NeuroImage 53 (2010) 943951

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NeuroImage
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i m g

Volition diminishes genetically mediated amygdala hyperreactivity

Dina M. Schardt a,, Susanne Erk a, Corinna Nsser a, Markus M. Nthen b,c, Sven Cichon b,c,
Marcella Rietschel d, Jens Treutlein d, Thomas Goschke e, Henrik Walter a,
a

Department of Psychiatry, Division of Medical Psychology, University of Bonn, Germany

Department of Genomics, Life & Brain Center, University of Bonn, Germany
Institute of Human Genetics, University of Bonn, Germany
d
Central Institute of Mental Health, Department of Genetic Epidemiology in Psychiatry, Mannheim, Germany
e
Department of Psychology, Technische Universitt Dresden, Germany
b
c

a r t i c l e

i n f o

Article history:
Received 18 September 2009
Revised 24 November 2009
Accepted 26 November 2009
Available online 5 December 2009
Keywords:
Serotonin transporter
Amygdala
Volition
Cognitive emotion regulation
fMRI

a b s t r a c t
Individuals carrying the short allele of a common polymorphism in the promoter region of the serotonin
transporter gene (5-HTTLPR) exhibit heightened amygdala responses to passive stimulation with aversive
emotional material. In turn, the level of amygdala activation in response to emotion can be decreased by
will, for example by using cognitive emotion regulation strategies. In the present study, 37 female subjects
(s-carriers: n = 21; l/l-homozygotes: n = 16) performed an emotion regulation task during functional
magnetic resonance imaging to determine whether cognitive emotion regulation can modulate the
genetically determined amygdala hyperreactivity in 5-HTTLPR short allele carriers. Our results demonstrate
that cognitive emotion regulation diminishes the difference in amygdala reactivity to threat-related stimuli
between 5-HTTLPR genotype groups. Furthermore, we also provide evidence that the effect of cognitive
regulation is mediated through altered coupling between the amygdala and prefrontal regulatory regions.
Our ndings demonstrate that while the presence of the 5-HTTLPR short allele leads to heightened
responses in the amygdala, cognitive regulation can modify genetically mediated effects upon brain function
by volitionally altering prefrontal-amygdala connectivity.
2009 Elsevier Inc. All rights reserved.

Introduction
Recent advances in imaging genetics have provided promising new
insights into the complex interplay of genes, neural processing and
behavior (Esslinger et al., 2009; Green et al., 2008; Meyer-Lindenberg
and Weinberger, 2006). For example, the neurotransmitter serotonin
(5-hydroxytryptamine, 5-HT) is critically involved in emotional
processing, stimulating imaging genetic studies of this neuromodulatory system (Hahn and Blakely, 2007). One well-described determinant of this system is the serotonin transporter (5-HTT), which
regulates 5-HT reuptake from the synaptic cleft (Hariri and Holmes,
2006). On the molecular level, genetic regulation of 5-HTT mRNA and
protein expression is inuenced by a common polymorphism found in
the 5-HTT linked promoter region (5-HTTLPR) of the serotonin
transporter gene SLC6A4. In the presence of at least one short (s)
allele, the level of 5-HTT mRNA and 5-HT reuptake in human
lymphoblastoid cells is approximately two-fold lower compared
with cells that are homozygous for the long (l) allele.

Corresponding authors. Current address: Institute of Neuroradiology, Hannover

Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Fax: +49 511 532
5876.
E-mail address: schardt.dina@mh-hannover.de (D.M. Schardt).
1053-8119/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuroimage.2009.11.078

Genetic variation within the serotonin transporter gene also

inuences psychological measures of negative emotionality: in
comparison to l/l-homozygotes, the s-carriers show higher neuroticism, harm avoidance, anxiety and depression (Ansorge et al., 2004;
Hoefgen et al., 2005; Lesch et al., 1996). In addition, it was assumed
that depression mainly developed in those risk allele carriers who had
suffered stressful life events (Caspi et al., 2003). The latter ndings
however could not be substantiated by two recent meta-analyses
(Munafo et al., 2008; Risch et al., 2009).
In contrast to the effects on behavior, the inuence of the serotonin
transporter genotype on neurophysiological parameters is more
consistent (Munafo et al., 2008). A number of recent studies showed
that the amygdala, a key region for emotional processing which is
densely innervated by serotonergic neurons (Hariri et al., 2006;
Hensler, 2006), is more reactive in s-carriers during the passive perception of negative emotions compared to l-homozygotes (Bertolino
et al., 2005; Hariri et al., 2002, 2005; Heinz et al., 2005; Pezawas et al.,
2005; Rhodes et al., 2007; Smolka et al., 2007). During passive
perception, the 5-HTTLPR genotype was also related to alterations in
the functional connectivity between the amygdala and the prefrontal
cortex (Heinz et al., 2005; Pezawas et al., 2005); additional structural
differences were found in the amygdala, the dorsolateral (dlPFC) and
ventrolateral (vlPFC) prefrontal cortex, the dorsomedial prefrontal
cortex (dmPFC) and the anterior cingulate cortex (Canli et al., 2005;

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D.M. Schardt et al. / NeuroImage 53 (2010) 943951

Heinz et al., 2005; Pezawas et al., 2005). The observed genotypedependent dysregulations regarding the structure, function and
connectivity within the amygdala-prefrontal emotion network are
thought to contribute to the increased negative emotionality observed
in s-allele carriers (Canli and Lesch, 2007; Hariri and Holmes, 2006).
These ndings clearly underscore the relevance of genetic variation
within the serotonergic system with respect to the central processing
of emotions, despite heterogeneous results regarding the inuence of
the 5-HTTLPR genotype on direct measures of 5-HTT central
availability. While some studies found that 5-HTT central availability
and mRNA levels in midbrain regions are inuenced by the serotonin
transporter genotype (Little et al., 1998; Reimold et al., 2007; van Dyck
et al., 2004), others failed to nd an association (Parsey et al., 2006;
Shioe et al., 2003). For example, a PET investigation with the
radiotracer [11C]McN 5652 found no effect of 5-HTTLPR genotypes
on serotonin transporter binding potential in the amygdala (Parsey
et al., 2006). This led the authors to conclude that 5-HTTLPR related
neural effects cannot be explained by altered serotonin transporter
binding but may be due to differences in methylation patterns or
changes in maximum velocity in the 5-HTT enzymatic reaction. Since
the association between the 5-HTTLPR and central measures of
emotional reactivity has however been reliably replicated, it also
seems reasonable to assume that a relative loss in serotonin
transporter function related to the short variant might have a
detrimental effect especially during the development of the neural
circuits involved in emotional regulation (Hariri and Holmes, 2006;
Parsey et al., 2006). Consistent with this notion, in rodents a disruption
in 5-HT functioning during early development entails alterations in
neural structure and function, such as for example lasting emotional
abnormalities (Esaki et al., 2005; Gaspar et al., 2003).
However, all previous investigations of 5-HTTLPR-dependent
effects on the neural processing of emotions have been restricted to
passive perception. In turn, the willful employment of cognitive
strategies is known to be effective in changing subjective and
physiological responses to emotional stimuli. Amygdala activation is
effectively reduced during e.g. labeling (Hariri et al., 2003), cognitive
reappraisal (Goldin et al., 2008; Ochsner et al., 2002, 2004) and
detachment (Beauregard et al., 2001; Levesque et al., 2004) through
topdown inuences from the dlPFC and vlPFC, the orbitofrontal
cortex (OFC) and the parietal cortex. Thus, the question arises if willful
emotion regulation is able to alter genetically determined differences
in amygdala reactivity. This question is not only relevant within
cognitive and clinical neuroscience but also touches questions related
to the power of conscious will. Recent years have witnessed a number
of studies trying to answer questions related to the power of
conscious will not by armchair philosophy but by conducting
empirical studies of the will using neuroscientic means (Bechara,
2005; Haggard, 2008; Soon et al., 2008; Walter, 2001; Wegner, 2004).
To investigate the effect that willful cognitive acts have on
genetically predisposed emotional processing, we conducted a functional magnetic resonance imaging (fMRI) study investigating the
passive perception and explicit regulation of negative and neutral
emotional material in female 5-HTTLPR short and long individuals.
Since on the one hand the s-allele of the serotonin transporter genotype
has been associated with amygdala hyperreactivity (Hariri et al., 2002,
2005), and on the other hand, amygdala BOLD signal is effectively
attenuated through cognitive emotion regulation (Beauregard et al.,
2001; Goldin et al., 2008; Levesque et al., 2004; Ochsner et al., 2002,
2004), this region was designated our a priori region of interest.
Our primary goal was to evaluate whether a willful effort, i.e. the
deliberate use of a cognitive emotion regulation strategy, can reduce
the genetically mediated amygdala hyperreactivity to aversive
emotional material. We used negative stimuli that evoked either
fear or disgust in order to evaluate whether s-allele carriers show
increased neural activation to the perception of aversive versus
neutral stimuli (Calder et al., 2001; Hariri et al., 2002). Since fear

stimuli are more relevant in the context of anxiety and depression, we

hypothesized that amygdala hyperreactivity in the short genotype
group is primarily related to the perception of fear and less to the
perception of disgust (Hariri et al., 2005; Lau et al., 2009; Schienle
et al., 2005). Regarding the effects of emotion regulation on 5HTTLPR-mediated amygdala hyperreactivity, two alternative assumptions arise: as prior work suggests that the dysfunctions within the
amygdala-prefrontal emotion network are caused by developmental
changes, the bias towards negativity might be unspecic with respect
to the presence or absence of willful efforts. In this case, the s-group
should show increased amygdala reactivity during passive perception
and less or even no amygdala attenuation during cognitive emotion
regulation compared with l/l-homozygotes. On the other hand,
cognitive emotion regulation might counteract the genetically
determined amygdala hyperreactivity, in which case we should nd
equal or even larger amygdala signal reductions during the regulation
of negative affect in s-carriers. To address these questions, we
compared the neural responses of the two genotype groups in both
the presence and absence of regulation.
Materials and methods
Subject data
Participants
Forty-four right-handed female university students of central
European descent with no history of neurological/psychiatric illness
or substance abuse participated in the study. Only female subjects
were studied to ensure comparability with previous studies on
cognitive emotion regulation which were also restricted to females
(Drabant et al., 2009; Eippert et al., 2009; Goldin et al., 2008; Ochsner
et al., 2002, 2004; Schaefer et al., 2002; Walter et al., 2009) and to avoid
confounding effects of genotype and gender (Barr et al., 2004; Mizuno
et al., 2006; Staley et al., 2006; Walderhaug et al., 2007). Written
informed consent was obtained, and subjects received a nancial
reward for their participation. The study protocol was approved by the
local ethics committee, in accordance with the Declaration of Helsinki.
Five participants were excluded due to technical problems or excessive
movement artifacts and two further participants were excluded
following failure of valid 5-HTTLPR genotyping. A total of 37
participants (mean SD: age M = 22.6 2.2) were included in the
analyses. For further assessment, neuroticism (N) scores from the
NEO-FFI (Costa and McCrae, 1992), Harm Avoidance (HA) from the TCI
(Cloninger et al., 1993) and habitual emotion suppression (S) and
reappraisal (R) scores from the ERQ (Gross and John, 2003) were
collected.
Genotyping
Venous blood samples obtained from all subjects were assessed
regarding the genetic variation of the promoter region of the
serotonin transporter gene. Genomic DNA was extracted from the
EDTA anti-coagulated venous blood samples using standard techniques. Polymerase chain reaction without multiplexing was conducted
following previously described methods (Wendland et al., 2006). In a
total volume of 20 l, 25 ng of genomic DNA was amplied in the
presence of 1 Promega PCR Master Mix (www.promega.com), with
oligonucleotide primers 5-HTTLPR-forw (5-FAM-TCCTCCGCTTTGGCGCCTCTTCC-3) and 5-HTTLPR-rev (5-VIC-TGGGGGTTGCAGGGGAGATCCTG-3), the sequences of which are given in Wendland et al.
(2006). To evaluate genotyping accuracy, 10% of the samples were
analysed in duplicate: amplication with the oligonucleotide primers
of Lesch et al. (1996), stpr5 (5-GGCGTTGCCGCTCTGAATGC-3) and
stpr3 (5-GAGGGACTGAGCTGGACAACCAC-3) and all yielded identical genotypes.
Based on previous reports (e.g. Hariri et al., 2002, 2005), participants were divided into two groups: individuals carrying one (s/l:

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945

n = 7) or two (s/s: n = 14) short alleles were assigned to the s-group,

while the l-group was formed by the long allele homozygous
individuals (l/l: n = 16). The two groups did not differ with respect
to age (t[35] = 0.84, p = 0.41), trait anxiety (STAI-T t[35] = 1.32,
p = 0.2) or depression scores (BDI t[35] = 0.05, p = 0.96).

tilted acquisition and low TE, ensures optimal signal detection

especially in the medial temporal cortex and amygdala by limiting
partial volume effects due to tissue borders in this region (Merboldt
et al., 2001; Weiskopf et al., 2006). A visual inspection of all data sets
conrmed excellent BOLD sensitivity in the amygdala.

Imaging

Data analysis

Functional task
The task completed by the participants during the scanning
session involved the natural experience of (perception) or active
detachment from (regulation) emotional responses following the
presentation of emotional stimuli in an event-related design successfully used in prior studies (Staudinger et al., 2009; Walter et al., 2009).
Participants viewed fear, disgust and neutral pictures from a
standardized stimulus set (Lang et al., 2008), matched for complexity,
content, color and brightness. Some additional pictures in the disgust
condition were collected by the authors to balance picture content
across conditions. During the perception condition, participants were
instructed to look at the picture and permit yourself to feel
whichever emotional response arises naturally, without trying to
alter it; in the regulation condition, they were to look at the picture
while detaching yourself from any emotional response which may
arise by adopting the position of a detached observer, who is not
affected by the scene presented in the picture. Participants were
trained outside the scanner until they felt condent to perform the
task during fMRI.
Thirty-two pictures of each valence were presented. Half of the
trials of each valence were presented with the perception instruction
and the other half with the regulation instruction. Subjects completed
four sessions of 24 trials each: two sessions included unregulated and
regulated fear and neutral trials; the other two sessions included
unregulated and regulated disgust and neutral trials. The sessions
containing fear and disgust were presented in alternating order; and
the order of sessions was counterbalanced across subjects (i.e. half of
the subjects started with a fear session, the other half started with a
disgust session). Within each session, trial presentation was pseudorandomized, with no more than two consecutive trials of the same
condition. A given trial began with an instruction of 2 s, followed by
8 s of picture presentation, and ended with a xation of 12 s during
which participants were instructed to relax.
After the scanning session, participants lled in a questionnaire
containing all 96 previously presented pictures. They rated the overall
valence of each picture (1very unpleasant to 9very pleasant) to
test whether aversive pictures were perceived as more negative than
neutral pictures. Additional ratings were obtained assessing the
overall compliance with the instructed detachment strategy (1
never used it to 9always used it) and the success at implementing
the strategy (1not at all successful to 9very successful). To
ensure that equal attention was paid to both regulated and
unregulated pictures, subjects also indicated which strategy they
remember to have followed during each picture's presentation.

Behavioral data
For the pleasantness and memory ratings, repeated-measures
ANOVAs were carried out to asses the effects of genotype (s-group,
l-group), regulation (perception [p], regulation [r]) and valence (fear
[F], disgust [D], neutral [X]). Regarding the compliance and regulation
success ratings, the effect of the 5-HTTLRP genotype was assessed
by independent-samples t-tests comparing the scores between the
s-group and the l-group. Statistical analyses of the behavioral data
were thresholded at p b 0.05. A GreenhouseGeisser correction was
applied where appropriate in the case of non-sphericity.

Functional image acquisition

During the functional image collection, visual stimuli were
presented via binocular video goggles (NNL, Bergen, Norway)
attached to the head coil and adjusted to t the subjects' vision.
Functional data were collected on a 3-T Siemens Trio with a gradientecho T2-weighted echo-planar imaging sequence. Thirty-one axial
slices covering the whole brain volume were acquired (repetition
time/echo time [TR/TE] = 2000/25 ms, ip angle = 90, eld of
view = 210, 64 64 matrix). Slice thickness was 3.3 mm with a gap
of 25 %, resulting in an in-plane resolution of 3.3 3.3 4.125 mm. The
slices were tilted by 45, following an imaginary line from the lower
boundary of the orbitofrontal cortex (BA 11) to the lower boundary of
the cerebellar nodule. The high in-plane resolution, along with the

Functional image processing

Before pre-processing with SPM2 (Statistical Parametric Mapping,
Wellcome Institute of Cognitive Neurology, London, UK), the rst
three volumes in each session were discarded to allow for T1
saturation. Functional imaging data analysis was carried out using
SPM2 for pre-processing, SPM5 for statistical analyses (Statistical
Parametric Mapping, Wellcome Institute of Cognitive Neurology,
London, UK) and Matlab 6.5.1 (MathWorks, Natick, MA, USA). The
images were slice-timed to correct for differences in slice acquisition
time, realigned to the rst volume of the rst session to correct for
head motion, spatially normalized to the standard EPI template and
smoothed with an 8-mm FWHM isotopic Gaussian kernel. Serial
autocorrelations were accounted for by a rst order autoregressive
model (AR1) and a high-pass lter with a cut-off of 128 s was applied.
Subsequent to pre-processing, a rst-level xed-effects model was
set up for each participant in SPM5: regressors were created for the
instruction phase, the experimental conditions and the xation period
in each of the four sessions. Two sessions for each subject contained
fear (permit fear [pF], regulate fear [rF]) as the aversive condition, while
the other two sessions contained disgust (permit disgust [pD], permit
regulate disgust [rD]) as the aversive condition. Boxcar regressors
representing the actual length of each condition in seconds (i.e. 2-s
instruction, 8-s picture presentation, 12-s xation) were convolved
with the canonical hemodynamic response function (HRF) implemented in SPM5. Movement parameters were also included in the model as
regressors of no interest, resulting in a total of 48 regressors.
Predetermined effects of each condition versus xation were
calculated using t-statistics (pF N x, pD N x, pX N x, rF N x, rD N x,
rX N x). The individual statistical images were then included in a
group-level random-effects model that accounts for both scan-to-scan
and subject-to-subject variability. At the group level, a three-factorial
2 2 3 design (genotype, regulation and valence) with nonsphericity correction to account for unequal variances was set up using
SPMs full-factorial model. To test whether the experimental
manipulation was successful, the effects of valence and condition
collapsed across genotypes were compared. Subsequently, the
inuence of valence and regulation was compared between genotype
groups. Masking of the a priori search region was performed with a
sphere of 12-mm diameter in the center of both amygdalae (x, y, z =
26, 4, 16). This region of interest was chosen based on the maxima
reported in previous, independent studies regarding the inuence of
the 5-HTTLPR genotype on the perception of aversive versus neutral
stimuli (Bertolino et al., 2005; Hariri et al., 2002; Heinz et al., 2005;
Pezawas et al., 2005). The t-statistics for each individual voxel
were considered signicant at p b 0.05 family-wise error (FWE)
corrected for multiple comparisons on the whole brain level and in
the amygdala.

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D.M. Schardt et al. / NeuroImage 53 (2010) 943951

Coordinates of cluster maxima were converted from MNI to

Talairach space (mni2tal-transform by Matthew Brett, www.mrc-cbu.
cam.ac.uk/Imaging/mnispace.html) for labeling according to the
Talairach and Tournoux (1988) atlas, but for reasons of accuracy,
the original unconverted MNI coordinates will be reported throughout (Chau and McIntosh, 2005; Poldrack et al., 2008).
Functional connectivity analysis
A post hoc psycho-physiological interaction (PPI) analysis was
carried out to elaborate on interactive effects of the 5-HTTLPR
genotype and the willful regulation of fear. In this analysis,
functional connectivity between a seed region and functionally
connected regions is assessed through signicant differences in
correlations depending on different tasks (Friston et al., 1997). To
this end, individual time series were extracted from the right
amygdala using the masking procedure described above. The right
amygdala was chosen as the seed region for this analysis because an
inuence of cognitive regulation on amygdala hyperreactivity to fear
was only found in the right but not in the left amygdala. The
physiological variable (amygdala ROI time-course), the psychological
variable (regulation versus perception of fear) and their interaction
term along with the movement parameters were entered into a rstlevel xed-effects model in SPM5. The resulting contrast weights of
the interaction term were then entered into a second-level ANOVA
to compare amygdala coupling between the s-group and the l-group.
Non-sphericity correction was applied to account for unequal
variances. Voxel level t-statistics were considered signicant at an
initial threshold of p b 0.005 uncorrected; for a priori regions of
interest, we used a small volume correction similar to the approach
reported in Eippert et al. (2009), with p-values 0.05 FWEcorrected for small volume. The peak coordinates for these
corrections were extracted from previous studies on the effects of
the 5-HTTLPR genotype or cognitive emotion regulation on the
amygdala. Correction of the vlPFC (Goldin et al., 2008) was achieved
using a sphere of 14-mm radius. Spheres with 10-mm radius for
correction of the sACC (Pezawas et al., 2005), rACC (Heinz et al.,
2005) and dmPFC (Ochsner et al., 2002) were applied. The diameters
of the spheres were chosen according to anatomical constraints. As
stated above, the original MNI coordinates of cluster maxima will be
reported.

s-carriers and l/l-homozygotes (compliance: t[35] = 0.30, p = 0.76;

success: t[35] = 1.46, p = 0.15).
Brain imaging results
Overall BOLD signal changes during perception and cognitive regulation
of negative cues
For the assessment of the overall effects of valence and regulation,
the neural responses to the perception of and the detachment from
aversive stimuli were compared collapsed across genotype groups.
Functional imaging data revealed a signicant (p b 0.05 FWEcorrected for multiple comparisons) main effect of valence in the
bilateral amygdala, where the BOLD signal was higher following both
fear and disgust stimuli as compared to neutral stimuli. The main
effect of the cognitive regulation of emotion yielded effective signal
attenuations in the bilateral amygdala, while signal increases were
observed in the dlPFC (BA 9), vlPFC (BA 47/10), the inferior (IPL, BA
40) and superior (SPL, BA 7) parietal lobule and the dmPFC (BA 8).
Additional information on activated regions can be obtained from
Table 1.

Personality scores
The 5-HTTLPR genotype groups did not differ regarding harm
avoidance or neuroticism scores (HA: t[35] = 1.55, p = 0.13; N:
t[35] = 1.66, p = 0.11). The habitual use of suppression was also
unaffected by 5-HTTLPR genotype (ERQ-S: t[35] = 0.63, p = 0.53).
However, the habitual use of reappraisal was more frequent in l/lhomozygotes compared with 5-HTTLPR s-allele carriers (t[35] =
2.42, p = 0.02).

Comparison of amygdala activation between 5-HTTLPR genotype groups

Neural responses of s-carriers and l/l-homozygotes were compared regarding the perception of fear, disgust and neutral stimuli
within our a priori region of interest in the bilateral amygdala (p b 0.05
FWE-corrected for multiple comparisons). Subsequently, the inuence of the cognitive regulation of fear and disgust on amygdala
reactivity was compared between s-allele carriers and the group of
l/l-homozygotes.
Amygdala hyperreactivity was observed in individuals with the
5-HTTLPR short variant. In s-allele carriers, the passive viewing of
pictures that induce fear entailed greater activation in the right
amygdala compared with neutral stimuli (x, y, z = 26, 4, 18,
t = 2.87, p = 0.037), while amygdala activation to the perception of
disgust versus neutral stimuli did not differ between the two 5HTTLPR genotype groups. Moreover, the bilateral amygdala of
s-carriers showed higher activation to fear compared with disgust
perception (left: x, y, z = 28, 8, 18, t = 2.77, p = 0.047; right:
x, y, z = 26, 4, 20, t = 2.97, p = 0.029). Despite the amygdala
hyperreactivity to fear, effective signal attenuation was observed in
response to detachment in individuals with the 5-HTTLPR short
variant (x, y, z = 26, 2, 12, t = 3.27, p = 0.013): s-carriers showed
larger signal reductions in the right amygdala during the regulation of
fear when compared to l/l-homozygous individuals (Fig. 1). Again, no
comparable effect was observed for the cognitive regulation of
disgust. For more information on activated regions, see Supplemental
material online.
In an exploratory analysis, we also found evidence that the
s-carriers' larger amygdala signal reduction during regulation is valence
specic when we lowered the threshold to an uncorrected p b 0.05 for
the a priori search region: the amygdala of s-carriers was signicantly
more attenuated during the regulation of fear compared with both
disgust ([pF N rF]N [pD N rD]) and neutral stimuli ([pF N rF] N [pX N rX]).

Manipulation check
Both fear and disgust stimuli were perceived as signicantly more
unpleasant in comparison with the neutral stimuli (valence: F[2,70]
= 402.58, p b 0.0001). Subjects attended the regulated stimuli well:
when asked to indicate the condition under which a picture had been
presented during fMRI, hit rates were higher for the two negative
conditions (valence: F[2,70] = 5.1, p b 0.01) and especially so for the
regulated stimuli (valence regulation: F[2,70] = 57.6, p b 0.0001).
The 5-HTTLPR genotype did not have any effects on pleasantness
ratings or hit rates (all p N 0.12).
The compliance with and success at implementing emotion
regulation was generally very high and did not differ between

5-HTTLPR effects on amygdala functional connectivity during cognitive

regulation
To assess amygdala functional connectivity during the cognitive
regulation of fear, we performed a psycho-physiological interaction
analysis (Friston et al., 1997). Since only the right amygdala showed a
5-HTTLPR effect during both perception and regulation of fear, this
region was chosen as the seed region for the psycho-physiological
interaction (PPI) analysis. Generally, detachment from fear modulated
the functional connectivity between the right amygdala and the
dlPFC (BA 9) and vlPFC (BA 47), the ventromedial PFC (vmPFC, BA
10), dorsal ACC (dACC), the medial OFC (mOFC, BA 11) and the dmPFC
(BA 6).

Results
Behavioral results

D.M. Schardt et al. / NeuroImage 53 (2010) 943951

947

Table 1
Overall effects of stimulus valence and regulation (s-allele carriers + l/l-homozygotes).
Regions

Fear N Neutral ([pF + rF] N [pX + rX])

Amygdala

Right/left

Thalamus
Middle temporal/fusiform gyrus

L
R
R
L

Inferior parietal lobule

R
L

Disgust N Neutral ([pD + rD] N [pX + rX])

Amygdala
Dorsolateral prefrontal cortex
Ventrolateral prefrontal cortex
Pre-supplementary motor area
Parahippocampal gyrus
Inferior parietal lobule
Cuneus/Middle occipital gyrus
Fusiform/lingual gyrus
Brainstem

L
R
R
L
R
L
L
L
R
L
R

Perception N Regulation ([pF + pD + pX] N [rF + rD + rX])

Amygdala
L
R
Thalamus
L
Medial orbitofrontal cortex
L
Insula
L
Cuneus/lingual gyrus
R
L
Cerebellum
L
Posterior cingulate cortex
R
Parahippocampal gyrus
R
L
Regulation N Perception ([rF + rD + rX] N [pF + pD + pX])
Dorsolateral prefrontal cortex
R
Ventrolateral prefrontal cortex
R
L
Dorsomedial prefrontal cortex
R
Pre-supplementary motor area
R
Frontal eye eld
R
Insula/precentral gyrus
R
Inferior parietal lobule
R
L
Superior parietal lobule
L
Cerebellum
L

Brodmann
area

37
36
39
40

9
47
6
35
40
19
19/37

11
13
18
18

Cluster size
(voxels)

t-score local
maximum

p-value
(FWE)

MNI coordinates
x

1
699
3
312
10

3.96
3.78
4.84
6.76
5.08
6.08
5.06

0.001
0.003
0.049
b 0.001
0.019
b 0.001
0.021

26
28
10
52
40
50
60

8
6
10
66
36
60
28

18
20
0
8
20
8
34

16
51
36
8
11
91
79
3336
24

3.94
5.47
5.29
5.66
5.74
5.04
5.19
5.46
5.35
9.21
5.40

0.002
b 0.001
0.008
0.002
0.001
0.023
0.012
0.004
0.006
b 0.001
0.005

22
22
50
32
10
16
62
26
36
42
8

2
2
6
30
6
28
30
78
78
64
24

18
16
30
10
68
4
40
26
18
6
12

3.49
3.54
4.98
5.33
4.44
7.96
7.62
7.12
6.39
6.15
5.4

0.007
0.006
0.029
0.007
b 0.001
b 0.001
b 0.001
b 0.001
b 0.001
0.005

28
16
2
2
32
26
24
6
12
8
24

0
8
6
54
14
92
92
44
48
40
28

18
14
6
12
22
2
4
0
6
0
6

8.55
8.29
7.31
5.15
7.28
5.42
6.5
9.17
5.53
4.88
5.53

b 0.001
b 0.001
b 0.001
0.014
b 0.001
b 0.004
b 0.001
b 0.001
0.003
0.043
0.003

34
38
32
4
24
44
40
56
56
16
36

40
48
46
28
6
14
16
52
52
60
50

32
4
8
40
68
44
4
40
44
62
46

2
24
85
1743
1109
1932

34
15

8/9
10
8
6
8
13/44
40
7

2075
496
12
387
33
289
1719
52
2
47

All coordinates are given in MNI space together with their t-scores. ROI: bilateral amygdala (x, y, z = 26, 4, 16), signicance threshold for ROI analyses p b 0.05 FWE-corrected
for small volume. Whole-brain analyses were thresholded at p b 0.05 FWE-corrected for multiple comparisons. pF: perception fear; pD: perception disgust; pX: perception neutral;
rF: regulation fear; rD: regulation disgust; rX: regulation neutral.

In a direct comparison between the 5-HTTLPR genotype groups,

cognitive regulation had a stronger modulatory inuence on functional connectivity between the amygdala and the bilateral vlPFC (BA
10/47), the left mOFC (BA 11), the subgenual (sACC, BA 25) and the
rostral (rACC, BA 32) anterior cingulate cortex in individuals carrying
at least one s-allele (Fig. 2). Detailed information on activation for this
comparison is reported in Table 2. In contrast, the l-group did not
exhibit regions of increased connectivity during cognitive regulation,
even when lowering the threshold to an uncorrected p b 0.05.
Discussion
Here, we show that heightened amygdala reactivity attributable to
the 5-HTTLPR short variant can be successfully attenuated by the
willful, cognitive regulation of emotions (Fig. 1), that this effect is
present for fear but not observed for disgust, and that it is mediated in
s-carriers by altered functional coupling between the amygdala and
the medial and ventrolateral prefrontal cortices (Fig. 2).

Both fear and disgust induced reliable activation in the amygdala

amongst other regions in comparison to emotionally neutral stimuli
(Calder et al., 2001; Phan et al., 2002). As expected from the growing
body of literature on increased amygdala reactivity to negative
emotional stimuli, s-carriers in our study also showed increased
amygdala activation during the passive perception of fear versus
neutral stimuli (Hariri et al., 2002, 2005). However, the same
s-carriers showed no amygdala hyperreaction to disgust, which
suggests that the effect of the serotonin transporter genotype on
amygdala activation might be modulated by the type of negative
emotion evoked by a stimulus; this notion is also supported by the fact
that the cognitive regulation of fear led to a larger decrease in
amygdala activation in the short allele group (Goldin et al., 2008;
Ochsner et al., 2002, 2004). However, these ndings speak against the
assumption that emotional regulation is generally compromised in
short allele carriers: the amygdala hyperreactivity to fear perception
in s-carriers was not present anymore during the cognitive regulation
of fear. It is unlikely that this effect is attributable to differences in

948

D.M. Schardt et al. / NeuroImage 53 (2010) 943951

Fig. 1. Increased amygdala signal attenuation during cognitive emotion regulation in 5-HTTLPR short allele carriers. (A) Individuals with the 5-HTTLPR short genotype exhibit greater
(p b .05 FWE-corrected for multiple comparisons) signal reduction in the right amygdala during the regulation of fear compared with l/l-homozygotes. (B) BOLD signal change values
(mean SEM) from the right amygdala ROI show that genotype-dependent group differences were driven by markedly increased activations during the perception but not the
regulation of fear in the s-group, while no such effect was observed for disgust or neutral stimuli. SEM: standard error of mean; pF: perception fear; pD: perception disgust; pX:
perception neutral; rF: regulation fear; rF: regulation disgust; rX: regulation neutral.

attention between the perception and the regulation conditions, as

subjects were able to correctly identify the majority of the regulated
pictures in a post-scan assessment, independent of their 5-HTTLPR
genotype. Moreover, we found evidence for a neurobiological
mechanism mediating this effect in the medial and the ventrolateral
prefrontal cortex. Although functional connectivity between the
medial prefrontal cortex and the amygdala was previously reported
to be decreased in s-carriers in an emotion paradigm without explicit
regulation (Pezawas et al., 2005), short allele carriers in the present
study were able to effectively down-regulate their hyperreactive
amygdalae. This effect was mediated by signicantly stronger
functional connectivity between the amygdala and the ventromedial
and ventrolateral prefrontal cortices during cognitive regulation.
Recent reports have described increased connectivity between the
amygdala and the dorsomedial, orbitolateral and ventrolateral
prefrontal cortices during willful emotion regulation (Banks et al.,
2007; Maren and Quirk, 2004; Wager et al., 2008). Increased
connectivity with the prefrontal cortex during cognitive regulation
might thus signal stronger inhibitory inuences on the amygdala
through direct afferent (rACC) or efferent (sACC, medial OFC)
anatomical connections (Paus, 2001; Stefanacci et al., 1996; Stein
et al., 2007).
How do the present ndings t in with the assumption of a
detrimental effect of the short allele on emotional regulation? The
present results emphasize that a clear distinction must be made
between implicit (automatic) and explicit (effortful) processing with
respect to the effects of the serotonin transporter genotype on neural
processing (MacDonald, 2008; Phillips et al., 2008; Satpute and
Lieberman, 2006). The decreased coupling between the amygdala and

the medial prefrontal cortex in s-carriers which has been described

previously (Pezawas et al., 2005) was observed when emotional
regulation was not an explicit task but occurred instead automatically.
In this paradigm, subjects either only viewed negative stimuli, or
matched them without the instruction to regulate. In contrast, our
task involved the explicit regulation of emotions, which is known to
lead to increased coupling of the amygdala and ventromedial (Banks
et al., 2007) and ventrolateral (Wager et al., 2008) prefrontal cortices.
Thus, the present results suggest that the willful effort to regulate
emotions can compensate for amygdala hyperreactivity in 5-HTTLPR
s-carriers (Fig. 1), a nding which is important also with respect to the
role of the will in controlling predetermined reactions (Haggard,
2008; Walter, 2001) since it suggests that willful acts can indeed
compensate for genetically mediated emotional sensitivity. If the
explicit, cognitive regulation of emotions diminishes the amygdala
hyperreactivity in s-allele carriers, how can we explain their supposed
bias towards negative emotionality? One possible explanation is that
s-carriers do not habitually access the full potential of cognitive
regulation, as suggested by the lower habitual reappraisal scores in
s-carriers in our study. An intensied experience of negativity in these
individuals might critically compromise the implementation of
cognitive regulation, particularly when stress is frequently experienced. The repeated exposure to adverse life events depletes cognitive
resources and makes successful emotion regulation more difcult
(Gross, 1999), which may consequently lead to an increase of
depression. At rst glance, this interpretation may seem difcult to
reconcile with the results of two recent meta-analyses which did not
nd an association between an elevated risk for depression and the
serotonin transporter genotype alone or in interaction with stressful

Fig. 2. Increased amygdala connectivity during cognitive emotion regulation in 5-HTTLPR short allele carriers. Increased functional coupling during the cognitive regulation of fear
(rF N pF) was observed in 5-HTTLPR s-allele carriers between the right amygdala seed region around x, y, z = 26, 4, 16, and the bilateral ventrolateral prefrontal cortex, left
medial orbitofrontal cortex, right premotor cortex as well as rostral and subgenual parts of the anterior cingulate cortex. The threshold for visualization is set to p b 0.005 uncorrected.

D.M. Schardt et al. / NeuroImage 53 (2010) 943951

949

Table 2
Regions showing greater amygdala connectivity during fear regulation (rF-pF) in 5-HTTLPR short allele carriers.
Regions

s-allele carriers N l/l-homozygotes

Ventrolateral prefrontal cortex

Pre-supplemental motor area

Medial orbitofrontal cortex
Dorsomedial prefrontal cortex
Subgenual anterior cingulate cortex
Rostral anterior cingulate cortex
Dorsal anterior cingulate cortex
Posterior cingulate cortex
Hypothalamus
Insula

Precuneus/cuneus

Right/left

Brodmann
area

Cluster size
(voxels)

t-score local
maximum

p-value
(uncorrected)

MNI coordinates
x

3.79
3.12
3.93
3.32
3.53
2.99
2.98
3.2
3.69
3.2
3.98
3.09
3.59
3.73
3.22
4.35
4.21
3.6
3.17
4.22
3.17

b0.001
0.002
b0.001
0.001
0.001
0.003
0.003
0.001
b0.001
0.001
b0.001
0.002
b0.001
b0.001
0.001
b0.001
b0.001
b0.001
0.002
b0.001
0.002

40
54
34
28
32
10
18
10
8
6
14
10
8
2
40
40
40
14
18
14
0

42
34
48
28
14
36
36
32
24
46
2
10
42
2
8
12
26
60
68
58
74

10
6
6
4
56
16
14
36
14
2
36
30
12
4
10
20
24
34
12
36
34

L
L
R
R
R
L

11
47
10
47
6
11

88
18
268
25
147
45

L
R
R
L
L
L
L
L
R
R
L
L
R
R

8
25
32
24
24
29

31
37
23
33

13
13
13
7/31
30
7
7

76
80
10
205
129
31
719

All coordinates are given in MNI space together with their t-scores. Whole-brain analyses were thresholded at b0.005 (uncorrected), 0.05 FWE-corrected for small volume.

life events (Munafo et al., 2008; Risch et al., 2009). However, while it
is well acknowledged that large and homogeneously assessed
samples are necessary to identify gene-environment interactions
(Hoefgen et al., 2005), the samples included in the above mentioned
meta-analyses are heterogeneous with respect to ethnicity, study
design, sample recruitment and assessment of stressful life events.
Moreover, the sample size of 1769 clinically depressed individuals
may still be too low to identify inuences of the serotonin transporter
genotype on a specic subgroup. Since depression is both clinically
and genetically heterogeneous, and the impact of stressful life events
on an individual at a given point in time is hard to determine, a
replication within the same study design is needed to reliably provide
or refute an association between the serotonin transporter genotype,
environmental factors and depression.
In the present design, however, we did not test the direct inuence
of the serotonin transporter genotype on depression per se; rather, we
examined quantitative measures of phenotypes of the cognitive
control of emotions which may underlie the etiology of depression.
The relevance of successful emotion regulation with regard to mood
disorders is underscored by reports of impaired cognitive emotion
regulation in clinically manifest depression. Whereas greater activation in the ventrolateral and ventromedial PFC was associated with
reduced amygdala activation in healthy controls, clinically depressed
individuals showed the reverse pattern, where greater reappraisal
effort was related to increased amygdala activation (Johnstone et al.,
2007). In our study, individuals with the risk genotype were able to
effectively attenuate amygdala hyperreactivity to fear-related stimuli
by means of cognitive regulation. This suggests that explicit strategies
may be benecial in attenuating genotype-dependent amygdala
activation, despite a less efcient automatic emotion regulation. In
fact, the efcacy of cognitive behavioral therapy for depressed or
depression-prone subjects might also rely on this mechanism
(Johnstone et al., 2007). Future investigations of 5-HTTLPR effects
on neural processing are needed to determine how explicit cognitive
as compared to automatic mechanisms of emotion regulation are
altered in depressed patients and whether explicit training in emotion
regulation strategies alters these effects.
Furthermore, the present sample comprised only female participants, while gender differences have been observed with respect to
the behavioral effects of the serotonin transporter genotype and

during cognitive emotion regulation. For example, in women, the 5HTTLPR short genotype led to a more cautious response style and more
negative mood during acute tryptophan depletion compared with
men (Walderhaug et al., 2007). In a Japanese sample, the effects of the
5-HTTLPR genotype on anxiety scores were also mediated by gender:
female ls heterozygous individuals showed greater anxiety scores than
ss or ll homozygous individuals, while in the male subgroup, the ss
homozygous individuals showed the highest anxiety scores (Mizuno
et al., 2006). McRae and colleagues (2008) reported that although
women did not differ from men with respect to the behavioral
outcomes of reappraisal, cognitive emotion regulation was associated
with greater prefrontal activation and lesser down-regulation in the
amygdala in women. The interpretation offered by the authors posits
that in men, cognitive emotion regulation may be associated with less
effort, possibly due to a greater use of automatic emotion regulation; a
conclusion which ts in well with the fact that the prevalence of
affective disorders is higher in women (Gater et al., 1998; Kessler et al.,
1993; Maciejewski et al., 2001; Wilhelm et al., 1997). Conversely,
previous studies of 5-HTTLPR-mediated amygdala activation to
negative emotional cues have not found evidence for gender-related
differences (Hariri et al., 2002, 2005), suggesting that higher amygdala
activation in short allele carriers is independent of gender. Moreover,
cognitive regulation led to decreased amygdala activation across both
genotype groups in our study, suggesting that the intended strategy is
effective in female subjects in general. Since our participants were not
recruited at a specic time during their menstrual cycle, while some
studies have found differences in emotional processing related to
hormonal changes (Amin et al., 2006; Goldstein et al., 2005; Pearson
and Lewis, 2005; Protopopescu et al., 2005), we cannot exclude that
our ndings are biased in this respect. However, the genotypedependent modulation of the amygdala activation was observed to be
highly signicant despite the fact that we did not control for menstrual
status, which suggests that hormonal changes play no or only a minor
role with regard to our ndings. Thus, the present results offer
important insights into the effects of cognitive emotion regulation on
5-HTTLPR-mediated amygdala function, while their limited generalizability warrants future studies that control for menstrual cycle as
well as a replication within males.
In conclusion, the results of the present study lend new insights
into our understanding of the complex interplay between willful

950

D.M. Schardt et al. / NeuroImage 53 (2010) 943951

efforts and genetic determination. They suggest that genetically

predisposed neural processing may be counteracted by willful actions.
Furthermore, the present ndings may partly explain why short allele
carriers of the 5-HTTLPR are prone to affective disorders in the
presence of stressful life events and why cognitive behavioral therapy
is effective in improving the willful regulation of emotions. Our
ndings generate a new set of research questions concerning the
neural, genetic and environmental factors involved in affective
disorders as well as effective mechanisms for coping with them.
Finally, they also lend support for the thesis that conscious will can
effectively counteract genetic determinants of emotional behavior.
Conict of interest
The authors report no potential conicts of interest.

Acknowledgments
This study was supported by the Volkswagen Foundation (grants
II/80777, II/80774) and the German Federal Ministry of Education
and Research (BMBF; grant 01GS08159) in the context of the National
Genome Research Network (NGFN plus). We thank Christine Schml
for her help in proofreading the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.neuroimage.2009.11.078.
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