The Benzodiazepine Alprazolam Dissociates Contextual Fear from Cued Fear in Humans as Assessed by Fear-potentiated Startle

Article (PDF Available)inBiological Psychiatry 60(7):760-6 · November 2006with66 Reads
DOI: 10.1016/j.biopsych.2005.11.027 · Source: PubMed
Abstract
The startle reflex is potentiated by aversive states. It has been proposed that phasic startle potentiation to a threat cue and sustained startle potentiation to contextual stimuli reflect distinct processes mediated by different brain structures. The present study tested the hypothesis that alprazolam would reduce the sustained startle potentiation to contextual threats but not the startle potentiation to a threat cue. Sixteen healthy subjects received each of four treatments: placebo, .5 mg of alprazolam, 1 mg of alprazolam, and 50 mg of diphenhydramine (Benadryl) in a crossover design. Participants were exposed to three conditions, including one in which predictable aversive shocks were signaled by a cue, a second in which shocks were administered unpredictably, and a third condition in which no shocks were anticipated. Acoustic startle were delivered regularly across conditions. Phasic startle potentiation to the threat cue in the predictable condition was not affected by alprazolam. In contrast, the sustained increase in startle in the predictable and unpredictable conditions was reduced significantly by the high dose of alprazolam. Startle responses to an explicit threat cue and to an aversive context are psychopharmacologically distinct, suggesting that they may represent functionally dissociable aversive states.
The Benzodiazepine Alprazolam Dissociates
Contextual Fear from Cued Fear in Humans as
Assessed by Fear-potentiated Startle
Christian Grillon, Johanna M.P. Baas, Daniel S. Pine, Shmuel Lissek, Megan Lawley, Valerie Ellis, and
Jessica Levine
Background: The startle reflex is potentiated by aversive states. It has been proposed that phasic startle potentiation to a threat cue
and sustained startle potentiation to contextual stimuli reflect distinct processes mediated by different brain structures. The present
study tested the hypothesis that alprazolam would reduce the sustained startle potentiation to contextual threats but not the startle
potentiation to a threat cue.
Methods: Sixteen healthy subjects received each of four treatments: placebo, .5 mg of alprazolam, 1 mg of alprazolam, and 50 mg
of diphenhydramine (Benadryl) in a crossover design. Participants were exposed to three conditions, including one in which
predictable aversive shocks were signaled by a cue, a second in which shocks were administered unpredictably, and a third condition
in which no shocks were anticipated. Acoustic startle were delivered regularly across conditions.
Results: Phasic startle potentiation to the threat cue in the predictable condition was not affected by alprazolam. In contrast, the
sustained increase in startle in the predictable and unpredictable conditions was reduced significantly by the high dose of alprazolam.
Conclusions: Startle responses to an explicit threat cue and to an aversive context are psychopharmacologically distinct, suggesting
that they may represent functionally dissociable aversive states.
Key Words: Alprazolam, anxiety, benzodiazepine, context, fear,
startle reflex
T
he startle reflex, a cross-species response to a sudden
intense stimulus, is sensitive to aversive states (Davis et al
1993). Rodents show robust startle potentiation to a
conditioned cue that signals an aversive event (e.g., a shock).
Evidence suggests that this effect is mediated by the central
nucleus of the amygdala (CeA). For example, lesions of the CeA
block fear-potentiated startle to a conditioned cue previously
paired with shock (Davis 1998). Although the CeA responds to
various types of stressors, this structure is not always critical for
fear-potentiated startle (Davis 1998) or for responses to stress in
general (Hammack et al 2004). Davis and his collaborators have
reported a series of studies showing no effect of lesions of the
CeA on startle potentiation caused by various stressors, such as
bright lights (i.e., light-enhanced startle), shock sensitization, and
corticotropin-releasing hormone injection (reviewed in Walker et
al 2003). Rather, another structure, the bed nucleus of the stria
terminalis (BNST), was found to mediate startle potentiation in
these conditions (Walker et al 2003). In an analysis of the
experimental situations that do or do not require the CeA and
BNST, Walker et al (2003) suggested that these structures were
involved in functionally different aversive states in rodents. It was
proposed that the CeA was crucial for the phasic form of
fear-potentiated startle to a predictable threat cue, whereas the
BNST was responsible for the more sustained form of startle
potentiation induced by unpredictable or unconditioned aver-
sive stimuli (Davis 1998; Gewirtz et al 1998; Walker and Davis
1997a). It was suggested further that the two aversive states
mediated by the CeA and the BNST in rodents were reminiscent
of fear and anxiety states in humans, respectively (Davis 1998).
According to this view, fear is a response to a clearly identifiable
danger that subsides shortly after the offset of a threat cue.
Anxiety is a more sustained form of general distress and anxious
apprehension in response to less identifiable cues (Barlow 2000;
Davis 1998; Lang et al 2000).
The distinction between a phasic and a more sustained form
of startle potentiation also has been made in humans (Cuthbert et
al 2003; Grillon et al 1991, 1997; Grillon and Davis 1997; Lang et
al 2000; Pole et al 2003). Startle is potentiated by an explicit threat
cue that signals an impending aversive event (e.g., a shock;
Grillon et al 1993; Hamm and Vaitl 1996), such as when phobic
individuals are confronted with their phobic objects (de Jong et
al 1996; Globisch et al 1999). More sustained forms of startle
potentiation can be found among individuals who are exposed
to stressful experimental settings (Bocker et al 2001; Grillon and
Ameli 1998; Pole et al 2003), when the experimental room is in
complete darkness (Grillon et al 1997), or after context condi-
tioning (Grillon and Davis 1997).
Consistent with the animal literature, phasic and sustained
forms of startle potentiation in humans appear to reflect distinct
processes. For example, individuals with posttraumatic stress
disorder (PTSD) or with panic disorder display normal startle to
explicit threat cues that signal a shock but display enhanced
startle reactivity in the experimental context in which the shocks
are administered (Grillon et al 1994, 1998b; Pole et al 2003).
Patients with PTSD also exhibit increased context conditioning
(Grillon and Morgan 1999) and increased facilitation of startle in
the dark (Grillon et al 1998a).
There currently is little information on the neurobiological
mechanisms that may differentiate phasic cued fear from more
sustained contextual anxiety in humans. Evidence for such a
neurobiological dissociation would be bolstered if one could
demonstrate that these two forms of aversive states are differen-
tially responsive to psychopharmacologic treatments. The main
objective of this study was to obtain such evidence by using the
benzodiazepine alprazolam.
From the Mood and Anxiety Disorder Program, National Institutes of Mental
Health, Bethesda, Maryland.
Address reprint requests to Christian Grillon, Ph.D., National Institutes of
Mental Health–Mood and Anxiety Disorder Program, 15K North Drive,
Building 15K, Room 113, MSC 2670, Bethesda, MD 20892-2670; E-mail:
christian.grillon@nih.gov.
Received July 12, 2005; revised October 27, 2005; accepted November 11,
2006.
BIOL PSYCHIATRY 2006;60:760 –7660006-3223/06/$32.00
doi:10.1016/j.biopsych.2005.11.027 © 2006 Society of Biological Psychiatry
There is an emerging literature on the effect of benzodiaz-
epines on fear-potentiated startle in humans. We have reported
results of four studies showing that oxazepam and diazepam did
not affect fear-potentiated startle to a threat cue (Baas et al 2002).
In contrast, other groups reported that alprazolam (Riba et al
2001), diazepam (Bitsios et al 1999), and lorazepam (Graham et
al 2005) reduced fear-potentiated startle in threat of shock
experiments. However, it is not clear whether the reduction in
fear-potentiated startle in these latter studies was caused by an
anxiolytic effect per se or was an artifact of the sedative effect of
benzodiazepines on baseline startle. A drug-induced reduction in
baseline startle reactivity may lead to an inaccurate measurement
of fear-potentiated startle (Grillon and Baas 2002; Walker and
Davis 2002b). Important methodologic differences between stud-
ies also may explain contradictory findings. For example, in two
studies the subjects were verbally informed of the threat and safe
conditions while the shock electrodes were being attached or
removed, and these studies were conducted in near darkness
(Bitsios et al 1999; Graham et al 2005). It has been argued that
both the shock electrodes and darkness are contextual stimuli
(Grillon and Ameli 1998; Grillon et al 1997; Walker and Davis
1997b), which may have increased contextual anxiety. Hence, it
is unclear whether the response that was effectively reduced by
diazepam in the Bitsios et al (1999) design constitutes a cued fear
response or contextual anxiety caused by placement of the shock
electrodes.
There is evidence to suggest that benzodiazepines affect
contextual anxiety. In rodents, benzodiazepines reduce baseline
startle. This reduction is a result not only of sedation but also of
an anxiolytic effect on contextual fear (Guscott et al 2000). Baas
et al (2002) reported a similar effect in humans. In addition, Baas
et al (2002) showed that diazepam reduced the facilitation of
startle in the dark in humans. The facilitation of startle in the dark
in humans is a sustained form of startle potentiation similar to the
light-enhanced startle in rodents (Grillon and Baas 2002), which
itself is alleviated by benzodiazepines (Walker and Davis 2002a;
de Jong et al 2002).
The present study improved on past studies by addressing
two main issues. First, we compared fear-potentiated startle
elicited by predictable and unpredictable shocks to clearly
dissociate phasic cued fear from sustained anxiety (Grillon et al
2004). Second, a sedative drug (diphenhydramine) that is not
used for anxiolysis was included to control for confounding
effects of sedation on startle reactivity (Grillon and Baas 2002;
Walker and Davis 2002b). The experimental design was based on
the observation that unpredictable aversive events increase
context conditioning (Grillon and Davis 1997; Odling-Smee
1975), which can be conceived of as a form of sustained anxiety.
In this design, subjects are presented with three conditions: no
shocks, predictable or signaled shocks, and unpredictable or
nonsignaled shocks. Previous results show two types of aversive
responses: (1) a phasic startle potentiation during the threat
signal relative to the absence of threat signal in the predictable
shock condition and (2) a sustained startle potentiation during
the predictable and the unpredictable shock condition compared
with the no-shock condition in the absence of specific cues
(Grillon et al 2004). For the remainder of this article, cued fear
will refer to the phasic startle potentiation to the explicit threat
signal, and contextual anxiety will refer to the sustained startle
potentiation in the absence of cues.
The main hypothesis of the study was that alprazolam would
not affect fear-potentiated startle to the threat signal in the
predictable shock condition (cued fear) but would reduce fear-
potentiated startle in the absence of cues in the predictable and
unpredictable shock condition (contextual anxiety). Each subject
was tested in four treatments: (1) placebo, (2) a low dose (.5 mg)
of alprazolam, (3) a high dose (1 mg) of alprazolam, and (4)
diphenhydramine (Benadryl, 50 mg). The two doses of alprazo-
lam were selected on the basis of a study that reported a
reduction of fear-potentiated startle by alprazolam (Riba et al
2001).
Methods and Materials
Participants
Participants were healthy volunteers who gave written in-
formed consent that had been approved by the National Insti-
tutes of Mental Health Human Investigation Review Board.
Inclusion criteria included the following: (1) no past or current
psychiatric disorders as per Structured Clinical Interview for
DSM-IV (SCID; First et al 1995), (2) no medical condition that
interfered with the objectives of the study as established by a
physician, and (3) no use of illicit drugs or psychoactive medi-
cations as per urine screen.
Participants underwent a screening session that consisted of a
SCID, a physical exam, and a shock workup procedure to
establish a level of shock that was “highly annoying but not
painful”. The mean intensity of the shock was 4.2 A, with a
range of 3–5 A. In addition, subjects were screened for baseline
startle reactivity with nine startle stimuli (40-ms duration, 103
dB). Subjects who displayed small startle responses (a mean of
less than 5 V over nine startle responses) or displayed no startle
response on at least one trial were not invited to participate in the
study. Four to 10 days after screening, participants returned for
the first of four testing sessions. Sixteen subjects (five were male)
with a mean age of 23.0 years (SD 4.7 y) ultimately were
included in the study. Mean scores on the state and trait portions
of Spielberger’s State and Trait Anxiety Inventory (Spielberger
1983) were 26.0 (SD 4.0) and 28.2 (SD 4.8), respectively.
Drug Manipulation
The treatments were placebo, .5 mg of alprazolam, 1 mg of
alprazolam, and 50 mg of diphenhydramine, tested in a double-
blind crossover design (within subjects). Treatment administra-
tion was performed according to a randomization table compris-
inga4 4 Latin square repeated four times.
Procedure
On the test day, subjects filled out a mood rating scale
(pretreatment) that evaluated subjective feelings of mental and
physical sedation. Next, they ingested a capsule containing one
of the active drugs or placebo. Subjects rested for 1 hour to allow
drug absorption, after which the procedure to apply measure-
ment- and shock-electrodes was started.
Details of the procedures are provided elsewhere (Grillon et
al 2004). During testing, subjects first were presented a habitua-
tion block consisting of nine startle stimuli delivered every 18 –25
sec to reduce excessive initial startle reactivity before the threat
study (data not reported). Participants then were given explicit
instructions regarding the conditions under which they would
and would not receive an aversive event. After the instructions,
the threat experiment began. The experiment consisted of three
different conditions: no shock (N), predictable shock (P), and
unpredictable shock (U), each lasting approximately 150 sec. In
the N condition, no shocks were delivered. In the P condition,
shocks were administered predictably, that is, only in the pres-
ence of a threat cue. In the U condition, the shocks were
C. Grillon et al
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unpredictable. In each 150-sec condition, an 8-sec cue was
presented four times. The cues were different geometric colored
shapes in each condition (e.g., a blue square for N, a red circle
for P, and a green star for U). The cues signaled the possibility of
receiving an aversive stimulus only in the P condition. They had
no signal value in the N and U conditions. Instructions were
displayed on a computer monitor to inform participants of the
current condition by displaying the following information
throughout the testing procedure: “no shock” (N), “shock only
during shape” (P), or “shock at any time” (U). During each
predictable and unpredictable condition, one shock was admin-
istered. When a shock was administered, it was delivered during
the cue in the predictable condition and in the absence of the
cues in the unpredictable condition. In each N, P, and U
condition, six acoustic startle stimuli were delivered, three during
intertrial intervals (ITI; i.e., between cues) and one during three
of the four cues, 5–7 sec after cue onset. The threat experiment
consisted of two recording blocks with a 5- to 10-min rest
between blocks. Each block consisted of three N, two P, and two
U conditions in one of the following two orders:PNUNUNP
orUNPNPNU.Each participant was presented with the two
orders, with half the participants starting with the P condition.
One shock was administered in each individual P and U condi-
tion, for a total of four shocks in the four P conditions and of four
shocks in the four U conditions. The shock was delivered 7.5 sec
after cue onset in the P condition. It was administered either 7 sec
or 10 sec after cue offset in the unpredictable condition. No
startle stimuli could follow a shock by less than 10 sec.
Subjects were asked to fill out the mood rating scale another
time during the interval between the two threat blocks. In
addition, after each recording block, subjects retrospectively
rated how anxious they felt in the presence and absence of the
cue in each condition (N, P, U) on an analog scale ranging from
0 (not at all anxious) to 10 (extremely anxious).
Stimuli and Physiological Responses
Stimulation and recording were controlled by a commercial
system (Contact Precision Instruments, London, United King-
dom). The acoustic startle stimulus was a 40-ms duration,
103-dB(A) burst of white noise with a near-instantaneous rise
time, presented binaurally through headphones. The eyeblink
reflex was recorded with electrodes placed under the left eye.
Amplifier bandwidth was set to 30 –500 Hz. The electric shock
was produced by a constant current stimulator and was admin-
istered on the right wrist.
Data Analysis
Peak amplitude of the blink reflex was determined in the 20-
to 100-msec time frame after stimulus onset relative to baseline
(average baseline EMG level for the 50 ms immediately preced-
ing stimulus onset) and was averaged within each condition. The
startle data and retrospective measures of subjective anxiety
were analyzed with analyses of variance (ANOVA) with repeated
measures. Preliminary analyses indicated no gender difference
for the startle and subjective measures. Hence, gender was not
entered as a factor in the data analysis. Given our specific a priori
hypotheses, separate comparisons were conducted to examine
cued fear and contextual anxiety. Cued fear was tested by first
calculating the difference scores between startle magnitude
during the cues and startle magnitude during ITI. These differ-
ence scores then were analyzed with two-way ANOVAs, with
treatment (placebo, diphenhydramine, low alprazolam, or high
alprazolam) and condition (N, P, or U) as repeated factors.
Contextual anxiety was evaluated by using the startle magnitudes
during ITI in each of the three conditions. This involved two-way
ANOVAs, with treatment (placebo, diphenhydramine, low alpra-
zolam, or high alprazolam) and condition (N, P, or U) as
repeated factors. Given a significant main effect of condition, the
presence of a linear trend in startle magnitude over N, P, and U
was tested. It was hypothesized that the linear trend would be
affected by alprazolam but not by diphenhydramine, compared
with placebo. These analyses were repeated on standardized
scores by using within-subjects t scores ([Z scores 10] 50).
Because similar results were obtained with the raw scores and
with the t scores for within-subjects comparisons, only results of
the raw scores are presented. The same analysis was conducted
for the retrospective subjective reports of anxiety. Alpha was set
at .05 for all statistical tests. Greenhouse-Geisser corrections
(GG-) were used for main effects and for interactions involving
factors with more than two levels.
Results
Startle Data
Cued Fear. The results in each condition and in each
treatment are presented in Table 1. Figure 1A displays the
magnitude of fear-potentiated startle (cue minus ITI). There was
a significant main effect of condition [F(2,30) 11.9, p .001,
GG-⑀⫽.65], reflecting greater fear-potentiated startle during the
cue in the U condition, compared to the N and P condition.
These effects were not affected by treatment, as reflected by a
lack of treatment main effect [F(3,45) .03, ns] and treatment
condition interaction [F (6,90) .3, ns].
Contextual Anxiety. The ITI data that were used to evaluate
contextual anxiety are shown in Table 1. Consistent with our
previous report (Grillon et al 2004), there was a linear increase in
startle magnitude from the neutral, to the predictable, to the
unpredictable condition [main effect of condition: F (2,30)
14.0, p .0009, GG-⑀⫽.7; linear effect of condition: F (1,15)
18.7, p .0009]. There also was a trend for a main effect of
treatment [F(3,45) 2.4, p .09, GG-⑀⫽.8]. However, this
effect was qualified by a significant treatment condition
interaction [F(6,90) 3.4, p .01, GG-⑀⫽.7] and by a treatment
condition linear effect [F (1,15) 5.7, p .03]. These
Table 1. Mean (SEM) Startle Magnitude (V) during the Cue and during ITI across Treatments and Conditions
Neutral Predictable Unpredictable
Cue ITI Cue ITI Cue ITI
Placebo 38.3 (9.7) 38.4 (10.8) 57.9 (11.9) 47.2 (11.1) 55.1 (12.1) 53.6 (12.0)
Diphenhydramine 28.0 (11.7) 26.8 (10.8) 42.2 (12.6) 32.2 (12.0) 40.1 (12.5) 40.3 (13.0)
Alprazolam, low 39.4 (10.0) 38.2 (10.6) 51.9 (12.3) 41.6 (10.0) 57.0 (12.3) 55.7 (12.8)
Alprazolam, high 23.9 (7.6) 25.5 (8.0) 37.0 (9.3) 26.3 (7.2) 33.0 (8.3) 30.3 (7.9)
ITI, intertrial intervals.
762 BIOL PSYCHIATRY 2006;60:760–766 C. Grillon et al
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interactions reflect differential effects of treatment on startle
potentiation during ITI. Follow-up tests focused on the increase
in startle magnitude from the neutral to both the predictable and
unpredictable conditions across treatments. This analysis was
implemented by calculating the difference scores for predictable
minus no-shock and unpredictable minus no-shock conditions
(Figure 2A). These difference scores were entered into a treat-
ment (placebo or high alprazolam) condition (P or U) ANOVA
yielding a main effect of treatment [F (1,15) 10.2, p .0009]
and no significant treatment condition interaction. The signif-
icant treatment main effect confirmed that the ITI startle poten-
tiation from the neutral to both the predictable and unpredictable
condition was reduced by high alprazolam.
The active control substance diphenhydramine was used to
examine whether the treatment effect could be attributed solely
to sedation. This was not the case. First, the treatment (placebo
or diphenhydramine) condition (P or U) ANOVA showed no
significant effect involving treatment. Second, the treatment
(high alprazolam or diphenhydramine) condition (P or U)
ANOVA showed a significantly lower potentiated startle in the
high-alprazolam condition compared to diphenhydramine
[F(1,15) 5.5, p .03]. The placebo, diphenhydramine, and
low-alprazolam treatment conditions did not differ significantly
from each other. Note that high alprazolam also reduced poten-
tiated startle compared with the low-alprazolam treatment con-
dition [F(1,15) 5.7, p .03].
Retrospective Ratings of Anxiety
Cued Fear. The anxiety rating scores are shown in Figure 1B.
There was a significant main effect of condition [F (2,30) 56.0,
p .0009, GG-⑀⫽.95], reflecting greater subjective anxiety
during the cue in the P condition, compared to the N and U
conditions. There was no significant main effect of treatment
[F(3,45) .61], but the treatment condition interaction was
significant [F(6,90) 2.4, p .04]. Because the comparison of
interest was anxiety ratings in the P condition across treatments,
follow-up tests contrasted anxiety scores between treatments in
the P condition in a one-way ANOVA. The results indicated no
reduction in anxiety with any of the treatments as reflected by a
nonsignificant treatment main effect [F (3,45) 1.9, p .1].
Contextual Anxiety. Consistent with the startle data, there
was a progressive increase in anxiety from the neutral, to the
predictable, to the unpredictable condition [F(2,30) 56.7, p
.0009, GG-⑀⫽.69; linear trend: F (1,15) 69.4, p .0009]. There
also was a main effect of treatment [F (3,45) 7.6, p .001, GG-
.83] that was caused by an overall reduction in anxiety with
diphenhydramine [F(1,15) 17.1, p .001], low alprazolam
[F(1,15) 4.8, p .04], and high alprazolam [F (1,15) 14.9, p
.002] treatments compared to placebo (Figure 2). However,
unlike the startle results, there was no treatment condition
interaction, indicating no significant difference in subjective
Figure 2. Responses during ITI (context) in each treatment. (A) Contextual
potentiated startle. Difference scores between startle magnitudes in the
threat conditions (predictable and unpredictable) and startle magnitudes in
the no-shock condition. *The increased startle from the neutral to the pre-
dictable and unpredictable conditions was significantly reduced (p .05) by
high alprazolam (alpraz), compared to the other treatments. (B) Subjective
anxiety. Difference scores between reported anxiety in the threat conditions
(predictable and unpredictable) and reported anxiety in the no-shock con-
dition. There was no significant difference among treatments.
Figure 1. Responses to the cues in each treatment expressed as a change
from baseline. (A) Cued fear-potentiated startle. Difference scores reflecting
cue minus ITI startle magnitudes in the no shock, predictable, and unpre-
dictable conditions. The main effect of condition was significant. *Signifi-
cantly (p .05) increased startle from ITI. There was no significant difference
among treatment. (B) Subjective anxiety. Difference scores reflecting startle
magnitudes in the presence minus absence of cues in each condition. The
main effect of condition was significant. *Significantly (p .05) increased
startle from ITI. There was no significant difference among treatments.
Alpraz,alprazolam.
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anxiety across conditions among the three active treatments
(Figure 2B).
Mental and Physical Sedation
The mental and physical sedation data from the mood rating
scale are shown in Table 2. Results were analyzed with treatment
(placebo, diphenhydramine, alprazolam low, or alprazolam
high) condition (baseline or posttreatment) ANOVAs. For both
mental sedation and physical sedation, there were significant
main effects of treatment [F (3,45) 6.5, p .001, GG-⑀⫽.78
and F (3,45) 5.4, p .003, GG-⑀⫽.92, respectively] and of
condition [F(1,15) 37.5, p .0009 and F (1,15) 44.0, p
.0009, respectively], as well as a significant treatment condition
interaction [F (3,45) 8.4, p .001, GG-⑀⫽.70 and F (3,45)
9.9, p .0009, GG-⑀⫽.79, respectively]. The interaction
reflected the fact that subjects exhibited greater increases in
physical and mental sedation from the baseline to the posttreat-
ment period with diphenhydramine, low alprazolam, and high
alprazolam compared with placebo (all p .05). There also was
a greater increase in mental and physical sedation with the
diphenhydramine and high-alprazolam, compared with low-
alprazolam, treatments (all p .05). Finally, there was no
significant difference in mental and physical sedation between
diphenhydramine and high-alprazolam treatments.
Discussion
The present study sought to establish a psychopharmacolog-
ical distinction by using the benzodiazepine alprazolam between
a phasic aversive response to a threat cue (cued fear) and a more
sustained aversive response associated with the experimental
context in which shocks are anticipated (contextual anxiety). The
experimental model to elicit these two aversive states was based
on preclinical data in rodents (Davis 1998) and on empirical
work in our laboratory that used the threat of predictable
(signaled) and unpredictable (unsignaled) shocks (Grillon et al
2004). Consistent with our hypothesis, alprazolam did not affect
phasic fear-potentiated startle to the threat cue but reduced the
sustained potentiation of startle in the predictable and the
unpredictable condition. This effect cannot be attributed to an
artifactual sedative effect of alprazolam on baseline startle
(Walker and Davis 2002b) for two reasons. First, baseline startle
responses, as well as mental and physical sedation, were affected
to the same extent by high alprazolam and diphenhydramine,
indicating that diphenhydramine was an appropriate nonspecific
active control substance. Yet, contextual startle potentiation in
the predictable and unpredictable conditions was reduced sig-
nificantly in the high alprazolam compared to the diphenhydra-
mine treatment. Second, if the reduction in contextual fear-
potentiated startle was a result of sedation, it is unclear why such
an effect would not have affected fear-potentiated startle to the
threat cue. These results suggest that alprazolam exerted a
differential effect on potentiated startle to an explicit threat cue
and to contextual cues.
It could be argued that the treatment effect of the high dose of
alprazolam on ITI startle in the predictable and unpredictable
condition was artifactually caused by a floor effect. This expla-
nation is unlikely because the floor effect for startle magnitude is
an absence of eyeblink response. In the present study, the
magnitude of startle in the high-alprazolam condition was clearly
above 0 (about 20 V). Alternatively, it could be argued that the
lack of effect of alprazolam on fear-potentiated startle to the cue
in the predictable condition was caused by a ceiling effect. This,
again, is unlikely. First, we analyzed the startle habituation data
(not shown) and found much larger responses during startle
habituation compared response to the threat cues in the predict-
able condition. For example, the group mean magnitude of the
first habituation startle in the placebo condition was 91 V,
which is larger than either the mean response to the first startle
in the predictable cue (74 V) or than the mean startle response
to all the cues in the predictable condition (57.9 V).
The present findings are consistent with preclinical and
clinical studies distinguishing cued fear from contextual anxiety.
Preclinical studies show that the CeA mediates phasic responses
to explicit threat cues and that the BNST mediates sustained
responses to contextual stimuli (Walker et al 2003). Clinical
investigations have reported normal fear-potentiated startle to an
explicit threat cue but elevated contextual fear-potentiated startle
in patients with anxiety disorders (Cuthbert et al 2003; Grillon et
al 1991, 1997; Grillon and Davis 1997; Pole et al 2003).
The present results also are consistent with animal data
showing that benzodiazepines reduce sustained forms of startle
potentiation. Walker and Davis (2002a) reported that the light-
enhanced startle effect was alleviated by the benzodiazepine
chlordiazepoxide. However, the lack of effect of alprazolam on
fear-potentiated startle to an explicit threat cue apparently con-
flicts with cued fear conditioning data in rodents (Hijzen et al
1995; Melia and Davis 1991). One important difference between
the present study and preclinical studies is the reliance of animal
studies on associative-learning processes such as fear condition-
ing. The reduced fear-potentiated startle after benzodiazepine
administration in animal models that rely on conditioning cannot
be attributed unambiguously to an anxiolytic effect as opposed
to an effect on learning or memory (Walker and Davis 2002a). In
addition, it is possible that the amount of drug used in animals is
not comparable to that used in humans. Nevertheless, a recent
fear-conditioning study in humans supports our findings. Scaife
et al (2005) found that diazepam blocked the acquisition but not
the expression of fear-potentiated startle to an explicit cue.
In terms of current results for subjective ratings of anxiety the
cue and contextual manipulations induced the same basic pat-
tern of results found in the startle data. In the predictable
condition, subjective anxiety was larger in the presence than in
the absence of the threat cue. Like the startle data, this increase
in subjective anxiety was unaffected by drug treatments. As for
the context effect, both startle magnitude and the subjective
ratings of anxiety to the context increased linearly from the
Table 2. Mean (SEM) Scores of Subjectively Reported Mental and Physical Sedation
Mental Sedation Physical Sedation
Pretreatment Posttreatment Pretreatment Posttreatment
Placebo 10.2 (1.0) 13.6 (1.7) 9.8 (1.1) 13.6 (1.7)
Diphenhydramine 11.0 (1.4) 22.6 (1.9) 0.0 (1.3) 22.7 (1.9)
Alprazolam, low 9.6 (1.0) 17.4 (1.4) 9.7 (.9) 17.4 (1.4)
Alprazolam, high 9.9 (1.2) 20.9 (1.6) 10.6 (1.0) 20.9 (1.6)
764 BIOL PSYCHIATRY 2006;60:760–766 C. Grillon et al
www.sobp.org/journal
no-shock to the predictable to the unpredictable conditions.
However, treatment effects on these subjective reports did not
follow the startle results; instead of a significant treatment
condition interaction, there was only a main effect of treatment.
This indicates that though the subjects were feeling less anxious
overall under all three drug treatments compared with placebo,
they did not report context-specific reductions in anxiety under
alprazolam. Apparently, startle was sensitive to subtle effects of
the treatments that were not accessible to subjective measures of
anxiety.
One obvious reason for this difference is that whereas startle
was used to probe contextual anxiety online, the subjective
anxiety measures were retrospective. The passage of time may
have obscured subtle differences in responding because of the
complexity of the design. The design included six different
conditions (three contextual manipulations, each with and with-
out the presence of a cue), for which subjective anxiety was
measured retrospectively. The retrospective subjective anxiety
data appeared to have been strongly influenced by sedation. This
is suggested by the fact that the pattern of retrospective ratings of
anxiety exactly paralleled the ratings of mental and physical
sedation (Table 1). Future studies may benefit from using an
online measure of subjective anxiety.
How could alprazolam reduce contextual anxiety? It is un-
likely that the present results were the result of a gross cognitive
deficit, such as memory impairment. Subjects did not have to
remember the contingency between threat and the different
conditions because the instructions were written on a monitor
screen throughout the duration of each condition. A purely
anxiolytic effect on sustained anxiety could be achieved, for
example via action at the level of structures involved in this type
of response (i.e., the BNST). Alternatively, alprazolam could
reduce anxiety by facilitating distraction from stressful cognitions
associated with the threat experiment. Distraction from stressful
cognitions may have been more likely to occur in the unpredict-
able condition when the threat was long lasting, compared to the
predictable condition, in which the threat cue signaled an
imminent danger, resulting in a reduction in contextual anxiety
but not phasic fear. Such a distraction could have been amplified
by drowsiness, but this is unlikely given that the sedative drug
diphenhydramine did not reduce the enhanced startle in the
unpredictable context.
The limitations of the present study must be considered when
interpreting the results. It is possible that alprazolam effects on
potentiated startle did not depend on the qualitative nature of the
induced emotion (e.g., fear vs. anxiety or explicit vs. contextual
cues), but on the intensity of the aversive reaction. According to
this view, benzodiazepines could reduce potentiated startle to
stimuli or situations that elicit little fear or anxiety, such as
contextual anxiety, but are ineffective when higher levels of fear
or anxiety are involved, such as during a threat cue. Such a
possibility should be tested experimentally by examining the
effect of benzodiazepines on threat cues that elicit different levels
of fear. However, the Scaife et al (2005) fear-conditioning study
may shed light on this issue. Startle potentiation to a cue is much
greater during a threat of shock experiment than during fear
conditioning (Grillon and Davis 1977; Grillon et al 1991). Yet
Scaife et al (2005) found that diazepam did not block the
expression of cued fear conditioning. Hence, a weak aversive
response is not necessarily affected by benzodiazepines. Another
limitation is that we used diphenhydramine as an active control
condition because of its sedative effect. The subjective data
suggest that diphenhydramine was mildly anxiolytic, and animal
data suggest that the histaminergic system may be implicated in
anxiety (Fish et al 2004; Privou et al 1998). Because benzodiaz-
epines have muscle relaxant properties, a muscle relaxant may
have been a more appropriate control drug.
The present results are consistent with the hypothesis of a
functional differentiation between phasic startle potentiation to a
threat cue and sustained startle potentiation to contextual threat
proposed by Davis (1998). Because of the translational nature of
startle studies, it is likely that human and animal models will
ultimately yield more insight into the neurobiology of cued fear
and contextual anxiety. Differentiating between cued fear and
contextual anxiety may help us identify the components of
aversive states and their relevance to pathologic anxiety. Assum-
ing that contextual anxiety is relevant to pathologic anxiety
(Grillon and Morgan 1999; Grillon et al 1998b), experiments in
animals and humans that manipulate contextual fear-potentiated
startle may be helpful for screening novel anxiolytics.
This research was supported by the Intramural Research
Program of the National Institutes of Mental Health.
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    • "No research to date has examined drug effects on contextual fear learning or fear generalization processes in PTSD patients, though preliminary experimental research suggests that acute glucose consumption may enhance retention of differential configural fear learning (Glenn et al. 2014). In healthy subjects, acute administration of 1 mg of the benzodiazepine alprazolam reduced sustained startle responding in both predictable and unpredictable " context " periods, but did not alter responding to discrete cues associated with predictable and unpredictable threat (Grillon et al. 2006 ). These findings tentatively suggest that acute benzodiazepine administration might reduce sustained contextual anxiety in PTSD patients, though they do not indicate treatment effects for sensitivity to unpredictable threat. "
    [Show abstract] [Hide abstract] ABSTRACT: The use of quantitative, laboratory-based measures of threat in humans for proof-of-concept studies and target development for novel drug discovery has grown tremendously in the last 2 decades. In particular, in the field of posttraumatic stress disorder (PTSD), human models of fear conditioning have been critical in shaping our theoretical understanding of fear processes and importantly, validating findings from animal models of the neural substrates and signaling pathways required for these complex processes. Here, we will review the use of laboratory-based measures of fear processes in humans including cued and contextual conditioning, generalization, extinction, reconsolidation, and reinstatement to develop novel drug treatments for PTSD. We will primarily focus on recent advances in using behavioral and physiological measures of fear, discussing their sensitivity as biobehavioral markers of PTSD symptoms, their response to known and novel PTSD treatments, and in the case of d-cycloserine, how well these findings have translated to outcomes in clinical trials. We will highlight some gaps in the literature and needs for future research, discuss benefits and limitations of these outcome measures in designing proof-of-concept trials, and offer practical guidelines on design and interpretation when using these fear models for drug discovery.
    Full-text · Article · Jun 2016
    • "Each condition comprises a specific context and a corresponding cue. While the N-condition serves as a control condition, in which the participants experience safety, the Pcondition elicits a phasic fear response to the cue, while in the Ucondition a sustained anxiety response is observed (Grillon, 2008; Grillon et al., 2006b; Grillon et al., 2004). While most studies so far used simple geometric shapes as cues and verbal instructions for the N, P, or U context, some studies employed virtual reality technology as a possibility to create environments of high external validity (Alvarez et al., 2011; Baas, 2013; Grillon et al., 2006a; Haaker et al., 2013; Lonsdorf et al., 2014 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Fear is elicited by imminent threat and leads to phasic fear responses with selective attention, whereas anxiety is characterized by a sustained state of heightened vigilance due to uncertain danger. In the present study, we investigated attention mechanisms in fear and anxiety by adapting the NPU-threat test to measure steady-state visual evoked potentials (ssVEPs). We investigated ssVEPs across no aversive events (N), predictable aversive events (P), and unpredictable aversive events (U), signaled by four-object arrays (30s). In addition, central cues were presented during all conditions but predictably signaled imminent threat only during the P condition. Importantly, cues and context events were flickered at different frequencies (15Hz vs. 20Hz) in order to disentangle respective electrocortical responses. The onset of the context elicited larger electrocortical responses for U compared to P context. Conversely, P cues elicited larger electrocortical responses compared to N cues. Interestingly, during the presence of the P cue, visuocortical processing of the concurrent context was also enhanced. The results support the notion of enhanced initial hypervigilance to unpredictable compared to predictable threat contexts, while predictable cues show electrocortical enhancement of the cues themselves but additionally a boost of context processing.
    Full-text · Article · Jun 2016
    • "The contrast of physiological responding selectively during predictable or unpredictable shock conditions with a neutral, no-shock condition has been proposed to map onto acute and potential threat constructs, respectively , within the RDoC negative valence system. The NPU task has been used extensively to study mood and anxiety disorders (Grillon et al., 2008Grillon et al., , 2009), addiction (Bradford, Curtin, & Piper, 2015; Hogle, Kaye, & Curtin, 2010 ), and the effects of pharmaceutical and recreational drugs (Grillon et al., 2006; Moberg & Curtin, 2009). The affective picture viewing task manipulates participants' affect with unpleasant, pleasant, and neutral pictures while measuring their concomitant physiological responses. "
    [Show abstract] [Hide abstract] ABSTRACT: The current study provides a comprehensive evaluation of critical psychometric properties of commonly used psychophysiology laboratory tasks/measures within the NIMH RDoC. Participants (N = 128) completed the no-shock, predictable shock, unpredictable shock (NPU) task, affective picture viewing task, and resting state task at two study visits separated by 1 week. We examined potentiation/modulation scores in NPU (predictable or unpredictable shock vs. no-shock) and affective picture viewing tasks (pleasant or unpleasant vs. neutral pictures) for startle and corrugator responses with two commonly used quantification methods. We quantified startle potentiation/modulation scores with raw and standardized responses. We quantified corrugator potentiation/modulation in the time and frequency domains. We quantified general startle reactivity in the resting state task as the mean raw startle response during the task. For these three tasks, two measures, and two quantification methods, we evaluated effect size robustness and stability, internal consistency (i.e., split-half reliability), and 1-week temporal stability. The psychometric properties of startle potentiation in the NPU task were good, but concerns were noted for corrugator potentiation in this task. Some concerns also were noted for the psychometric properties of both startle and corrugator modulation in the affective picture viewing task, in particular, for pleasant picture modulation. Psychometric properties of general startle reactivity in the resting state task were good. Some salient differences in the psychometric properties of the NPU and affective picture viewing tasks were observed within and across quantification methods.
    Full-text · Article · May 2016
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