psych science correctedproofs

Research Article

Genetic Gating of Human FearLearning and ExtinctionPossible Implications for Gene-Environment Interaction inAnxiety DisorderTina B. Lonsdorf,1,2 Almut I. Weike,3 Pernilla Nikamo,4 Martin Schalling,4 Alfons O. Hamm,3,5 and

Arne Ohman1,2,5

1Department of Clinical Neuroscience, Psychology Section, Karolinska Institutet; 2Stockholm Brain Institute, Stockholm,

Sweden; 3Department of Clinical and Biological Psychology, Ernst Moritz-Arndt University of Greifswald; 4Department of

Molecular Medicine and Surgery, Neurogenetics Unit, Karolinska Institutet; and 5Center for the Study of Emotion and

Attention, University of Florida

ABSTRACT—Pavlovian fear conditioning is a widely used

model of the acquisition and extinction of fear. Neural

findings suggest that the amygdala is the core structure for

fear acquisition, whereas prefrontal cortical areas are

given pivotal roles in fear extinction. Forty-eight volun-

teers participated in a fear-conditioning experiment,

which used fear potentiation of the startle reflex as the

primary measure to investigate the effect of two genetic

polymorphisms (5-HTTLPR and COMTval158met) on

conditioning and extinction of fear. The 5-HTTLPR

polymorphism, located in the serotonin transporter gene,

is associated with amygdala reactivity and neuroticism,

whereas the COMTval158met polymorphism, which is

located in the gene coding for catechol-O-methyltrans-

ferase (COMT), a dopamine-degrading enzyme, affects

prefrontal executive functions. Our results show that only

carriers of the 5-HTTLPR s allele exhibited conditioned

startle potentiation, whereas carriers of the COMT met/

met genotype failed to extinguish conditioned fear. These

results may have interesting implications for understand-

ing gene-environment interactions in the development and

treatment of anxiety disorders.

Pavlovian conditioning is the most basic of all paradigms for the

study of associative learning. It provided a substantial portion of

the empirical basis for the classical theories of learning, which

formed the core of behaviorist psychology. The famous early

work on fear conditioning in ‘‘Little Albert’’ (Watson & Raynor,

1920) pioneered the analysis of fear and anxiety in terms of

learning, an enterprise that is still vigorously pursued (e.g.,

Lissek et al., 2005; Mineka & Zinbarg, 2006; Ohman & Mineka,

2001). Despite the iconic status of Pavlovian conditioning as an

epitome of learning and environmentalism, we show in this ar-

ticle that Pavlovian fear conditioning and extinction, as mea-

sured by fear-potentiated startle reflexes, are modulated by

common genetic polymorphisms in the serotonin and dopamine

systems, respectively.

Pavlovian fear conditioning imbues a relatively neutral

stimulus (the conditioned stimulus, or CS) with fear-evoking

properties as the result of its co-occurrences with an aversive

event (the unconditioned stimulus, or US) that threatens the

well-being of the organism. As a result of fear conditioning,

therefore, remnants of traumatic events will stick to trauma-

exposed individuals as components of fear memories evoked by

stimuli associated (more or less consciously) with traumas.

Accordingly, fear conditioning has carried a heavy explanatory

burden in psychological theories of anxiety disorders such as

phobias (e.g., Ohman, Dimberg, & Ost, 1985; Seligman, 1971),

panic disorder (e.g., Bouton, Mineka, & Barlow, 2001), and

posttraumatic stress disorder (e.g., Orr et al., 2000). Extinction

(i.e., the waning of fear as a result of the CS being presented in

the absence of the US) has obvious implications for the treatment

of phobias and other anxiety disorders (Anderson & Insel,

2006). Indeed, it inspired the exposure therapies that have

provided highly effective treatments of anxiety disorders (Barlow,

Address correspondence to Arne Ohman or Tina B. Lonsdorf, Sec-tion of Psychology, Department of Clinical Neuroscience, KarolinskaInstitutet, SE-171 77 Stockholm, Sweden, e-mail: [email protected] [email protected].

PSYCHOLOGICAL SCIENCE

Volume ]]]—Number ]] 1Copyright r 2009 Association for Psychological Science

2002). In direct support of the common assumption that fear

conditioning is a potential mechanism of anxiety disorder, meta-

analyses indicate that patients diagnosed with anxiety disorder

show more rapid fear conditioning and slower extinction than

control subjects (Lissek et al., 2005).

Important progress has been made in understanding the neural

mechanisms of Pavlovian fear conditioning (see reviews by Davis

& Whalen, 2001; Fanselow & Poulos, 2005; LeDoux, 2000).

Briefly summarized, research has shown that cortical and sub-

cortical sensory pathways converge on the lateral nucleus of the

amygdala, which houses the molecular machinery for forming

associations between the CS and the US (Fanselow & Poulos,

2005; LeDoux, 2000). As a result of this associative process, the

CS may activate the central nucleus of the amygdala, which

recruits various fear responses through pathways to the striatum,

diencephalon, midbrain, and brainstem (Davis & Whalen, 2001).

The neural basis of extinction is less well understood, but both

animal (e.g., Quirk & Gehlert, 2003) and human (e.g., Phelps,

Delgado, Nearing, & LeDoux, 2004) data suggest that extinction

depends on structures in the medial prefrontal cortex.

Behavior genetic studies suggest that about one third of the

variance in human fear conditioning (Hettema, Annas, Neale,

Kendler, & Fredrikson, 2003) and the risk to develop anxiety

disorders (Gordon & Hen, 2004) can be attributed to genetic

factors. Given that a polymorphism in the serotonin transporter

(5-HTT) gene (SLC6A4) has been convincingly related to

amygdala reactivity (for a meta-analysis, see Munafo, Brown, &

Hariri, 2008), this polymorphism is a likely candidate to be

involved in conditioned fear. A 43-bp insertion/deletion in the

5-HTT promoter, referred to as 5-HTTLPR, yields a long (l) and

short (s) allele, the latter of which reduces 5-HTTexpression and

serotonin uptake by close to 50% (Lesch et al., 1996). In ad-

dition to having a relationship to the amygdala, this ‘‘low ac-

tivity’’ s allele is associated with higher neuroticism scores (for a

meta-analysis, see, e.g., Sen, Burmeister, & Ghosh, 2004).

The potentially pivotal role of the prefrontal cortex in ex-

tinction points to a candidate gene for extinction processes,

the gene coding for catechol-O-methyltransferase (COMT),

which degrades released extracellular dopamine. The COMT

gene harbors an interesting single nucleotide polymorphism:

COMTval158met. Carriers of the met allele have a 4-fold

reduction in enzyme activity compared with carriers of the val

allele, and as a result have higher extracellular dopamine levels,

particularly in prefrontal areas (for a review, see, e.g., Bilder,

Volavka, Lachman, & Grace, 2004). The met allele enhances

prefrontal cognition and working memory (for a review, see, e.g.,

Bilder et al., 2004) and has been associated with negative mood

states (e.g., anxiety, dysphoria) and impaired pain regulation.

Furthermore, the met allele is related to higher responsiveness

in, and connectivity between, brain areas involved in the evo-

cation and regulation of negative affective responses—in par-

ticular, the amygdala, hippocampus, and prefrontal areas (for a

review, see, e.g., Heinz & Smolka, 2006).

The purpose of this study was to use psychophysiological

indices of fear to examine whether these polymorphisms are

related to fear conditioning and extinction in humans. Given

some degree of heritability of both fear conditionability and

anxiety disorders, such relationships could provide mechanisms

behind the gene-environment interaction in anxiety disorder.

First, because of the associations between the 5-HTTLPR s

allele and amygdala reactivity, and between the amygdala and

fear conditioning, we hypothesized that carriers of this allele

would exhibit enhanced fear acquisition. Second, we hypothe-

sized a selective effect of the COMT met allele on extinction

because of the prefrontal focus of the actions of COMT and the

suggested role of this brain area in extinction and other forms of

emotion regulation.

Startle blink potentiation and skin conductance responses

(SCRs) were used as measures of conditioning. Fear potentiation

of the startle reflex is one of the most useful indices of defensive

response mobilization (Lang, Davis, & Ohman, 2000). Neurally,

it reflects the influence of direct and indirect connections from

the amygdala to the primary startle-reflex pathway in the

brainstem (Davis & Whalen, 2001). In contrast to the SCR,

which can be dissociated from amygdala activations (Tabbert,

Stark, Kirsch, & Vaitl, 2006) and appears to primarily reflect

cognitive contingency learning (Lovibond & Shanks, 2002),

startle potentiation appears to index a basic, affective level of

fear conditioning largely independent of higher cognition (Hamm

& Weike, 2005; Ohman & Mineka, 2001).

METHOD

Participants

Eighty-one students of the University of Greifswald were re-

cruited by advertisements, filled in an informed-consent form

(approved by the ethics committees at the Karolinska Institutet

and the University of Greifswald), and donated 20 ml of blood for

DNA extraction and genotyping. Thirty-four of these students

completed the experiment before being genotyped, and 47 stu-

dents were genotyped before being invited to participate in the

experiment. Out of the latter 47 students, 29 were selectively

invited to participate in order to balance out the allele fre-

quencies and sex distribution in the experimental sample. Thus,

63 volunteers participated in the experiment (both participants

and the experimenter were blinded to genotype) and were paid

h15.

We excluded 6 (3 male, 3 female) participants from the data

analysis because of technical problems and 9 (3 male, 6 female)

participants because they could not correctly report the CS-US

contingency at the end of the conditioning phase (see Proce-

dure). Lack of awareness, which was unrelated to our specific

genotype groups, typically compromises SCRs, but not startle

conditioning (Hamm & Weike, 2005; Lovibond & Shanks,

2002), and thus would likely introduce an irrelevant source of

variance. The final sample comprised 48 participants.

2 Volume ]]]—Number ]]

Genetic Gating of Fear Learning and Extinction

Genotyping

DNA extraction from whole blood was performed using standard

methods (Autopure LS system, Gentra Systems, Minneapolis,

MN; see Lindblom & Holmlund, 1988). For 5-HTTLPR geno-

typing, primers (Thermo Scientific, Ulm, Germany) flanking the

5-HTTLPR of the 5-HTT gene (forward 50-TGAATGCCAG

CACCTAACCCCTAA-30 and reverse 50-GAATACTGGTAGG

GTGCAAGGAGA-30) were used. Polymerase chain reactions

(PCRs) were carried out using the following cycling conditions:

an initial 5-min step at 95 1C; followed by 10 cycles consisting of

denaturation at 95 1C for 1 min, annealing at 65 1C (decrease of

0.5 1C with each cycle) for 1 min, and elongation at 72 1C for 1

min; followed by 39 cycles consisting of denaturation at 95 1C

for 45 s, annealing at 60 1C for 1 min, and elongation at 72 1C for

1 min; followed by a final step at 72 1C for 4 min. Reactions were

performed in 10 � Reaction buffer (Finnzymes, Espoo, Fin-

land), 1.5-mM MgCl2, 20 ng of genomic DNA, 0.75 ml of dNTPs

(Larova, Teltow, Germany; 10 mM), 0.625 ml of each primer

(10 mM), 0.0375 ml of 7-deaza-dGTP (Roche, Mannheim, Ger-

many), and 0.5 ml of TaqPolymerase (Dynazyme, Finnzymes,

Espoo, Finland). The PCR products (10 ml) were separated into

short (336 bp) and long (379 bp) fragments by electrophoresis on

2.5% agarose gels with 2% normal agarose (Certifiedt Molec-

ular Biology Agarose, Bio Rad, Hercules, CA) and 0.5% low-

melting agarose (Sea Plaques GTG Agarose, Cambrex Bio

Science, Rockland, ME) with ethidium bromide in TRIS-Borat-

EDTA-Buffer (TBE). After 2.5 hr of electrophoresis in TBE,

the products were visualized by ultraviolet illumination.

COMTval158met (rs4680) genotype was determined on an

ABI HT7900 (Applied Biosystems, Foster City, CA) using the

TaqMans allelic discrimination (50 nuclease assay; Livak, 1999).

Stimulus Materials

Four different color pictures depicting male faces were selected

from the Karolinska Directed Emotional Faces (Lundqvist,

Flykt, & Ohman, 1998) to serve as CSs (two pictures depicted

neutral expressions, and two depicted angry facial expres-

sions1). Which picture was coupled to the US, the valence of the

expressions presented (each participant saw only neutral faces

or only angry faces), and the stimulus sequence used (eight

different sequences) were balanced across the genotype groups;

Table 1 shows the distribution of the variables of interest in the

final sample of 48 participants. The pictures (visible size: 126 cm

� 93 cm) were projected onto a screen approximately 2 m in

front of the participant using a projector (Sanyo PLC-XU86) that

was situated in an adjacent room.

The US was a 500-Hz monopolar DC-pulse electric stimula-

tion applied above the participant’s right ankle in a 10-ms train

of 1-ms single pulses. It was generated by a commercial stim-

ulator (Grass Instruments S48K, West Warwick, RI), isolated

(SIU5), and transmitted via a constant-current unit (CCU1) to a

bipolar electrode (F-E10S2). The mean physical intensity cho-

sen during the intensity adjustment for the experiment (see

Procedure), the sensitization effect (increase in startle and SCRs

after US intensity adjustment), and unconditioned SCRs to the

US did not differ between the 5-HTTLPR groups or between the

COMTval158met genotype groups.

Startle probes were 50-ms bursts of 95-dB[A] white noise (rise

time< 1 ms). They were presented binaurally over Sony (MDR-

CD 170) headphones.

Physiological Recordings

Startle responses were measured by recording electromyo-

graphic (EMG) activity over the orbicularis oculi muscle be-

neath the left eye using miniature Ag/AgCl surface electrodes.

The raw EMG signal was amplified and filtered through a 30-Hz

high-pass filter (Coulbourn S75-01) and a 400-Hz low-pass filter

(Kemo KEM-VBF8-03; Beckenham, Kent, United Kingdom),

rectified, and integrated with a time constant of 10 ms. Skin

conductance was recorded using Hellinge Ag/AgCl standard

electrodes placed adjacently on the hypothenar eminence of the

right hand (see Weike, Schupp, & Hamm, 2007, for a more

detailed description of the recording procedures).

Procedure

Experimental Groups

Carriers of the 5-HTTLPR s allele (s/s or s/l genotype) were

combined to form the s-allele-carrier group and were compared

with l/l homozygotes. Similarly, carriers of the COMT val allele

were combined to form the val-allele-carrier group and com-

pared with met/met homozygotes2 (see Table 1 and Table 2 for

further description of these groups).

Day 1 (Fear Acquisition)

The experimental procedure on Day 1 included a baseline phase

(5 min of familiarization with the lab situation and presentation

of four startle probes for habituation), individual adjustment of

the US to a level described as ‘‘highly annoying but not painful,’’

and conditioning. During conditioning (see Fig. 1a), participants

1CS valence was not included in the statistical analyses because of a non-significant Stimulus � Valence interaction for startle responses and SCRmagnitudes (see the Data Analysis section for further information on the factorsin these analyses).

2During conditioning and extinction, carriers of one 5-HTTLPR s allele(s/l genotype) and carriers of two 5-HTTLPR s alleles (s/s genotype) showedcomparable CS1 potentiation, but significantly larger CS1 potentiation thanthe l/l homozygotes. The three COMT genotype groups did not differ from eachother in CS1 potentiation during conditioning. During extinction, the met/metgroup showed larger CS1 potentiation than both the heterozygotes and the val/val group. However, the latter trend did not reach significance, likely because ofthe small sample size for this contrast (n 5 9 for met/met and n 5 12 for val/val). Thus, we considered it empirically justified to combine carriers of at leastone 5-HTTLPR s allele into a single group and to combine carriers of at leastone COMT val allele into a single group.

Volume ]]]—Number ]] 3

T.B. Lonsdorf et al.

viewed each of two CS pictures nine times, in a mixed order.

Each picture was presented for 6 s. One of the pictures (CS1)

was always paired with the 10-ms US, which occurred simul-

taneously with the offset of the picture (100% reinforcement,

delay conditioning); the other picture (CS�) was never coupled

to the US. Acoustic startle probes were presented 4 or 5 s after

picture onset for six of the nine presentations of each CS and

during six intertrial intervals (ITIs; ITI 5 10–18 s). Participants

were instructed to attend to the pictures, but no information

about the CS-US contingencies was given. The conditioning

phase ended with a standardized postexperimental awareness

interview (cf. Bechara et al., 1995), so we could assess aware-

ness of the CS-US contingency. Participants also retrospectively

rated the US, acoustic startle probe, CS1, and CS� for valence

and arousal using the Self-Assessment Manikin (Lang, 1980);

these ratings did not differ between the 5-HTTLPR groups or

between the COMTval158met genotype groups.

Day 2 (Extinction)

The experimental procedure on Day 2 (approximately 24 hr

later; see Fig. 1b) included a baseline phase (presentation of four

startle probes for habituation) and extinction. During extinction,

the CS1 and CS� were presented 18 times each without

administration of any further USs. Startle probes were presented

4 or 5 s after picture onset for 12 of the 18 presentations of each

CS and during 12 ITIs. Again, participants were instructed to

attend to the pictures, but no information about the CS-US

contingencies was given. After completing the experiment,

participants were debriefed and paid.

Data Reduction and Response Definition

The magnitude of the startle eyeblink (in microvolts) was mea-

sured from onset to peak, as described previously by Weike et al.

(2007). Blink magnitudes were normalized using z-standard-

ization and converted to T scores to ensure that all participants

contributed equally to the group means. The T-score calculation,

50 1 (z� 10), results in a distribution with an overall mean of 50

and a standard deviation of 10 for each participant.

SCR magnitude (in microsiemens) was scored as the largest

response occurring 0.9 to 4.0 s after picture onset. Logarithms

were computed for all values, to normalize the distribution (Ven-

ables & Christie, 1980), and these log values were range-corrected

(individual score/individual maximum response) to account for

interindividual variability (Lykken & Venables, 1971).

Startle and SCR measurements that showed recording arti-

facts or excessive baseline activity were discarded. Thirty-four

of the 3,120 startle measurements were discarded (1.1%; 0–6

per participant), as were 70 of the 3,360 SCR measurements

(2.1%; 0–13 per person).

Data Analysis

Data were analyzed separately for the conditioning and extinc-

tion phases using SPSS 15 for Windows. Fear-potentiated startle

was measured by subtracting the mean magnitude of startle

responses elicited by probes during ITIs from the mean mag-

nitude of startle responses elicited by the same probes presented

during the CSs. We calculated the mean startle potentiation to

the CS1 and the CS� using individually standardized T-score

differences. Furthermore, we calculated scores for mean CS

discrimination by subtracting the mean T-score startle magni-

tude elicited during the CS� from the mean T-score startle

magnitude elicited during the CS1. To make sure that T scores

reflected the group differences in stimulus effects, rather than

baseline differences, we examined group differences in raw-

score startle responses to ITI probes separately. SCR condi-

tioning was assessed as mean CS discrimination.

We performed repeated measures analyses of variance (ANOVAs)

with stimulus (CS1 vs. CS� vs. ITI for startle; CS1 vs. CS�for SCR) as a within-subjects variable and 5-HTTLPR genotype

(s carriers vs. l/l homozygotes) or COMTval158met genotype (val

carriers vs. met/met homozygotes) as a between-subjects vari-

able (N 5 48). If an ANOVA revealed a significant main effect

for genotype or a significant Stimulus � Genotype interaction,

simple contrasts (CS1 potentiation, CS� potentiation, and CS

discrimination) were calculated to specify this effect. For an ex-

TABLE 1

Mean Age (in Years) and Sex Distribution of the Participants and

Assignment of Stimuli to the Different Genotype Groups

Genotype group nMeanage

Sex:CS

valence:male/female

angry/neutral

CS1:#09/#28

5-HTTLPR

s-allele carriers 30 23.7 (0.5) 15/15 12/18 16/14

l/l homozygotes 18 24.3 (0.6) 10/8 8/10 10/8

COMTval158met

val-allele carriers 39 24.0 (0.4) 22/17 16/23 22/17

met/met homozygotes 9 23.7 (1.0) 3/6 4/5 4/5

Note. For age, standard errors of the means are given in parentheses. The‘‘CS valence’’ column indicates how many participants in each group viewedfaces with angry expressions and how many viewed faces with neutral ex-pressions. The ‘‘CS1’’ column indicates the number of participants for whomPicture 09 (in the Karolinska Directed Emotional Faces; Lundqvist, Flykt, &Ohman, 1998) was coupled with the unconditioned stimulus and the number ofparticipants for whom Picture 28 was coupled with the unconditioned stimulus.

TABLE 2

Distribution of Genotypes in the Sample

COMTval158met

5-HTTLPR

Totals-allele l/lcarriers homozygotes

met/met homozygotes n 5 6 n 5 3 n 5 9

val-allele carriers n 5 24 n 5 15 n 5 39

Total n 5 30 n 5 18



ploratory within-subjects analysis in s-allele carriers, the same

analyses were applied with COMT genotype as a between-subjects

factor (N 5 30). We adopted a significance level of .05, and

Greenhouse-Geisser adjustments of degrees of freedom were used

when appropriate. We report Zp2 as the estimate of effect size.

RESULTS

A 3 � 2 ANOVA revealed a significant interactive effect of

stimulus and 5-HTTLPR genotype on startle blink responses

during conditioning, F(2, 92) 5 3.78, p 5 .026, Zp2 ¼ :076, in

the absence of significant group differences in blink response

during the ITI (raw-score magnitudes in microvolts), F(1, 46) 5

1.07, p 5 .307. Contrasts showed that 5-HTTLPR s-allele car-

riers exhibited significantly stronger startle potentiation to the

CS1 than did l/l homozygotes, p 5 .01, Zp2 ¼ :132 (Fig. 2a);

these two genotype groups did not differ significantly in CS

discrimination or CS� potentiation. Within-group analyses re-

vealed robust CS1 and CS� potentiation and significant CS

discrimination in s-allele carriers, whereas the l/l group did not

show significant CS1 or CS� potentiation or significant CS

discrimination. COMTval158met genotype did not affect fear-

potentiated startle during conditioning (Fig. 2c).

During extinction, a significant Stimulus � 5-HTTLPR

Genotype interaction was again found, F(2, 92) 5 8.80,

p < .001, Zp2 ¼ :161, in the absence of group differences in

startle response magnitudes during the ITI (uncorrected mag-

nitudes in microvolts). As during conditioning, 5-HTTLPR

s-allele carriers showed significantly more CS1 potentiation

than did l/l homozygotes ( p < .001, Zp2 ¼ :221). In addition,

the l/l group showed stronger CS� inhibition than did the s-

allele carriers (Fig. 2b). CS discrimination was comparable in

the two groups and significant for both.

In contrast to the conditioning phase, the extinction phase

showed a significant Stimulus � COMT Genotype interaction,

F(2, 92) 5 5.09, p 5 .008, Zp2 ¼ :100, in the absence of

significant group differences in blink response during the ITI

(uncorrected blink magnitudes in microvolts). The met/met

homozygous group showed clearly more pronounced CS1 po-

tentiation than did the val-allele carriers (p 5 .005,Zp2 ¼ :159;

Conditioning

a

b

CS– CS+ CS–CS+ CS–CS+

Startle ProbeUS

6 s 6 s 6 s 6 s6 s 6 s

Extinction (24 hr later)

CS– CS–CS+ CS–CS+ CS+

6 s 6 s 6 s 6 s 6 s 6 s

Fig. 1. The fear-conditioning paradigm. During conditioning (a), participants viewed each of two con-ditioned stimuli (CSs) nine times, in a mixed order. One of the pictures (CS1) was always paired with the10-ms unconditioned stimulus (US); the other picture (CS�) was never coupled to the US. Acoustic startleprobes were presented 4 or 5 s after picture onset for six of the nine presentations of each CS and duringsix intertrial intervals (ITIs). During extinction (b), the CS1 and CS� were presented 18 times eachwithout administration of any further USs. Startle probes were presented 4 or 5 s after picture onset for 12of the 18 presentations of each CS and during 12 ITIs.



Fig. 2d); CS discrimination and CS� potentiation were com-

parable in these two groups. Furthermore, although CS1 po-

tentiation remained at the same level as during conditioning for

the met/met group, it decreased to a nonsignificant level for val-

allele carriers.

SCRs showed reliable CS discrimination in the absence of ge-

notype effects during both conditioning and extinction. This result

suggests that the participants, irrespective of genotype, success-

fully learned the CS-US contingencies on a cognitive level.

As the l/l group did not show reliable startle potentiation during

conditioning, only s-allele carriers were selected for an exploratory

within-subjects analysis assessing the effect of COMTval158met

genotype on extinction (see Table 2 for the distribution of

COMTval158met genotype groups within 5-HTTLPR s-allele

carriers). As in the overall analysis, COMTval158met genotype

did not modulate startle responses during conditioning (Fig. 2e).

However, during extinction, a significant Stimulus � COMT Ge-

notype interaction was found, F(2, 56) 5 6.00, p 5 .004,

Zp2 ¼ :177, in the absence of significant differences in startle

responses during the ITI (uncorrected magnitudes in microvolts).

Those s-allele carriers who were homozygous for the COMT met

allele showed significantly more pronounced CS1 potentiation

than did those s-allele carriers who also carried a val allele (p 5

.002, Zp2 ¼ :302), even though significant CS1 potentiation was

observed for both groups (Fig. 2f). No significant differences in CS

discrimination or CS� potentiation were found.

Analyses of SCRs within s-allele carriers revealed significant

CS discrimination during conditioning, and a trend for CS

10

a

b

c

d f

e

8

6

4

2

0

–2

10

8

6

4

2

0

–2Sta

rtle

Blin

k M

agni

tude

(Diff

eren

ce F

rom

ITI;

ΔT S

core

s)S

tartl

e B

link

Mag

nitu

de(D

iffer

ence

Fro

m IT

I; ΔT

Sco

res)

s-allelecarriers

long/long met/met val-allelecarriers

met/met val-allelecarriers

*****

***

***

***

*

*

Conditioning:5-HTTLPR s Carriers

Extinction:5-HTTLPR s Carriers

Conditioning:5-HTTLPR

CS+ CS–

Extinction:5-HTTLPR

*

***

*

10

8

6

4

2

0

–2

10

8

6

4

2

0

–2s-allelecarriers

long/long met/met

Conditioning:COMTval158met

Extinction:COMTval158met

* ******

val-allelecarriers

met/met val-allelecarriers

**10

8

6

4

2

0

–2

10

8

6

4

2

0

–2

**

Fig. 2. Potentiation of startle-response magnitudes as a function of genotype and stimulus. Black bars show the difference between magnitude of thestartle response elicited during the CS1 (the conditioned stimulus coupled to the unconditioned stimulus) and magnitude of the startle responseelicited during the intertrial interval (ITI); white bars show the difference between magnitude of the startle response elicited during the CS� (theconditioned stimulus never coupled to the unconditioned stimulus) and magnitude of the response elicited during the ITI. Results are shown for 5-HTTLPR genotype groups (a) during conditioning and (b) during extinction, for COMTval158met genotype groups (c) during conditioning and (d)during extinction, and for COMTval158met genotype groups within 5-HTTLPR s-allele carriers (e) during conditioning and (f) during extinction.Error bars represent standard errors. Asterisks indicate significant differences, np < .05, nnp < .01, nnnp < .001.



discrimination during extinction, in the absence of an effect of

COMTval158met genotype.

DISCUSSION

In summary, our results suggest the rather strong conclusion that

the 5-HTTLPR and COMTval158met polymorphisms gate fear

learning and extinction, respectively, as measured by fear-

potentiated startle. On the one hand, only carriers of the

5-HTTLPR s allele acquired potentiated startle reactions to

environmental stimuli associated with an aversive event through

Pavlovian conditioning. On the other hand, homozygosity for the

COMT met allele selectively blocked the ability to extinguish

conditioned fear when the CS no longer was predictive of the US.

Our exploratory analysis on s-allele carriers alone confirmed on

a within-subjects basis that the combination of a 5-HTTLPR s

allele and COMT met-homozygosity conferred an enhanced risk

for acquiring fear that resisted extinction.

These findings undermine the commonly held (but mistaken)

belief in an impenetrable barrier between genes and environ-

ment. According to this belief, genes are inherited, intraorgan-

ismic, causal factors, and the environment provides the arena for

learned influences on behavior. Our results imply a more dy-

namic relationship by suggesting that genes may act through the

environment by making carriers of particular gene combinations

more likely than other individuals to easily pick up and retain

fear of stimuli associated with threat and trauma. Thus, people

carrying at least one 5-HTTLPR s allele and two COMT met

alleles are likely to expand their sets of fear- and anxiety-

evoking stimuli through facilitated fear conditioning and poor

extinction. This process might be further accelerated by stim-

ulus generalization and second-order conditioning, and hence

such individuals may end up fearful of many stimuli that they are

exposed to in their everyday environment. As a consequence,

they might have frequent experiences of negative affect, which is

a core characteristic of neuroticism (Clark, Watson, & Mineka,

1994). This could explain the relationship between the 5-

HTTLPR s allele and neuroticism. If the fear elicited by many

stimuli is intense enough to promote coping attempts in the form

of avoidance, the result could be the restrictions in life options

that characterize people with anxiety disorder. This could also

explain why negative affect is a risk factor for anxiety disorder

(Clark et al., 1994).

The two polymorphisms we studied will act synergistically in

this process, one by promoting fear acquisition, and the other by

slowing extinction. Nevertheless, they are independent of each

other, as shown by the double dissociation, with the 5-HTTLPR

polymorphism affecting acquisition but not extinction of fear,

and the COMT polymorphism affecting extinction but not ac-

quisition.

It is noteworthy that our conclusions were based on the results

of conditioned startle potentiation and were not valid for SCRs.

Our results show that the amount of CS1 startle potentiation

differed between the 5-HTTLPR genotype groups and between

the COMTval158met genotype groups during acquisition and

extinction, respectively, whereas CS1/CS� differentiation was

comparable between the two groups (the finding for s-allele

carriers echoes results with anxiety-disorder patients; Lissek

et al., 2005). Consistent with the lack of differences between

groups in discriminative startle potentiation to the CS1 and the

CS�, SCRs showed reliable CS1/CS� discrimination that was

independent of genotype. The dissociation between our startle

and SCR results in response to the CS1 probably reflects a

selective impact of 5-HTTLPR and COMTval158met on a basic

emotional level of fear that is manifested in startle potentiation,

but not in conditioned SCRs (e.g., Hamm & Weike, 2005;

Ohman & Mineka, 2001).

However, in an earlier study (Garpenstrand, Annas, Ekblom,

Oreland, & Fredrikson, 2001) that measured only SCRs, s-allele

carries were significantly overrepresented among individuals

showing very good fear conditioning. The discrepancy between

our results and those reported by Garpenstrand et al. may be

attributed to different research strategies: Whereas the latter

authors selected their participants on the basis of conditioning

performance and compared the genotypes of people who ex-

hibited extremely good and poor conditioning, we followed the

hypothesized causal sequence and grouped participants a priori

by genotype and subjected them to a fear-conditioning proce-

dure.

The results for the COMTval158met genotype appear con-

sistent with the tonic-phasic dopamine hypothesis for COMT

(Bilder et al., 2004). Thus, the low-activity met allele should

facilitate cognitive stability but jeopardize the cognitive

flexibility (updating or resetting working memory content) need-

ed to adapt to the change in conditioning contingencies during

fear extinction. This emotional perseveration, observed as a

failure of extinction in the met/met group, most likely reflects

impaired cognitive control over emotional reactions. Our results

are thus in line with the fact that the COMT met allele has been

associated with both cognitive control and anxiety proneness.

Although our data suggest that the 5-HTTLPR s allele serves

as a gate for fear conditioning and that carrying a COMT val

allele serves as a gate for fear extinction, the generality of these

conclusions across experimental conditions and measures re-

mains to be determined. For example, one interesting possibility

is that our results depended on our use of facial stimuli as CSs

(Canli & Lesch, 2007). Another potentially critical factor is the

intensity of the US. For ethical reasons, our US was of moderate

intensity; therefore, the present findings should not be taken to

imply that homozygosity for the 5-HTTLPR l allele precludes

fear conditioning regardless of circumstances. Similarly, more

prolonged extinction or the intense CS exposure used in single-

session treatment of specific phobia (Ost, 1997) may overcome

the apparent limitation to extinction we observed among met

homozygotes. Examining these limitations, as well as realizing

the promise of a deeper understanding of the dynamics inherent



in vulnerability-stress conceptualizations of anxiety disorder,

must await future research.

Acknowledgments—This work was supported by grants from

the Swedish Science Research Council, the Nordic Research

Council for the Humanities and Social Sciences (NOS-HS)

Nordic Centre of Excellence in Cognitive Control, and the Na-

tional Institute of Mental Health Center for the Study of Emotion

and Attention to A.O., and by a grant from the Swedish Research

Council to M.S. T.B.L. was supported by the German Academic

Exchange Service (Deutscher Akademischer Austauschdienst).

We thank Heino Mormann for technical assistance, Carmen

Hamm for blood sampling, and Andreas Olsson and Armita

Golkar for comments on earlier versions of this manuscript.

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