Serotonin Transporter Genotype (5-HTTLPR):

Effects of Neutral and Undefined Conditions

on Amygdala Activation
Andreas Heinz, Michael N. Smolka, Dieter F. Braus, Jana Wrase, Anne Beck, Herta Flor, Karl Mann,
Gunter Schumann, Christian Büchel, Ahmad R. Hariri, and Daniel R. Weinberger
Background: A polymorphism of the human serotonin transporter gene (SCL6A4) has been associated with serotonin transporter expres-
sion and with processing of aversive stimuli in the amygdala. Functional imaging studies show that during the presentation of aversive
versus neutral cues, healthy carriers of the short (s) allele showed stronger amygdala activation than long (l) carriers. However, a recent report
suggested that this interaction is driven by amygdala deactivation during presentation of neutral stimuli in s carriers.

Methods: Functional MRI was used to assess amygdala activation during the presentation of a fixation cross or affectively aversive or
neutral visual stimuli in 29 healthy men.

Results: Amygdala activation was increased in s carriers during undefined states such as the presentation of a fixation cross compared with
emotionally neutral conditions.

Conclusions: This finding suggests that s carriers show stronger amygdala reactivity to stimuli and contexts that are relatively uncertain,
which we propose are stressful.

Key Words: 5-HTT genotype, amygdala, baseline, emotion, fixation stimuli with the fixation condition. This pattern was explained by
cross, functional MRI the finding that s carriers showed relative decreases in amygdala
activation when contrasting neutral stimuli with fixation. Thus,
the direct contrast of negative versus neutral stimuli revealed an

I
n a recent report, Canli et al. (2005) employed functional
magnetic resonance imaging (fMRI) to assess the interaction
between a variable number of tandem repeats (VNTR)
genetic variation of the transcriptional control region (5-HTTLPR)
of the human serotonin transporter (5-HTT) gene (SLC6A4) and
amygdala activation elicited by affective stimuli. As is common
practice in blood oxygen level dependent (BOLD) fMRI studies,
subjects were exposed to alternating presentations of affective
stimuli (neutral, positive, and negative words) and a fixation
baseline condition (crosshair). This design allowed them to
examine both the main effects of the 5-HTTLPR on amygdala
activity related to affective words (e.g. negative word vs. fixation)
as well as the differential effects of the polymorphism on valence
specific amygdala activity (e.g. negative word vs. positive word).
Their analyses suggested that genotype-dependent differences in
amygdala activation are driven not by relatively increased acti-
vation to negative stimuli but rather relatively decreased activa-
tion to neutral stimuli, in carriers of the 5-HTTLPR short (s) allele.
More specifically, they found that amygdala activity was
relatively greater in s carriers when contrasting negative stimuli
with neutral stimuli but not when directly contrasting negative
From the Department of Psychiatry (AH, JW, AB), Charité University Medi-
cine Berlin, Campus Charité Mitte; Central Institute of Mental Health
(MNS, HF, KM), Mannheim; Department of Psychiatry (DFB), and the
Department of Systemic Neuroscience (CB), NeuroImage Nord, Univer-
sity Clinic Hamburg-Eppendorf, Germany; Institute of Psychiatry (GS),
London, United Kingdom; Department of Psychiatry (ARH), University of
Pittsburgh, Pennsylvania; National Institute of Mental Health (DRW),
National Institutes of Health, Bethesda, Maryland.
Address reprint requests to Daniel R. Weinberger, M.D., Genes, Cognition
and Psychosis Program, National Institute of Mental Health, National
Institutes of Health, Room 4S-235, 10 Center Drive, Bethesda, MD 20892;
E-mail: weinberd@mail.nih.gov.
Received January 24, 2006; revised June 29, 2006; accepted August 21, 2006.
effect of the s allele only because of the relative decrease in
amygdala activity during the processing of neutral stimuli. Inter-
estingly, there was no effect of the 5-HTTLPR reported on the
amygdala response to positive stimuli in contrast to either
fixation, neutral, or negative stimuli. These observations differ
from previous studies, which reported that, compared with
homozygote carriers of two long (l) alleles, healthy carriers of the
short (s) allele exhibit stronger amygdala activation during the
presentation of aversive or threatening stimuli versus neutral
stimuli (Bertolino et al. 2005; Hariri et al. 2002; Hariri et al. 2005;
Heinz et al. 2005). However, these prior studies either were not
designed to examine (Bertolino et al. 2005; Hariri et al. 2005) or
did not report (Heinz et al. 2005) the differential impact of the
5-HTTLPR on amygdala activation elicited by negatively and
positively valenced stimuli in comparison to a fixation baseline
condition.
Earlier studies have established that carriers of the s allele
display reduced 5-HTT protein expression and function (Lesch et
al. 1996; Little et al. 1998) and are at relatively increased risk to
develop major depression, especially after exposure to stressful
life events (Caspi et al. 2003; Eley et al. 2004; Kaufman et al.
2004). Given the increased vulnerability of s carriers for negative
mood states (Munafo et al. 2005; Schinka et al. 2004; Sen et al.
2004), it is important to further explore the specific nature of the
amygdala hyperactivity associated with this genetic variant to
better understand the biological pathways mediating this genet-
ically driven effect on mood. In particular, do the current
functional imaging results support the conclusion that s carriers
display relatively increased amygdala activation to affectively
negative stimuli as suggested by earlier work (Bertolino et al.
2005; Hariri et al. 2005; Heinz et al. 2005), or relatively reduced
activation to affectively neutral cues as suggested by Canli et al.
(2005)? The unexpected finding of Canli et al. may reveal not
only important insights as to the nature of 5-HTTLPR effects on

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doi:10.1016/j.biopsych.2006.08.019 © 2007 Society of Biological Psychiatry
1012 BIOL PSYCHIATRY 2007;61:1011–1014
amygdala function but also the general effects of the commonly
employed fixation baseline in fMRI studies.
The critical issue in resolving this question may involve the
nature of the stimuli used in these studies and their explicit
versus implicit emotional valence. As described previously, a
passive resting or fixation cross baseline condition under the
conditions of fMRI scanning may not be as emotionally neutral as
it appears, because it involves unpredictable mental states (Gus-
nard and Raichle 2001; Stark and Squire 2001) and environmental
confinement and noise, and the relatively unconstrained experi-
ence of the subjects during the presentation of baseline condi-
tions may be more uncertain and ambiguous than familiar
unambiguous stimuli (e.g. neutral words). It is conceivable that
this unstructured experience may be particularly pronounced in
s carriers who are possibly more threat sensitive and reactive
than l allele homozygotes (Lesch et al. 1996; Mazzanti et al.
1998). In addition, recent studies have demonstrated that the
human amygdala may be most sensitive to stimuli depicting or
conveying uncertain (e.g. neutral facial expressions) or ambigu-
ous (e.g. surprised facial expressions) information about the
environment (Adams et al. 2003; Kim et al. 2003, 2004). Thus, the
amygdala response of subjects genetically predisposed to being
relatively more threat sensitive (i.e., s carriers) may be most
pronounced during conditions of relatively increased ambiguity
and uncertainty (e.g. fixation) in comparison to structured,
explicit, and familiar information (i.e. neutral words). In order to
further examine this potential explanation, we reassessed our
data (Heinz et al. 2005) and directly compared amygdala activa-
tion to fixation baseline in contrast with affectively neutral stimuli
(Heinz et al. 2005).
Methods and Materials
Twenty-nine male volunteers (mean age 40 ⫾ 8 years, all
right-handed) participated in this study and provided informed,
Figure 1. Average signal change in left (upper row) and right (lower row) amygdala elicited by the contrast “affectively neutral stimuli versus fixation cross”
(left), “affectively negative stimuli versus affectively neutral stimuli” (center), and “fixation cross versus affectively neutral stimuli” (right) as function of 5-HTT
genotype. Note the pronounced activation increase in bilateral amygdala in s- versus ll-carriers for both affectively negative versus neutral and fixation cross
versus neutral stimuli.
www.sobp.org/journal
A. Heinz et al.
written consent according to the Declaration of Helsinki. All
subjects were of central European ancestry. The study was
approved by the Ethics Committee of the University of Heidel-
berg. Standardized clinical assessment with the Structured Clin-
ical Interview (SCID I and II) was performed to exclude axis I
and II psychiatric disorders according to DSM IV and ICD 10
(First et al. 1997, 2001). We assessed the genotype of the
promoter of the 5-HTT gene with polymerase chain reaction
(PCR) and oligonucleotide primers. Nine subjects were homozy-
gous for the long and nine subjects for the short allele of the
promoter of the 5-HTT gene; 11 heterozygote subjects carried
one short and one long allele of the 5-HTT gene; subjects were
not significantly different with respect to age, education, marital
status, State-Trait Anxiety Inventory (STAI; Spielberger et al.
1970) or depressed mood (Beck Depression Inventory; Beck et
al. 1961).
As emotional stimuli, we used a set of standardized affectively
unpleasant, pleasant, and neutral pictures (18 per category).
Using a standardized rating system (Bradley and Lang 1994) subjects
experienced the negative pictures as more arousing (5.1 ⫾ 2.8
versus 2.7 ⫾ 1.8; p ⫽ .004) and affectively less pleasant (1.7 ⫾ .8 vs.
6.2 ⫾ 1.2; p ⬍ .001) than the neutral pictures. Ratings of valence
and arousal were not related to genotype. The stimuli were
presented for 750 msec in an event-related design in random
order and an intertrial interval (fixation cross) between 9.9-19.8
sec. Participants were instructed to passively view the stimuli,
because even simple rating tasks can influence amygdala activa-
tion elicited by aversive pictures (Taylor et al. 2003). Only male
healthy volunteers were included in the present study to exclude
gender-related effects and reduce heterogeneity.
Scanning was performed with a 1.5T magnetic resonance
scanner (Magnetom VISION, Siemens, Erlangen, Germany) and a
standard echo planar imaging sequence (24 slices, 4 mm thick-
ness, repetition time (TR) 3.3 sec, echo time (TE) ⫽ 66 msec, flip
A. Heinz et al.
Table 1. Average Signal Change in Left and Right Amygdala Elicited by the Contrasts “Affectively Neutral Stimuli Versus Fixation Cross” and “Affectively
Negative Stimuli Versus Affectively Neutral Stimuli” as Function of 5-HTT Genotype
Left
Neutral – Fixation Negative – Neutral
Mean SD Mean SD
LL .037 .081 ⫺.001 .094
SL ⫺.007 .087 ⫺.001 .055
SS ⫺.055 .079 .104 .073
angle ⫽ 90°) with an in-plane resolution of 64 ⫻ 64 pixels (field
of view 220 mm). Data were analyzed with Statistical Parametric
Mapping (SPM2; www.fil.ion.ucl.ac.uk/spm/) by modeling the
different conditions (negative pictures, neutral pictures, and
fixation cross baseline) as explanatory variables within the
context of the general linear model. To detect the association
between genotype and fMRI activation in the amygdala on a
voxel-by-voxel basis, the contrast images of all subjects (signal
change of neutral pictures vs. fixation cross as well as negative
vs. neutral pictures) were included in a second level random
effects analysis with SPM. In vitro studies suggested that the
5-HTTLPR s allele is dominant (Lesch et al. 1996; Little et al. 1998);
therefore, genotype groups (ll vs. sl and ss) were compared using
independent-samples t-tests. Masking was done with a sphere of 12
mm diameter in the center of both amygdalae (Montreal Neurolog-
ical Institute (MNI) coordinates: x, y, z ⫽ ⫾ 24, -3, -15). All
thresholds were set to p ⬍ .05.
Results
Consistent with the recent report of Canli et al. (2005), we
observed a 5-HTTLPR genotype-dependent increase in the activa-
tion of both amygdalae not only when comparing aversive versus
neutral stimuli (as reported before: see Hariri et al. 2002, 2005;
Bertolino et al. 2005; Heinz et al. 2005) but also when comparing
fixation versus neutral stimuli (left amygdala: (MNI: x, y, z) ⫽ (-21,
-6, -18), t (27) ⫽ 1.94; right amygdala: (MNI: x, y, z) ⫽ (24, -6, -15),
t (27) ⫽ 1.93; Figure 1 and Table 1).
Discussion
This study shows that unconstrained processing of an affectively
undefined symbol such as a fixation cross in the potentially anxio-
genic (narrow, dark and loud) environment of an MRI scanner
elicited increased amygdala activation in s carriers compared with
relatively more concrete visual processing of affectively neutral and
familiar stimuli. This finding further underscores the general impor-
tance of carefully considering the nature of baseline conditions in
affective neuroscience studies (Gusnard and Raichle 2001; Stark and
Squire 2001). Unlike measurement of cerebral blood flow with
positron emission tomography, BOLD fMRI does not allow for
measurement of an absolute baseline magnitude of activation, and
is thus always dependent on relative responses to varying cognitive
or affective demands. While it could be argued that s carriers may
display decreased amygdala activation during the presentation of
affectively neutral stimuli in comparison to fixation baseline, the
similarity of 5-HTTLPR genotype-dependent response profiles for
fixation (in a potentially stressful context) versus affectively neutral
stimuli as well as affectively aversive versus neutral stimuli (Heinz et
al. 2005) suggests that the fixation baseline in the MRI scanner
environment may be a relatively more aversive or anxiogenic
context, particularly for s carriers with potentially increased vulner-
BIOL PSYCHIATRY 2007;61:1011–1014 1013
Right
Neutral – Fixation Negative – Neutral
Mean SD Mean SD
.024 .061 ⫺.024 .080
⫺.038 .088 .001 .063
⫺.022 .058 .088 .090
ability for anxiety-related traits such as threat sensitivity (Lesch et al.
1996).
Altogether, these observations suggest that 5-HTTLPR s carriers
are generally more reactive to their environments and respond
with increased amygdala activation not only to clearly aversive
stimuli but also to undefined and ambiguous cues, especially
in anxiogenic contexts. The common link between these
conditions/stimuli is the underlying need to martial resources
(both behavioral and physiologic) to meet impending or
potential environmental challenges. As such, the 5-HTTLPR may
bias mood and temperament by increasing the reactivity of the
amygdala to stimuli and contexts that are relatively uncertain and
unbounded. This hypothesis can be explored in further studies
that directly manipulate the degree of uncertainty of the stimulus
material.
This research was supported in part by the Deutsche For-
schungsgemeinschaft (He 2597/7-3, Sm 80/1-1) and the Na-
tional Genome Research Network NGFN-1 (FKZ:01GS0117).
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