Supplemental material
Manuscript: The functional -1019C/G HTR1A polymorphism and mechanisms of fear
Supplementary Figure 1. Flow chart
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Supplementary Figure 2. The conditioning paradigm
The time course of the fMRI paradigm consisted of three phases: familiarisation (F), acquisition
(A) and extinction (E) 30, each subdivided into an early and a late phase 27. Different neutral
stimuli (yellow/blue spheres and violet/green squares) were used in parallel versions to account
for repeated exposure to the experiment in the pre-post-design (t1, t2). Each sphere/square was
visually presented for 2000 ms with a variable inter-stimulus interval (ISI) of 4.785 to 7.250 sec.
An unpleasant white noise was used as the US and presented for 100 ms. The volume of the US
was individually adapted (between 70 and 110 dB) to be unpleasant for the participant. The
same individual adapted volume level of this first measurement was also used for the second
measurement (t2). During the acquisition phase, one sphere/square was paired
pseudorandomly with the US (thus becoming CS+), while the other sphere was not (thus
becoming CS-). We used a partial reinforcement strategy in which 50% of the CS+ were paired
with the US and 50% were not. Only those trials without US were analysed during acquisition to
avoid overlap with neuronal activation directly related to the presentation of the US. The
presentation of the US occurred 1900 ms after the onset of the CS+. Thus, both stimuli were coterminated. The US was not presented during the extinction phase.
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Control fMRI analysis
As control analysis, we tested whether changes in activation (t1 vs. t2) could also be observed in
a healthy subjects group (n=42, for a more detailed group description, see27), which obtained no
CBT. In the healthy subjects group, we found no significant activation changes in relevant
regions such as the SMA, Insula and IFG, as supported by ROI analyses with the activation map
illustrated in Fig. 4 (see also Tab. 2). By contrast, whole brain analyses revealed that activation
changes are restricted to regions of the parietal lobe and the fusiform gyrus (see Supplementary
Fig. 3, top). Therefore, the only overlap between activation changes in healthy subjects and the
results of the gene by CBT interaction were found in the bilateral parietal lobe (right MNIxyz=26,
-30, 72, t=3.43, 197 voxels; left MNIxyz=-26, -30, 72, t=3.35, 308 voxels; Supplementary Fig. 3,
bottom). These data support the assumption that activation changes in relevant regions such as
the SMA, Insula and IFG observed for the CC group are not based on general changes due to
repeated exposure to the task and scanning environment. Thus, activation change in the SMA,
Insula and IFG might represent specific CBT effects.
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Supplementary Fig. 3. Activation change in the healthy control group
Supplementary Fig. 3 illustrates activation change (t1>t2) for the conditioned response (CS+>CS-)
during early acquisition in the healthy control group 27. Whereas the upper panel (Fig. 3A) displays
the whole brain effect in healthy subjects, the panel below (Fig. 3B) reflects its overlap with the
gene (CC vs. GG) by intervention (t1 vs. t2) interaction in the patient groups.
Supplementary Fig. 4. Activation change in the late acquisition phase
Supplementary Figure 4 illustrates activation changes (t1 vs. t2) in the late acquisition phase, in
predominantly bilateral parietal and temporal brain regions.
-5Behavioural avoidance test: Supplementary results
Since CC and CG genotype carrier did not differ in any of the performed analyses below, BAT
results for the comparison between the G allele homozygotes and C allele carriers (CC and CG)
were comparable to the results of the comparison between GG and CC.
Effect of HTR1A: Risk genotype was significantly associated with acute flight behaviour
before therapy (t1): GG genotype carriers compared to C allele carriers escaped more often
during exposure to the test chamber (χ² = 4.05, P=0.04; see Fig. 2A). Univariate analysis of
variance with genotype (GG carriers vs. C carriers) and behaviour (escapers vs. non-escapers) as
between-subjects variables revealed significant interaction effects between genotype and
behaviour on reported anxiety during anticipation period (F(1,241)=7.69, p<.01) and exposure
period (F(1,241)=9.88, p<.01). Post hoc analysis revealed that C allele carriers who showed
escaping behaviour during the exposure period already reported significant more anticipatory
anxiety compared to non-escaping patients at the anticipation period (Behaviour
F(1,178)=25.54, p<.001;) while anticipatory anxiety between escaping and non-escaping G allele
homozygotes was comparable (Behaviour F(1,63)=0.11, p=.75; see Fig. 2B). This suggests that
pronounced self-reported anticipatory anxiety preceded escape behaviour only in the C allele
group. During exposure reported anxiety was significantly increased in escaping patients as
compared to non-escaping patients in both, C allele carriers (Behaviour F(1,178)=104.83, p<.001)
and G allele homozygotes (Behaviour F(1,63)=12.88, p<.01). However, escaping C allele carriers
reported significantly higher anxiety than G allele homozygous escapers (Genotype
F(1,55)=12.50, p<.01) while no significant difference between genotypes was observed in nonescaping patients (Genotype F(1,186)=.92, p=.34; see Fig. 2B).
-6References (numbers refer to the main text)
30.
Reinhardt I, Jansen A, Kellermann T, Schüppen A, Kohn N, Gerlach AL, et al. Neural
correlates of aversive conditioning: development of a functional imaging paradigm for
the investigation of anxiety disorders. Eur Arch Psychiatry Clin Neurosci 2010; 260(6):
443-453.
27.
Kircher T, Arolt V, Jansen A, Pyka M, Reinhardt I, Kellermann T, et al. Effect of cognitivebehavioral therapy on neural correlates of fear conditioning in panic disorder. Biol
Psychiatry 2013; 73(1): 93-101.