety (fear-potentiated startle) during task performance and minimize 
the detrimental effect of anxiety on performance. 
 
 
 
Fig. 1: N-back performance during threat and safe: performance was impaired during 
threat vs. safe in low-load task, but not in the high load task (Vytal et al., 2012 ). 
 
 

 
 
 
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Fig. 2: Fear-potentiated startle (i.e., difference in startle reactivity between the threat and 
safe conditions) during the view condition (no task) and during the n-back task (Vytal et 
al., 2012) 
Effects of MPH on Cognition and Anxiety 
 
Methylphenidate primarily acts by blocking the dopamine transporter (DAT), which 
removes excessive dopamine from the synaptic cleft, resulting in increases of 
extracellular dopamine levels (Volkow et al., 2002). It also blocks norepinephrine 
transporter (NET) but is viewed more generally as an indirect dopamine agonist. 
It is the first‐line medication treatment of attention deficit hyperactivity disorder 
(ADHD). In this clinical context, MPH decreases symptoms of inattention and 
hyperactivity (Faraone and Buitelaar, 2010). 
 
MPH on Working Memory: 
As reviewed below, most of the experimental and clinical evidence supports a beneficial 
effect of MPH on working memory. 
Generally, MPH has been shown to enhance the efficiency of cognitive processing 
(Swanson et al., 2011; Tomasi et al., 2011; Volkow et al., 2008). A large body of work 
evidences MPH positive effects on attention tasks (see review (Swanson et al., 2011). 
The literature on the effects of single dose MPH  on working memory is scarcer and 
mostly involved treatment of children and adolescents with ADHD. However, findings 
tend to evidence improved performance (see reviews (Swanson et al., 2011). For 
example, MPH single dose (for children >25kg, 3 single MPH doses 10 mg, 15 mg, and 
20 mg) improved auditory–verbal WM. Of note, this study was conducted in ADHD 
children with and without anxiety. MPH-related WM performance improvement was not 
seen in the comorbid ADHD-anxiety group (Bedard and Tannock, 2008). A similar 
finding was reported in ADHD children with and without anxiety receiving single MPH 
doses of 0.3, 0.6 and 0.9 mg/kg (Tannock et al., 1995). Because of the dearth of studies 
examining the MPH effects on WM in clinical anxiety, or even high anxiety state, it is 
difficult to interpret the role of comorbid anxiety in ADHD children as decreasing the 
efficacy of stimulants on WM improvement. One study in healthy adults reported that a 
single 40mg MPH dose improved performance on a visual–spatial WM task, particularly 
in those adults with lower baseline WM performance (Mehta et al., 2000a). Taken 
together, these data suggest a beneficial effect of MPH on working memory, but this 
effect may be dependent on individual factors, including baseline WM capacity and 
clinical anxiety. 
 
MPH on Anxiety: 
As reviewed below, there is a dearth of studies of the effect of MPH on anxiety. The few 
studies reviewed below suggest that MPH might have some anti-anxiety effects. 
However, these studies in humans are weak and are confounded by the ADHD status of 
the participants. From a clinical perspective, MPH is not used to treat anxiety, and there 
is no consensus that MPH would influence anxiety in patients with ADHD who are 
treated with MPH. Here, we will be able to test the direct effect of MPH on anxiety using 
the fear potentiated startle measure in the absence of WM. 

 
 
 
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Animal models suggest that MPH at 3mg/kg may have anti-anxiety effects (Zhu et al., 
2010). Similarly, MPH seems to reduce state anxiety in ADHD patients clinically treated 
with MPH (e.g., (Barrickman et al., 1995; Bouffard et al., 2003). However, this effect 
may be secondary to improved cognition. To our knowledge, no human studies have 
examined MPH single doses on anxiety, not only clinical anxiety, but also anxiety state. 
Accordingly, no studies have assessed MPH effects on startle reflex or fear-potentiated 
startle reflex. The closest works concern pre-pulse inhibition or exposure to positive and 
negative emotionally evocative distractors. The findings are confounded by the clinical 
population (ADHD) and the use of emotionally salient distractors (Ashare et al., 2010). 
The latest study reported normalization of startle response to these evocative stimuli after 
MPH treatment in ADHD adults. 
Based on this literature review, we predict that single MPH dose compared to placebo 
will improve WM memory performance in a safe condition, but even more so in the 
induced-anxiety condition by reducing attention to the threat context in favor of 
performance on the task (Fig. 3). In addition, this will lead to reduced anxiety state 
during the threat context in the MPH vs. placebo condition (Fig. 4).  
 
 
 
Fig. 3: Predicted effects of MPH on WM (arbitrary units, au) working memory 
performance compared to placebo in a safe and threat context (y axis represents WM 
accuracy, higher number representing better performance). MPH is expected to reduce 
the detrimental effect of induced-anxiety on performance.  (Difference is the difference 
score threat minus safe). 
 
0
2
4
6
8
10
12
14
PLC
MPH
WM performance (au)
Safe
Threat
Difference

 
 
 
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Fig. 4: Predicted effects of MPH on fear-potentiated startle (difference score threat minus 
safe) compared to placebo in the absence of task and during the low load WM task. 
Performing the task is expected to reduce the magnitude of fear-potentiated startle. MPH 
is expected to 1) have no or minimal effect on startle reactivity or fear-potentiated startle 
in the no task condition but 2) reduce fear-potentiated startle in the WM task condition.  
 
Effects of PRO on Cognition and Anxiety 
Propranolol hydrochloride (PRO) is a synthetic beta-adrenergic receptor-blocking that 
crosses the blood-brain barrier. It is primarily used for its peripheral effects in the 
treatment of hypertension, cardiac arrhythmia, essential tremor and migraine (Gleiter and 
Deckert 1996 pharmacopsychiat). It is also a first line of treatment of stage fright (James 
et al., 1978; Neftel et al., 1982), test anxiety (Faigel, 1991) and post-traumatic stress 
disorder (Pitman et al., 2002; Reist et al., 2001; Taylor and Cahill, 2002). Whether PRO 
also improves cognitive performance in tandem with reducing stage fright is unknown 
(e.g., (Beversdorf et al., 2002)). 
 
PRO on WM: 
Overall, the review below indicates the absence of or the potential for weak negative 
effects of PRO on WM. We will be able to test this cognitive effect by assessing the 
effect of PRO on WM in the safe condition. 
A few studies of acute administration of single doses of PRO suggest impaired memory 
performance in healthy adults. A 25mg PRO single dose administered to young healthy 
volunteers (16 subjects per drug condition) has been shown to slow reaction time in a 
numerical working memory task (Müller et al., 2005). This was particularly true for 
individuals with low state anxiety rating at baseline. The trend for a preferential slowing 
of manipulation times in less anxious subjects by PRO is in accordance with an inverted 
U-model suggested for performance on working memory (Kimberg et al., 1997; Mattay 
et al., 2000) and task-related brain activity (Mattay et al., 2003). It can be assumed that 
0
50
100
150
200
PLC/no task MPH/no task
PLC/task
MPH/task
Startle magnitude (microV)
Safe
Threat
Fear-potentiated startle

 
 
 
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less anxious subjects have lower endogenous noradrenaline levels and only this group’s 
performance will fall under the left threshold after PRO, whereas the more anxious 
subject have higher noradrenaline levels and their performance stays within the normal 
range, even after PRO treatment. Consistent with the results of Muller et al’s study, PRO 
affected cognitive functions positively in high-anxiety subjects and negatively in low-
anxiety subjects, when subjects were required to give a brief speech (Hartley et al., 1983).  
 
For completeness, a body of literature describes the influence of PRO on the memory of 
emotional stimuli, the most consistent effect being the blockage of the memory advantage 
conferred by the emotional load associated with stimuli (Cahill et al., 1994; Reist et al., 
2001). Conversely, PRO fails to show any effect on memory of neutral material. This 
selective negative effect on memory for emotionally-laden stimuli has been ascribed to 
the blockade of the peripheral somatovegetative feedback to the CNS, as described by the 
‘somatic-marker hypothesis’ (Bechara et al., 2005). However, PRO’s central effects on 
beta adrenergic receptors within the prefrontal cortex cannot be excluded.  
 
In summary, findings with acute PRO administration in humans at doses of 25 mg to 180 
mg report most consistently either no effects or negative effects on memory performance. 
No studies have yet examined how the blockade of somatovegetative responses to 
fear/anxiety affects working memory. However, PRO improves WM performance in the 
presence of negative distractors (unpleasant pictures), an effect that is partially mediated 
by cortisol and that has been attributed to a lessening of the arousal component of the 
emotional distractors (Oei et al., 2010). Based on the hypothesis of the “distracting” role 
of vegetative inputs and physiological arousal, we expect improved working memory 
during stress under PRO. 
 
PRO on anxiety: 
The studies reviewed below evidence the efficacy of PRO in reducing the physiological 
markers of anxiety (e.g,, during stage fright), and, in turn, mitigate the negative emotional 
response to threat situations or stimuli. There are no controlled studies showing increased 
anxiety under PRO. 
As already mentioned, PRO is currently used to treat test-anxiety (Faigel, 1991) and 
acute stage fright (Brantigan et al., 1979).  Propanolol reduces the physiological 
correlates of anxiety (i.e., anxious arousal), including startle EMG (Morgan et al., 1993), 
by blocking changes in anxiety-related autonomic reactivity. Accordingly, the beneficial 
effects on acute anxiety have been related mainly to PRO’s peripheral action that blocks 
somatovegetative changes associated with stress, i.e., palpitations, sweating, shortness of 
breath. However, it is not clear whether this blockade of anxiety-related physiological 
arousal is solely based on peripheral effects. Of note, PRO is much less efficient in 
affecting the cognitive component of anxiety (worry) (Gottschalk et al., 1974; 
Papadopoulos et al., 2010), perhaps suggesting that its main effects on anxiety are not 
centrally mediated. Accordingly, we expect reduced fear-potentiated startle and 
subjective response to threat under propanol. 
 
Based on this literature review, we predict that a PRO single dose  administration, 
compared to placebo, will not (or minimally) affect WM memory performance in a safe 

 
 
 
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condition, but will block or reduce the negative effect of threat in the induced-anxiety 
condition (Fig. 5). In addition, PRO, compared with PLA, will reduce startle in the threat 
condition (Fig. 6).   
 
 
 
Fig. 5: Predicted effects of PRO on working memory performance compared to placebo 
in a safe and threat context (y axis represents WM accuracy, higher number representing 
better performance). PRO is expected 1) to have no or minimal effect on performance in 
the safe condition, but 2) to reduce the detrimental effect of induced-anxiety in the threat 
condition.  (Difference is the difference score threat minus safe).  
 
 
 
 
0
2
4
6
8
10
12
PLC
PRO
WM performance (au)
Safe
Threat
Difference
0
50
100
150
200
PLC/no task PRO/no task
PLC/task
PRO/task
Startle magnitude (microV)
Safe
Threat
Fear-potentiated startle

 
 
 
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Fig. 6: Predicted effects of PRO on fear-potentiated startle (difference score threat minus 
safe) compared to placebo in the absence of task and during the low load WM task. 
Performing the task is expected to reduce the magnitude of fear-potentiated startle. PRO 
is expected to reduce fear-potentiated startle in the no task and the task conditions. 
 
The 40 mg single dose propranolol did not have any significant effects on either measures 
of anxiety or cognitive performance variables when compared to placebo in step-1 of this 
study. Given the lack of significant findings, the propranolol portion of the study is now 
complete and will not be included in the step-2 fMRI study. 
 
In step-2 fMRI, the single dose of methylphenidate 20 mg did not produce anticipated 
physiological effects (i.e. increased heart rate), nor changes in neural activity that 
survived correction for multiple-comparisons. A recent study by Schmidt et al. noted that 
methyplphenidate (60 mg) enhanced cognitive performance more than other tested 
stimulants (Schmidt et al 2017). Thus, a follow-up substudy to the step-2 fMRI will test 
neural activity on a higher dose (60mg). The follow-up study will continue to test the 
initial aims and hypotheses of the step-2 fMRI study.  
 
Results of the 1st version of the Protocol with 20 mg MPH 
 
Results of the behavioral study have been published (Ernst, M., et al. 2016). "The effects 
of methylphenidate and propranolol on the interplay between induced-anxiety and 
working memory." Psychopharmacol 233: 3565-3574.). Findings were twofold: (1) 
Methylphenidate blocked anxiety interference only on the 3-backWM performance, while 
propranolol and placebo had no effects on anxiety-WM interference, and (2) contrary to 
hypothesis methylphenidate did not reduce anxiety-potentiated startle. The fMRI data are 
still being analyzed but there is no clear effect. 
 
 
 
 
2. Study objectives 
 
The objectives of the study are to examine the effects of single dose administration of 
MPH on (1) induced-anxiety (measured with the startle reflex) evoked by the threat of 
shock, (2) performance on working memory, and (3) the cerebral fMRI blood-oxygen-
level dependent (BOLD) responses.   
 
We will test the hypotheses (see also Figures above) that:  
• Methylphenidate 
o Prevents or minimizes the impairment in WM caused by the threat of 
shock 
o Does not affect anxiety evoked by the threat of shock per se (threat of 
shock at rest), but enhances the anxiety-reducing effect of performing a 
task (threat of shock while performing a WM task) 
 

 
 
 
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3. 
Subjects 
a) Description of study population: 
The subjects will be healthy adult male and female volunteers ages 18-50. There 
will be 2 groups, MPH, and placebo (PLA) for step 1 behavioral study and step-2 
fMRI study. The target number of completers for the step-1 behavioral  study will 
be N=28 per group for a total of 56 completers. Subjects who drop out will be 
replaced. We anticipate that about 15%-20% of subjects (N=10) will either drop 
out from the study or will have poor data due to low startle reactivity or problem 
with the recording of the data. The total accrual number for step-1 will be 66 (see 
power analysis). 
 
For step-2, there will be two groups: MPH and PLA. The target number of 
completers for the fMRI study will be N=30 per group for a total of 60 
completers. Subjects who drop out will be replaced. We anticipate that about 
25%-30% of subjects (N=15) will either drop out from the study or will have poor 
data due to low startle reactivity or problem with the recording of the data. As a 
result the total accrual number for step-2 will be  75 subjects (see power analysis). 
The total accrual for the follow-up step-2 fMRI study will also be 75 subjects with 
a target of 60 completers (n = 30 per group). 
 
The accrual ceiling will be 300 healthy volunteers.   
 
NIH, but not NIMH, employees may participate. NIH employee participation is 
guided by intramural institute policy. 
 
b) Inclusion criteria 
• Ages 18-50 
• Males and females 
• Subjects give their own consent 
 
c) 
Exclusion criteria 
• Clinically significant prior exposure to medications, that based on the 
investigator’s judgment, may impact the study, such as Ritalin (MPH). 
• Any significant medical or neurological problems (e.g. cardiovascular illness, 
respiratory illness, neurologic illness, seizure, etc.) 
• Raynaud syndrome 
• IQ < 80 
• Sinus bradycardia (P<45), or tachycardia (P>90)  
• Significant ECG abnormality (i.e., greater than first-degree block etc.) as 
determined by investigators judgment 
• High or low blood pressure (SBP>140 or SBP<90; SDP<50 or SDP>90)  
• A first-degree family history of mania, schizophrenia, or other psychoses 
based on verbal reports 
• Significant past psychopathology (e.g., hospitalization for psychiatric 
disorders, recurrent depression, suicide attempt, psychoses) 

 
 
 
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• Current psychiatric disorders according to DSM-V 
• Current alcohol or substance use disorder  
• History of moderate or severe alcohol or substance use disorder 
• Current use of psychotropic medication 
• Impaired hearing 
• Pregnancy or positive pregnancy test 
• Neurological syndrome of the wrist (e.g., carpal tunnel syndrome) for shocks 
to be delivered on affected arm. 
• Breastfeeding 
• Significant lab abnormalities (i.e., CBC with differential, acute care and 
mineral panel, hepatic panel, thyroid panel) 
• Positive urine toxicology screen  
• You have been in another study with an experimental medication within the 
previous month 
• For physiological/clinic participants: Small startle reactivity (a change in 
EMG activity that is less than 3 times the baseline EMG activity) 
• Current use of anti-acid medications 
• Employee of NIMH or an immediate family member who is a NIMH 
employee. 
• For fMRI participants: Any medical condition that increases risk for fMRI: 
- Any metal implants (clips, screws, plates, pins, etc) or metal fragments 
cause by injuries or metal working 
- Any sort of medical implants (aneurysm clips, pacemaker, insulin pump, 
Hickman line, etc.) 
- Permanent eye liner and tattoos above the neck 
- Patients who have difficulty lying flat on their back for up to 90 min in the 
scanner 
- Participants who are uncomfortable in small closed spaces (have 
claustrophobia) and would feel uncomfortable in the MRI machine 
 
4. Study Design and Methods 
 
a) Study overview 
 
This protocol will examine the effect of PLA and MPH in step-1 psychophysiological 
study and step-2 fMRI study on WM during experimentally-induced anxiety in a double-
blind between-subject design. The study involves 1 visit lasting up to 6 hours. The 
increased dose of MPH follow-up psychophysiological and fMRI studies will be 
conducted similarly to the initial studies. 
 
First, we will measure individual startle responses for the psychophysiological study 
only. We do not use auditory startle as a threat condition for the fMRI study due to the 
noise of the fMRI scanner. Because there is a large inter-individual variability in startle 
response, and some individuals do not show reliable startle responses we will complete 
an auditory startle test prior to the study tasks. We need to make sure that we study only 

 
 
 
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individuals who show reliable startle response. Thus, the criterion to be eligible for the 
psychophysiological drug study is a measurable startle response for all 9 startles used 
during the startle test visit. A measurable startle response should be 3 times the baseline 
EMG activity.  
 If participants passed the startle test (all 9 measurable startle responses) they will move 
on to the drug portion of the visit.   
 
Participants will be assigned to one of two treatments, 1) PLA,  or 2) 60 mg MPH. For 
participants in the behavioral study, they will complete study tasks in the outpatient 
clinic. For participants in the fMRI substudies, they may be instructed to lay in the fMRI 
machine for up to 90 minutes while completing the study tasks. For both the behavioral 
substudies and fMRI substudies, the tasks will consist of performing a working memory 
task under safe conditions and under threat conditions (anticipation of shock). Startle will 
be used to examine subjects’ anxiety level.  
 
b) Recruitment methods 
 
All recruitment materials (paper or web ads, flyers and listserv announcements) will be 
IRB approved prior to use.  
o Advertisements will be placed in publications such as university newspapers, city 
newspapers or magazines and local gazettes. We will also post flyers on the NIH 
campus as well in community sites such as local eateries, small businesses, and 
public libraries.  
o Many universities are now paperless, and we may post web ads on their electronic 
newsletters.  We may advertise on local buses. We will use the Public Relations 
and Public Liaison (PRPL) list of volunteers. Lastly, recruitment efforts will 
include listservs provided by PRPL as appropriate for our target audience. An 
example is the Club PCR site which is part of the Recreation and Welfare (R&W) 
Association targeting young scientists on the NIH campus.    
o 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 also be made available at 
outreach exhibits, speaking engagements, and professional meetings with 
approval of the venue or in accord with their policy.  They may be given directly 
to those requesting study information.  
o Postcards may be sent using commercially-available mailing lists via direct mail. 
The postcards will identify the source of the mailing list.  
o 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. 
o We may also identify potential healthy volunteers through the NIMH protocol 
titled “Recruitment and Characterization of Research Volunteers for NIMH 
Intramural Studies.” 

 
 
 
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o 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. 
o There is no direct solicitation of employees/staff by supervisors nor co-workers.  
We understand that all recruitment materials, including those specific for NIH 
(e.g. for IRTA list-serv) must be IRB approved. 
 
c) Screening methods  
 
 
There are three steps before participants are able to consent to the drug study. 
1- Phone Screening: There will be an initial phone screening to explain the study and 
exclude subjects meeting exclusion criteria. Individuals who appear to qualify for 
inclusion will be invited for the second step, the Medical and Psychiatric Screening. 
2- Protocol 01-M-0254 screening (Medical and Psychiatric Screening): This screening 
comprises a psychiatric interview (SCID), a physical exam, questionnaires and computer 
tasks, all covered under protocol 01-M-0254.  Participants who are cleared to continue 
are invited to the third step, the Startle test. 
 
 
d) Drug Study design & procedure 
 
Subjects will participate in a single testing session in our laboratory in the Clinical 
Center. The scans will be performed in the NMR center. In step-1, they will be randomly 
assigned to either PLA or MPH (60 mg) to achieve 2 groups of 28 completers each (see 
section ii. Description of the various components). In step-2, they will be assigned to 
either PLA or MPH (60 mg), and each group will include 30 completers. In follow-up 
studies to the step-1 behavioral and step-2 fMRI, the subjects will take either PLA or 
60mg of MPH. Each group will include 30 completers for both follow-up studies. The 
design is double-blind, i.e., neither the participant nor the investigators will know what 
drug the subject is taking. 
 
At the start of the study visit for the follow-up behavioral study, each participant will 
have a startle test. The participants are tested 9 times with the startle procedure. They 
need to have a measurable startle for each of the 9 measurements to be continue onto the 
study itself. A measureable eyeblink response is defined as a change in EMG activity that 
is 3 times baseline EMG activity calculated as the average of the 20 ms that precedes the 
delivery of the startle stimulus. EMG values below this level will be considered as no 
startle response. The startle test will not be performed for the follow-up fMRI study.  
 
Participants will also receive electric shocks to try out the shocks. They will be given 3-5 
sample shocks to determine the level of shock for the experiments. 

 
 
 
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The timeline is presented first, and the various procedures are detailed afterwards. 
 
 
i. 
Study visit 
 
• Subject signs the study consent  
• Urine pregnancy for females  
• Completes questionnaires to qualify cognitive styles (i.e., attention control, 
BRIEF) 
• Self-administered questionnaires 
o Spielberger state anxiety #1 
o Mood Rating Scale #1 
• Blood pressure, heart rate, VAS #1 
• Placement of the recording electrodes 
• Assessment of baseline startle #1 
• Drug administration at time 0 min 
• Self-administered questionnaires (~80 min after ingestion) 
o Spielberger state anxiety #2 
o Mood Rating Scale #2 
• Blood pressure, heart rate, VAS #2 
• Assessment of baseline startle # 2 
• Threat of shock experiment (~90 min post drug administration) 
• Placement of shock electrodes 
• Shock work up procedure 
• Instructions 
• WM under shock threat / safety block 1 
• WM under shock threat / safety block 2 
• Blood pressure, heart rate and VAS #3 
• Questionnaires including a debriefing of the experiment 
• End of experiment; recording of side effects and release of subjects by a  
nurse or MD.  
 
 
 
 
ii. 
fMRI study visit 
 
• Subject signs the study consent  
• Urine pregnancy test for women  
• Completes questionnaires to qualify cognitive styles (i.e., attention control, 
BRIEF) 
• Self-administered questionnaires 
o Spielberger state anxiety #1 
o Mood Rating Scale #1 

 
 
 
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• Blood pressure, heart rate, VAS #1 
• Placement of the recording electrodes 
• Assessment of baseline startle #1 
• Drug administration at time 0 min 
• Self-administered questionnaires (~80 min after ingestion) 
o Spielberger state anxiety #2 
o Mood Rating Scale #2 
• Blood pressure, heart rate, VAS #2 
• Assessment of baseline startle # 2 
• Subject enters the fMRI machine 
• T12 Threat of shock experiment (~90 min post drug administration) 
o Placement of shock electrodes 
o Shock work up procedure 
o Instructions 
o Resting State task 
o WM under shock threat / safety block 1 
o WM under shock threat / safety block 2 
• Subject exits the fMRI machine 
• Blood pressure, heart rate and VAS #3 
• Questionnaires including a debriefing of the experiment 
• End of experiment; recording of side effects and release of subjects by a  nurse or 
MD. 
 
ii. 
Description of the various components 
 
Psychopharmacological challenges: 
 
This is a double-blind study. Neither the participant nor the investigators will know what 
drug the subject is taking. The blinding is done by the pharmacy. Both compounds will 
be placed in capsules of similar appearance. The NIH pharmacy will randomize the 
administration of drugs across subjects. 
 
 
Dose, Timing and Side Effects 
MPH 
A 60 mg oral dose is selected because of its documented effectiveness in improving 
cognitive efficiency in adults. This dose complies with the maximum FDA approved 
daily dose, and single dose MPH studies have used 60 mg without any serious adverse 
events (e.g., Schmidt et al 2017, Hysek et al 2014). 
 
A delay of 90 minutes between dose administration and cognitive testing will be used to 
optimize MPH effects, based on MPH pharmacokinetics. Peak plasma levels of 
methylphenidate are reached after 1 to 2 hours, and plasma half-life is 2 to 4 hours 
(Gualtieri et al., 1982).  
 

 
 
 
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The most common side effects associated with Ritalin are rapid heart rate, palpitations, 
nervousness, restlessness, insomnia, dry mouth, constipation, nausea, diarrhea, loss of 
appetite, headache, irritability, and elevation of blood pressure. MPH cardiovascular and 
subjective effects will be monitored regularly. Cardiovascular measures will include heart 
rate and blood pressure, which will be recorded prior to drug administration, and then 
every ½ hour. Subjective ratings will be collected prior to drug administration, prior to 
cognitive testing, and at the end of the study, and they will consist of ratings on visual 
analogue scales (Bond and Lader, 1974). 
 
 

 
 
 
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Design 
A between-group repeated measures design will be used. Subjects will be assigned 
randomly, in double blind to one of three drugs (PLA or MPH) in step-1 and in step-2. 
The NIH pharmacy will establish the random order within females and males.  
 
fMRI: The study may be done using Magnetic Resonance Imaging (MRI). MRI is 
painless and safe. Participants with any magnetic metal implants (such as pacemakers) 
will be excluded. The participant will lie on a table inside the scanner for up to 90 
minutes while doing the task. During the scan, the participant may be asked to lie still for 
up to 90 minutes at a time. While in the scanner the participant will hear loud knocking 
noises and will be fitted with earplugs or earmuffs to muffle the sound. The participant 
will be able to communicate with the MRI staff at all times during the scan and may ask 
to be moved out of the machine at any time. 
 
Resting State Procedure: In the fMRI scanner, subjects may be asked to lie still with 
their eyes open and stare at a fixation cross for 10 minutes. They will be asked to remain 
as still as possible and not fall asleep. 
 
 
Psychophysiological procedures and threat of shock 
• Startle stimulus & response 
The startle reflex will be elicited with a 102 dB white noise (40-ms duration) delivered 
via headphone. The eyeblink component of the startle reflex will be recorded binaurally 
with two AgCl electrodes placed under one eye. Eyeblink responses will be scored in the 
20-120 ms window following the onset of the startle stimulus. This is included in the 
behavioral study only. The auditory startle will not be done in the fMRI scanner.  
 
• Autonomic measures 
In addition to the acoustic startle reflex, we will also record the heart rate and the skin 
conductance for exploratory purposes. These measures will not add discomfort to the 
subjects. The heart rate will be monitored with two disposable electrodes on the ribcage 
midway between the waist and the armpit. The skin conductance will be measured using 
two (Ag-AgCl) electrodes in conjunction with a .05M NaCl electrolyte. Electrodes will 
be placed on the distal phalange of the index and second finger of the left hand.  
 
• Electric shock 
The shocks will be delivered through two disk electrodes located on fingers, arm, or 
wrist. The shock will have an intensity between 1.0-5 mA and duration of 100-300 ms. 
Shock will be delivered at a level that is judged by the subject as unpleasant but not 
painful. Study shock levels will be determined during participant screening prior to the 
first experimental session.In very rare occasions, subjects have experienced symptoms 
that may be related to the shock. A participant with a condition called “cubital tunnel 
syndrome,” a repetitive motion injury similar to carpal tunnel syndrome, indicated 

 
 
 
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worsening of his syndrome over the months subsequent to his participation. Another 
participant reported pain in her arms for several hours after testing. The pain was no 
longer present the next day. It is unclear whether these symptoms were due to the shocks. 
Nevertheless, subjects with neurological symptoms of the wrist and arms will be 
excluded from the study.  
 
The experiment will consist of alternating periods (e.g., 1-3 min) of safety and shock 
threat conditions during which subjects will perform the N-back WM task.  
 
N-Back WM task 
Stimuli are presented one at a time on a screen. Participants are instructed to remember 
one, two, or three stimuli back from the current stimulus on the screen as we have done in 
the past (Vytal et al., 2012).  
 
Questionnaires: 
We will use various questionnaires to assess the subjective effect of treatment on mood, 
anxiety, and sedation, and the perceived aversiveness of the shocks during testing using 
visual analog scales. These include State-Trait Anxiety Inventory (Spielberger, 1983) and 
the Mood Rating Scale (Bond and Lader, 1974). 
 
The non-analogue measures from these reports 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) for the purposes of this study.  Participants may 
enter their responses while at NIH using a wireless-device interface to access the NIH-
intranet secure CTDB. No new questionnaires will be used with CTSS and there are no 
actionable items with use of CTSS requiring real-time monitoring. 
 
e. End of participation 
Participants are discharged after an assessment and clinically cleared by a physician or 
nurse.  As we work only with healthy volunteers, there are no other follow up procedures 
barring adverse effects.  Adverse effects are followed by the medically responsible 
physician or nurse practitioner and medical care will be provided if needed.  Otherwise, 
the participant is discharged at that time.  
 
Individual research results are not shared with the participants.  Medical information will 
be shared with the primary provider upon request if there are abnormal findings upon 
physical exam or from clinical laboratory findings.  
 
5. Management of Data and Samples 
a. Storage 
Phone screenings with personal identifying information will be kept in a secure private 
office locked at all times.  When participants are admitted the phone screenings are 
placed into a binders and moved to a locked cabinet. 
 
All electronic records will be kept confidential to the extent permitted by law.  
Participants’ names and other personal identifying information will be stored in 

 
 
 
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electronically secured databases at the study sites.  These databases are password 
protected and only study personnel will be given a password.  Results will be published 
as group data without the use of characteristics that would identify individual subjects.   
 
b. 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. Consent for sharing was added with Amendment H.  Repositories receiving 
data and/or samples from this protocol may be open-access or restricted access.  
 
Data will be stripped of identifiers and may be coded (“de-identified”) or unlinked from 
an identifying code (“anonymized”). When coded data is shared, the key to the code will 
not be provided to collaborators, but will remain at NIH.  Data may be shared with 
investigators and institutions with an FWA or operating under the Declaration of Helsinki 
(DoH) and reported at the time of continuing review.  Sharing with investigators without 
an FWA or not operating under the DoH will be submitted for prospective IRB approval.  
Submissions to NIH-sponsored or supported databases and repositories will be reported 
at the time of Continuing Review.  Submission to non-NIH sponsored or supported 
databases and repositories will be submitted for prospective IRB approval. 
 
Required approvals from the collaborating institution will be obtained and materials will 
be shipped in accordance with NIH and federal regulations.    
 
 
 
6. Additional Considerations 
 
a. Research with investigational drugs or devices.   
Acoustic startle and shock device used in this protocol are considered non-significant risk 
(NSR) devices and will only be used within published guidelines.  
   
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.  
 
Methylphenidate: On 10/2/13, we received IND exemption for the drug’s use in this 
protocol.  
 
b. Gene Therapy. N/A 
 
7. Risks/ discomforts 
 

 
 
 
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a. Study medications 
 
Methylphenidate:  Adverse effects include non-specific side effects (e.g., stomach ache, 
headache, nausea, dizziness), as well as nervousness and insomnia. Decreased appetite is 
also common. Occasional palpitations, blood pressure and pulse changes (both up and 
down) have been reported. The 60mg dose in the step-2 follow up study is on the higher 
end of dosing. Subjects are more likely to experience cardiovascular side effects such as 
palpitations, tachycardia, and a rise in blood pressure than those getting lower doses.  
Sudden death has also been reported in chronic treatment with MPH, but a causal link 
with MPH has not been established. 
(http://www.fda.gov/drugs/drugsafety/ucm277770.htm). No sudden death has ever been 
reported after a single dose of MPH. 
b. Testing: 
 
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 procedures rely on the same physical properties of brain tissue.  
Potential risk of heart rhythm disturbances exists for patients with a history of heart 
rhythm abnormalities or those who have certain types of pacemakers. A substantial risk 
to persons who have metallic objects inside their bodies exists, as the magnet in the 
scanner can cause these to move. Pregnant women should not undergo MRI because of 
the possible harmful effects to the fetus. fMRI scanner is very noisy for this reason 
participants wear ear plugs, or headphones that are designed to reduce the noise impact of 
the scanner. The enclose space of the scanner can be uncomfortable. Participants have 
access to a panic button at all times and can press this to stop the scan and be removed 
from the scanner. 
 
 
Psychophysiological recording: The psychophysiological measures that will be obtained 
are non-invasive, requiring the administration of no needles, drugs, or dyes. Little 
discomfort is expected. During electrode placement, the possibility of skin irritation from 
contact with the saline electrode paste exists. However, this is unlikely as the salt 
concentration of the paste is similar to that of human sweat. The risk is equivalent to that 
of an EEG recording. 
 
Auditory startle stimulus: The auditory stimuli that will be used in the startle studies are 
40-ms duration 102 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 US. The 
short duration (40 ms) of these sounds makes them safe (i.e., there is no danger of 
hearing impairment). 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. 
 

 
 
 
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Electric shock: The shocks will be delivered through two disk electrodes located on the 
median nerve, on the forearm, or on fingers. The shock will have an intensity of up to 5 
mA and duration of 50-100 ms, as in our published studies. The PI has extensive 
experience with shocks. The shock is generally described by subjects as anxiogenic and 
moderately painful. The mean rating of aversiveness on a scale of 1 (not painful at all) to 
10 (extremely painful) is about 5. Over 95% of subjects who experienced the shock chose 
to participate in the experiment.   
 
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. 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. 
 
c. Procedures to minimize risks 
MRI: The potential risks related to MRI will be minimized as follows: 1) Claustrophobia 
associated with MRI will be reduced by explaining the nature of the procedure in detail 
prior to subject enrollment; and 2) a possible history of any intraocular, intra aural, 
intracranial, or intrathoracic metal will exclude the subject from the study. Earplugs will 
be given to each subject to wear during the scan to minimize discomfort and prevent any 
adverse effects on hearing resulting from the scanning procedure. Females of 
childbearing potential will undergo either serum or urine pregnancy testing to rule out 
pregnancy no more than 24 hours prior to each MRI session before the scanning 
procedures are initiated. During MRI scanning, the subject can communicate with the 
control room personnel via an intercom at the operating console. Thus, the subject can be 
removed immediately from the MRI scanner, if necessary. 
 
8. Subject safety monitoring 
 
The study timeline includes regular monitoring of blood pressure and heart rate as well as 
mental state. Subjects will be informed that they can withdraw from the study whenever 
they wish. Sample shocks will be administered prior to the study. At this point, subjects 
will be explicitly asked if they wish to continue. The experiment will also be stopped for 
any subject who exhibits signs of distress during any phase of the study. Subjects will be 
constantly monitored via closed circuit video by the AI. A credentialed staff member 
(RN, NP or MD) will assess the subject at the conclusion of the study or at any time if 
significant side effects develop.  The subject will be withdrawn at anytime if unable to 
follow the rules for participation in this study or fails to meet exclusion criteria.  

 
 
 
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9. Outcome measures 
 
The primary outcome measures include (1) anxiety measures:  amplitude of the startle 
reflex; (2) cognitive measures: accuracy and reaction time on the WM task. In the 
functional magnetic resonance imaging (fMRI) substudy, the primary outcome measure 
includes the event-related haemodynamic response related to neural activity in the brain.  
 
 
Secondary outcome measures include heart rate and skin conductance and responses to 
questionnaires.  
 
10. Statistical Analysis 
 
Analysis of the data: Startle and performance data will be analyzed with ANOVAs with 
repeated measures using Treatment (2 for step-1  ), condition (stress, no stress) and Load 
(low, high) as factors.   
 
This design will help us detect and control potential effects of PRO on cognitive function, 
and, conversely, of MPH on anxiety. Specifically, to measure Drug effects on cognitive 
function, we will use a repeated measures ANOVA with Treatment (MPH, PLA) as the 
between-subjects factor and condition (stress, no stress) and Load (low, high) as the 
within-subjects factors on WM accuracy. To examine Drug effects on anxiety, we will 
conduct a repeated measures ANOVA with  Treatment (MPH, PLA) and condition 
(stress, no stress) on startle EMG in the absence of task. This analysis will permit us to 
detect and control the effects of MPH on anxiety. For the fMRI analysis, only 2 drugs 
(MPH, PLA) will be analyzed following a similar approach as above. Specifically, to 
measure drug effects on anxiety, we will conduct a repeated measures ANOVA with 
treatment (MPH, PLA) and condition (threat, no threat) on startle EMG in the absence of 
task. No analyses will be conducted using both data from step-1 and data from step-2 
data. Step-1 and Step-2 studies yield independent results. 
 
Analysis of functional imaging data involves a series of initial steps including 
realignment (i.e., correction for head movement). Hypotheses regarding task-associated 
changes in the BOLD signal will initially be tested by analyzing task-related 
hemodynamic responses in five regions-of-interest (ROI) selected based on hypothesized 
areas of activation: the amygdala, the striatum, 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 10 regions (5 
regions in each hemisphere). 
 

 
 
 
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In addition, a voxel-wise analysis will be performed to investigate activations outside of 
these regions. 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. 
 
Hypotheses regarding the resting state study will be tested by analyzing the intrinsic 
connectivity of the networks involving the nodes (ROIs) examined in the task-based 
fMRI study. These nodes include the amygdala, striatum, dorsal anterior hippocampus, 
anterior insula, and anterior cingulate. The state of these networks at rest may inform 
their sensitivity to the pharmacological challenges and help predict their efficiency in 
mediating task performance. 
 
Power analysis:  
The main expected outcome measure is a decreased in the anxiety-potentiated startle 
following methylphenidate. Thus, we base the sample size calculation on the power 
analysis for obtaining an effect size sufficiently large to conclude to a significant 
difference between groups of the anxiety-potentiated startle among drug conditions.  
To run the power analysis, we are using data from our initial study, Ernst et al (2016)  . 
The mean and SD of anxiety-potentiated startle was 7 and 4.8 T-scores, respectively. We 
expect MPH to reduce anxiety-potentiated startle by about 50%, corresponding to an 
effect size (Cohen’s d) of .72. Given an effect size of 0.72 and a power of 0.8, a total of 
56 subjects or 28 per group will be needed to detect a significant difference in anxiety-
potentiated startle between the two treatment groups. Given that we expect approximately 
15-20% of subjects to have bad EMG recording, unanalyzable or small startle responses, 
or poor WM performance, we will recruit 68 subjects (34 per group).  
 
Regarding the fMRI study of Step-2, we request the same number of subjects as in our 
initial Protocol, i.e., 30 per group, for each of the drug group. Given that we expect 
approximately 15-20% of subjects to have bad recording or poor WM performance, we 
will recruit 70 subjects (35 per group).  
 
 
11. Human Subjects Protection 
 
This study will be conducted in healthy adult males and females.  
a) Subject Selection: Subject selection will be equitable and will include both men 
and women between the ages of 18-50 of diverse racial and ethnic backgrounds.  
As people get older the probability that they startle decreases. Because startle is 
our main independent measure it is important to test subjects who are likely to 
startle in order to collect analyzable data.  Based on our experience, we made the 
decision that 50 would be the upper age limit to ensure usable data and thus 
justify the more than minimal risk. 

 
 
 
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b) Justification for exclusion of children: Children are excluded because of the 
aversive nature of the tests and the use of drugs.  This is a more than minimal risk 
study with no prospect of direct benefit. 
c) Justification for exclusion of vulnerable subjects.  This study will include healthy 
volunteers only. All subjects must be able to provide their own consent. This 
study is above minimal risk and we do not want to enroll participants who do not 
understand the risk/benefit ratio of the study, particularly when there is no benefit 
to the participants. Vulnerable subjects will not be included because this is a more 
than minimal risk study, with no direct benefit.  
For this reason, we exclude people with IQ lower than 80.  We exclude and rule 
out pregnant women via a pregnancy test because of the unknown effects of the 
shocks and of the drugs on the developing fetus.  
We will exclude NIMH employees to prevent bias from previous or current 
professional relations.  
Protections for employees and staff participating in this study include 1) assuring 
that the participation or refusal to participate will have no effect, either beneficial 
or adverse, on the subject’s employment or position at the NIH, 2) giving 
employees and staff who are interested in participating the “NIH Information 
Sheet on Employee Research Participation” prior to obtaining consent, and 3) 
assuring that there will be no direct solicitation of employees or staff. This study 
collects sensitive information (e.g. drug and alcohol use, specific medical 
diagnoses). The PI will train study staff regarding obtaining and handling 
potentially sensitive and private information about co-workers through staff 
discussions and written branch/section procedures.  Information about sensitive 
information (e.g. drug and alcohol use, specific medical diagnoses) will be in the 
participant’s NIH medical record. 
 
d) Justification of sensitive procedures:  N/ 
e) Safeguards for vulnerable populations:  
Neither participation nor refusal to participate as a subject will have an effect, 
either beneficial or adverse, on the participant’s employment or position at NIH. 
The NIH Information Sheet on Employee Research Participation will be available 
to employees considering enrollment. 
 
12. Consent documents and process 
 
a) 
Designation of those obtaining consent 
Qualified staff for obtaining study consent only includes Dr. Monique Ernst 
and Emily Page, FNP. 
Consent procedures  
b) 
Informed screening consents for the study will be obtained in-person at the 
initial study visit, prior to any screening.  Informed study consent for the study 
will be obtained in-person after screening.  This will be conducted in a private 
room  
c) 
Consent documents 

 
 
 
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The consent form contains all required elements.  There is one consent form 
for the Step-1 clinic study and one for the fMRI study. 
 
13. Data and Safety monitoring 
 
a) Data and Safety monitor 
Data and safety will be monitored by the Principal Investigator. 
b)  Data and safety monitoring plan 
The principal investigator is responsible for participants in this protocol and will 
report all serious adverse events to the NIMH Clinical Director and the NIMH IRB 
within the guidelines set by the standards for clinical research within the NIH 
intramural research program. The PI will review data and safety parameters at least 
annually. The PI will document the data and safety review in the research records and 
at the time of continuing review. 
 
a)  Criteria for stopping the study or suspending enrollment 
The study will be stopped if there is any Serious Adverse Event related to the 
research.   
The PI and IRB will determine if changes are needed for the research to continue or if it 
will be closed.  
 

 
 
 
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14. Quality Assurance 
 
a. Quality Assurance Monitor 
Quality assurance will be monitored by the PI, the research team and the 
NIMH Office of Regulatory Compliance (ORO).    
 
b. Quality Assurance Plan 
ORO monitors intramural research studies to ensure compliance with GCP, 
organizational policies and regulations.  Audit frequency is determined by the ORO SOP 
based on the study level of risk.  Results of ORO audits are provided to the PI, The 
Clinical Director and the CNS IRB. This study will undergo audits at least once every 
three years and for cause. 
 
15. Adverse event and unanticipated problem reporting 
 
Reportable events for this protocol will be tracked and reported in compliance with 
Policy 801. 
 
16. Alternatives to participation 
 
The alternative to participating in this study is not to participate. 
 
17. Privacy 
 
All research activities will be conducted in as private a setting as possible.  
 
 
20. Confidentiality 
 
a. For research data 
We will actively protect confidentiality of the subjects and the data in each step. 
Information will be stored using a confidential case number, and no identifiers (name, 
address, etc.) will be used that could allow direct linking of database information to 
individual subjects. Only study investigators will have access to the data. 
b. For stored samples 
N/A 
c. Special Precautions:  N/A 
 
21. Conflict of Interest 
 
a) Distribution of NIH Guidelines 
NIH guidelines on conflict of interest have been distributed to all investigators.  
b) Conflict of Interest 
There are no conflicts-of-interest to report. 
 

 
 
 
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22. Technology Transfer 
 
A tech transfer agreement (2019-0418) is in place between Dr. Ernst and Dr. Saggar. The 
agreement will be included in the data sharing spreadsheet. 
 
23. Research and Travel Compensation 
 
Volunteers will be compensated for time and research-related inconveniences based on 
NIH standards for time devoted to research projects. Travel time or expenses are not paid.  
NIH employees or staff who participate during work hours must have permission from 
their supervisor. NIH employees or staff must either participate outside of work hours or 
take leave in order to receive compensation. 
 
VOLUNTEER PAYMENT SCHEDULE 
 
Procedure - Psychophysiology 
Duration    
Amount  
Outpatient Visit for study session Drug, shock 
5-8 hrs 
$200 
TOTAL 
Up to 8 hrs 
$200 
 
Procedure - fMRI 
Duration    Amount  
Outpatient Visit for study session drug, fMRI ,shock 
5-8 hrs 
$210 
TOTAL 
Up to 8 hrs 
$210 
 

 
 
 
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