International Journal of Psychophysiology xxx (2014) xxx–xxx

INTPSY-10870; No of Pages 7

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International Journal of Psychophysiology

j ourna l homepage: www.e lsev ie r .com/ locate / i jpsycho

Genetic influences on the acquisition and inhibition of fear

Julia Wendt a,⁎, Jörg Neubert a, Katja Lindner a, Florian D. Ernst b, Georg Homuth b,Almut I. Weike a, Alfons O. Hamm a

a Department of Psychology, University of Greifswald, Germanyb Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, University of Greifswald, Germany

⁎ Corresponding author at: Department of PsycholoFranz-Mehring-Strasse 47, 17487 Greifswald, Germany. T

E-mail address: julia.wendt@uni-greifswald.de (J. Wen

http://dx.doi.org/10.1016/j.ijpsycho.2014.10.0070167-8760/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Wendt, J., et al., Gdx.doi.org/10.1016/j.ijpsycho.2014.10.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 July 2014Received in revised form 16 September 2014Accepted 10 October 2014Available online xxxx

Keywords:Fear learningFear inhibition5-HTTLPRCOMT Val158MetStartle blink response

As a variant of the Pavlovian fear conditioning paradigm the conditional discrimination design allows for adetailed investigation of fear acquisition and fear inhibition. Measuring fear-potentiated startle responses, weinvestigated the influence of two genetic polymorphisms (5-HTTLPR and COMT Val158Met) on fear acquisitionand fear inhibition which are considered to be critical mechanisms for the etiology and maintenance of anxietydisorders. 5-HTTLPR s-allele carriers showed a more stable potentiation of the startle response during fearacquisition. Homozygous COMT Met-allele carriers, which had demonstrated delayed extinction in previousinvestigations, show deficient fear inhibition in presence of a learned safety signal. Thus, our results providefurther evidence that 5-HTTLPR and COMT Val158Met genotypes influence the vulnerability for the developmentof anxiety disorders via different mechanisms.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Pavlovian fear conditioning involves a procedure in which a biolog-ically neutral stimulus is associated with an aversive unconditionedstimulus (UCS) such as pain or dyspnea. As a consequence, the formerlyneutral stimulus (conditioned stimulus; CS) comes to elicit a species-specific defensive behavior (e.g., freezing) and supporting physiologicaladjustments (e.g., increase in autonomic arousal). Fear conditioning hasbeen used as a laboratory tool to study fear and anxiety across species,also uncovering the neural networks that are involved in the acquisitionand extinction of fear (Davis et al., 2010; HammandWeike, 2005;Miladet al., 2006). The startle reflex and its modulation is probably one of themost promising indicators for the study of defensive behavior acrossspecies. In rodents the startle response is reliably potentiatedwhen elic-ited during a conditioned cue that has previously been associatedwith apainful aversive UCS (Davis, 1992). Converging evidence now suggeststhat the amygdala with its many efferent projections represents one ofthe key structures involved in such fear potentiation of the startle reflex.In humans, the blinking of the eyelid, the most reliable and fastest com-ponent of the startle reflex is recorded (Blumenthal et al., 2005). It hasbeen repeatedly demonstrated that the eyeblink component of the star-tle reflex is also potentiated in humans when elicited during cues thathave previously been paired with aversive moderately painful UCSs(Hamm et al., 1993; Hamm and Vaitl, 1996; see Hamm and Weike,

gy, University of Greifswald,el.: +49 3834 863719.dt).

enetic influences on the acq

2005 for a review). Animal data show that bilateral lesions of the centralnucleus of the amygdala block fear-potentiated startle (Hitchcock andDavis, 1986, 1991). Accordingly, fear-conditioned startle potentiationwas not completely abolished but significantly impaired in patientswith unilateral lesions of the amygdala after resections of the anteriorpart of the temporal lobe due tomedically intractable temporal lobe ep-ilepsy (Weike et al., 2005). Supporting these findings, fear-potentiatedstartle was completely blocked in a group of four female subjects withUrbach–Wiethe disease from the Northern Cape of South Africa, al-though half of these subjects had declarative knowledge about the CS–UCS contingencies (Klumpers, 2012).

Recent behavioral genetic studies suggest that acquisition but alsoextinction of fear-potentiated startle is modulated by genetic polymor-phisms. Hariri and colleagues (Hariri, 2011; Munafò et al., 2008)showed that a polymorphism in the promoter region of the human se-rotonin transporter (5-HTT) gene (SLC6A4) is related to amygdala activ-ity evoked by threat cues. Carriers of the short-(s) allele (a 43 bpdeletion) of the 5-HTT promoter (5-HTTLPR) show increased amygdalaactivation during viewing of fearful faces compared to long-(l) allelecarriers. Based on these findings, Lonsdorf et al. (2009b) tested fear-potentiated startle in 5-HTTLPR s-allele carriers and l-homozygotes.During acquisition, fear-conditioned startle potentiation was signifi-cantly stronger in s-allele carriers than in l/l carriers. The same geneticmodulation of fear-potentiated startle was found when participantswere instructed that the aversive UCS would follow one of two cues(Klumpers et al., 2012). Depending on certain variations of othergenes (neuropeptide S receptor: Glotzbach-Schoon et al., 2013; cortico-tropin releasing hormone receptor 1: Heitland et al., 2013) 5-HTTLPR

uisition and inhibition of fear, Int. J. Psychophysiol. (2014), http://

1 Only participants who were aware about CS–UCS contingencies were included in theanalysis since deficits in fear inhibition have been found before in unaware subjects usingthe conditional discrimination paradigm (Jovanovic et al., 2006).

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s-allele carrier also show increased context conditioning. Taken togeth-er, these findings indicate that 5-HTTLPR s-allele carriers seem to bemore prone to form excitatory CS–UCS associations.

The catechol-O-methyltransferase (COMT) gene codes the activityof the enzyme COMT responsible for the O-methylation of catechol-amines and contains a number of polymorphisms including Val158Met(Männistö and Kaakkola, 1999). Since carriers of the COMT Met-alleleshow reduced enzymatic activity, synaptic dopamine availabilityis higher in Met-carriers (Tunbridge et al., 2006). Homozygous Met-allele carriers show reduced extinction of fear potentiated startle(Lonsdorf et al., 2009b) as well as deficient inhibition of the fearresponse to the unreinforced CS- during safety learning (particularlyin PTSD patients; Norrholm et al., 2013). Deficient extinction recall hasrecently also been found in Met/Met mice (Risbrough et al., 2014).Together, these findings support the hypothesis that homozygous COMTMet-allele carriers show deficits in forming new inhibitory associationsafter the previously conditioned cue is no longer followed by the aversiveUCS.

After extinction training, the CS holds two qualitatively different as-sociations, excitatory associations with the UCS per acquisition trainingand inhibitory associations per extinction training (cf. Myers and Davis,2004), which are difficult to disentangle. Davis and coworkers thereforeused a conditional discrimination paradigm (Jovanovic et al., 2005;Myers and Davis, 2004) which allows for the systematic comparisonof excitatory and inhibitory learning bymeans of two different elementsof a compound (A and B) that provide the information whether a thirdstimulus (X) is paired with an aversive UCS or not (AX+/BX−). In asubsequent summation test (AB) one can test, if the presence of theconditioned safety signal (B) does indeed result in an inhibition of thefear response elicited by the conditioned danger signal (in this case fearresponses to AB should be decreased relative to those to AX+). Here,our goal is to investigate the geneticmodulation of the acquisition and in-hibition of the fear response as indexed by fear-potentiated startle as ameasure of defensive behavior and skin conductance as a measure of au-tonomic arousal. Based on previous findings, we investigated the influ-ence of the 5-HTTLPR and the COMT Val158Met polymorphisms on fearlearning and fear inhibition.

We expected that 5-HTTLPR s-allele carriers would show increasedfear-potentiated startle (AX+ N BX−). As described above, homozy-gous COMTMet-allele carriers were expected to show reduced inhibito-ry learning which was operationalized as reduced inhibition of startlepotentiation in response to AB compared to AX+ trials. To test the hy-pothesis that the presumed lack of response inhibition in homozygousCOMT Met-allele carriers is indeed due to a specific deficit in learninginhibitory CS–UCS associations, we additionally introduced a novel stim-ulus C conducting a second test phase (AC). According to Pavlov (1927),stimulus C is referred to as an external inhibitorwhose inhibiting strengthis characterized as an attention-related phenomenon (Myers and Davis,2004; Pavlov, 1927) and should be generally less pronounced than thatof the conditioned inhibitor B (Jovanovic et al., 2005). That is, homozy-gous COMTMet-allele carrierswere expected to showan inhibition deficitin AB but not in AC trials.

2. Method

2.1. Participants

Students of the University of Greifswald aged between 18 and40 years were recruited via notice boards or internet advertisement.Exclusion criteria were mental or neurological disorders, regular intakeof medication affecting the central nervous system, color blindness, lossof hearing, and being of non-Caucasion descent. Overall, 412 partici-pants filled out questionnaires and donated a blood sample. Based ontheir genotypes, 114 individuals whose current and lifetime mentalhealth was verified by way of the Structured Clinical Interview forDSM-IV (SKID; Wittchen et al., 1997) participated in the laboratory

Please cite this article as: Wendt, J., et al., Genetic influences on the acqdx.doi.org/10.1016/j.ijpsycho.2014.10.007

experiment. For three participants, the experiment was terminateddue to personal or technical reasons. Five participants were excludedfrom data analysis because they reported to be unaware of CS–UCS con-tingencies after finishing the experiment1 (see procedure). Thus, thefinal sample comprised 106 participants. Of these, 57 subjects werehomo- or heterozygous carriers of the 5-HTTLPR s-allele and 35 werehomozygous COMT Met158Met carriers (see Table 1 for more detailedinformation about the genotype groups). Subjects received coursecredit or up to 30 euros depending on the extent of their participa-tion. Before participation, all subjects signed a written informedconsent for the study, which was approved by the Ethics Committeeof the University of Greifswald.

2.2. Genotyping

DNA extraction from whole blood as well as typing of polymor-phisms was performed at the Department of Functional Genomics ofthe University Medicine Greifswald. For typing of the 5-HTTLPR short/long promoter polymorphism, PCR was performed essentially as de-scribed by Lonsdorf and coworkers (Lonsdorf et al., 2009b) using theprimer pair described in this study (forward primer: 5'-TGAATGCCAGCACCTAACCCCTAA-3′; reverse primer: 5′-GAATACTGGTAGGGTGCAAGGAGA-3′) with some minor modifications. In detail, the touchdownPCR was performed using a final MgCl2 concentration of 3 mM,0.75mMdNTPs, 20–50 ng template DNAper assay, a 10× Reaction Buff-er composed in equal parts by 10× KCl-containing Taq Buffer and 10×(NH4)2SO4-containing Taq Buffer (Thermo Scientific), and 1 U Taq Poly-merase (Thermo Scientific). Subsequently, the obtained PCR products(short allele: 336 bp; long allele: 379 bp) were size-separated by elec-trophoresis using 2.5% agarose-TBE gels, visualized by ethidium bro-mide staining, and documented using a GeneFlash gel documentationsystem (Syngene). Typing of the tri-allelic 5-HTTLPR polymorphismwas performed exactly as described earlier (Lonsdorf et al., 2009a).COMTVal158Met (rs4680) SNP genotypeswere determined using an ap-propriate TaqMan® assay (C_25746809) and an ABI HT7900 PCR Sys-tem (both Applied Biosystems).

2.3. Materials, design, and procedure

Visual cues were four differently colored geometric shapes (bluepentagon, pink triangle, yellow trapezoid, and black star) which werepresented alone or in combination (see below) for 7 s on a screen 2 min front of the participant. The UCS was a 10 ms train of 500 Hz singleelectrical pulses (1 ms) generated by a commercial stimulator (S48K;Grass Instruments, West Warwick, RI) and applied to the participants'left ankle. The intensity of the UCSwas adjusted for each participant in-dividually to a level that was described as “highly unpleasant, but notpainful”. The mean physical intensity of the UCS was 16.16 mA (SD =6.88) and did not vary between genotypes (5-HTTLPR: F b 1; COMTVal158Met: F = 1.26, n.s.). The acoustic startle probe was95 dB(A) white noise presented binaurally via headphones (MDR-CD170; Sony, Cologne, Germany) for 50 ms.

During acquisition, geometric shapes were presented in pairswhich were either followed by the UCS (AX+, 100% reinforcement)or not (BX−). The order of reinforced and unreinforced trials wasbalanced between participants. During the test phases, A and Bwere presented together (to test for conditioned inhibition) or A waspaired with a novel stimulus C (to test for external inhibition). X wasalways the black star, and the assignment of A, B, and C to one of the re-maining geometric shapes was balanced between participants. Acousticstartle probes were presented 5.5 or 6.5 s after every cue onset (seeFig. 1A). The inter-trial intervals (ITIs) varied between 11 and 23 s.

uisition and inhibition of fear, Int. J. Psychophysiol. (2014), http://

Table 1Mean age and sex distribution in 5-HTTLPR and COMT Val158Met genotype groups.

Genotype group N Mean age in years (SD) Sex: male/female

5-HTTLPRs-allele carriers 57 23.2 (2.9) 31/26l-allele homozygotes 49 24.0 (3.3) 24/25

COMT Val158MetMet-allele homozygotes 35 23.1 (2.6) 19/16Val-allele carriers 71 23.9 (3.3) 36/35

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Upon arrival in the laboratory, each participant signed an informedconsent form and was asked to give a urine sample to conduct a rapiddrug test (Drug Control Integra Cup; HediMed). Then sensors forphysiological data recording and the electrode for electrical stimulationwere attached. In an initial habituation phase, six startle probes werepresented without any visual foreground stimuli, then visual cues A, B,and X were presented four times each without any UCSs; startle probeswere administered 5.5 or 6.5 s after picture onset and additionallysix times during ITIs. Then the UCS intensity was adjusted within fivetrials. The following procedure was segmented into four parts (seeFig. 1B): (1) during conditioning 12 AX+ presentations co-terminatedwith the UCS, while 12 BX− presentations were never paired withthe UCS. The order of conditioned stimuli presentation was pseudo-randomized with the constraint of no more than two consecutive pre-sentations of the same shape pair; (2) during the first test phase, Awas presented eight times in a compound with B (for half of the partic-ipants) or with a novel stimulus C (for the other half of the participants)without any UCS; (3) during re-conditioning four AX+ presentationsagain co-terminated with the UCS, while four BX− presentationswere never paired with the UCS; and (4) during the second testphase, A was presented in compound with either B or C (whichever

Fig. 1. (A) Timing of visual cues, startle probes, and UCS presentations. Acoustic startleprobes followed cue onset by either 5.5 or 6.5 s in 100% of the trials. (B) Procedure ofconditioning, re-conditioning, and test phases.

Please cite this article as: Wendt, J., et al., Genetic influences on the acqdx.doi.org/10.1016/j.ijpsycho.2014.10.007

was not presented in the first test phase). The order of AB and AC testphases was balanced between participants. For ethical reasons, thefinal test phase was followed by an extinction phase in which AX aswell as A, B, and Xwere presented several timeswithout reinforcement.

After finishing the experiment, participants were asked severalquestions to determine their awareness of CS–UCS contingencies. Theinterview started with a free recall question (“Did you know whenyou were going to receive an electro-tactile stimulus?”). Then multiplechoice questions about the association of the UCS with each of the geo-metric shapes were asked which applied only to those experimentalphases in which electro-tactile stimuli were presented (e.g. “The pre-sentation of an electro-tactile stimulus followed the pentagon (1)most-ly yes, (2) mostly no, and (3) not possible to answer.”). Participantswere classified as aware if they either answered the free recall questioncorrectly (91%) or were able to correctly identify the AX+ throughoutthe further interview (in total 95.5%; cf. Weike et al., 2007). Therewere no differences in awareness between genotype groups. To mini-mize a possible moderating effect of hormone levels on the associationof genotype and conditioned inhibition of fear, women participated inthe experiment only between the 2nd and 4th day of theirmenstruationcycle to ensure low levels of female sexual hormones and their releasingfactors (Montag et al., 2008).

2.4. Physiological recordings

2.4.1. Electromyography (EMG)EMG activity was recorded over the left orbicularis oculi muscle to

measure the eyeblink component of the startle response. Two Ag/AgClminiature surface electrodes (4 mm diameter; Grass Products) filledwith electrolyte (Marquette Hellige) were attached beneath the lefteye. A Coulbourn S75-01 bioamplifier served to amplify the raw EMGwith a 30 Hz high-pass filter and a 400 Hz Kemo-VBF8-03 low-passfilter. Digital sampling was set to 1000 Hz from 100 ms prior to until400 ms after onset of the startle probe. The EMG signal was filteredthrough a 60 Hz high-pass filter and was rectified and integrated(time constant: 10 ms) offline using a digital filter.

2.4.2. Electrodermal activity (EDA)EDAwas recorded from the hypothenar eminence of the palmar sur-

face of the participant's right hand. A Coulbourn S71-22 skin conduc-tance coupler provided a constant 0.5 V across two Ag/AgCl standardelectrodes (8mmdiameter; Gelimed) filled with a 0.05M sodium chlo-ride electrolytemedium. The analog signalwas sampled continuously ata rate of 10 Hz.

2.5. Data reduction and response definition

2.5.1. Startle blink magnitudesThemagnitude of the startle eyeblink was scored offline using a com-

puter algorithm (Globisch et al., 1993) that automatically identified laten-cy of blink onset and peak amplitude in microvolt. Only blinks starting20–120 ms after probe onset and peaking within 150 ms were scored.No detectable blinks were scored as zero responses. Five participantswith excessive baseline activity or recording artifacts (N20%) were ex-cluded from the data analysis. For the remaining participants, trials withexcessive baseline activity or recording artifacts were rejected (5.9%)and replaced with the mean response of all subjects in that trial (cf.Blumenthal et al., 2005). The number of rejected trials did not differ be-tween genotype groups (5-HTTLPR and COMT Val158Met: both Fs b 1).Blink magnitudes were standardized for each participant using a z-scoretransformation. The standardized responses of each participant werethen converted to T-scores [50 + (z × 10)]. To test for fear acquisition,AX+ and BX− mean scores were calculated for the conditioning phaseas well as for the re-conditioning phase. To test for fear inhibition, an av-erage AX+ score covering the last four trials of conditioning and the fourre-conditioning trials were compared with the first four trials of the AB

uisition and inhibition of fear, Int. J. Psychophysiol. (2014), http://

3 Additional categorical analysis revealed that more homozygous COMTMet-allele car-

4 J. Wendt et al. / International Journal of Psychophysiology xxx (2014) xxx–xxx

and AC test phases.2 Potentiation scores were calculated as differencescores from ITI startle responses.Withfive additional participants exclud-ed due to technical problems (e.g., detachment of electrodes) the finalsample for startle blink magnitude analysis comprised 96 participants.

2.5.2. Skin conductance responses (SCRs)SC responses to AX+, BX−, AB, and AC were scored as the largest in-

crease between 0.9 and 4.0 s after stimulus onset (first interval response)using a computer program (Globisch et al., 1993). A logarithmic transfor-mation was applied to normalize the distribution (Venables and Christie,1980) and the log valueswere range corrected bydividing each individualscore by the participant's maximum response to reduce inter-individualvariability unrelated to the task (Lykken and Venables, 1971). Mean SCRscores were calculated as described for startle blink magnitudes. Threeparticipants were excluded from SC response analysis due to technicalproblems (final sample: N = 103).

2.6. Data analysis

To test for genotype group differences during acquisition, repeatedmeasures analyses of variance (ANOVAs) with condition (AX+ vs. BX−vs. ITI for startle responses, AX+ vs. BX− for SCRs) as a within-subjectvariable and 5-HTTLPR (s-allele carriers vs. l-allele homozygotes) orCOMT Val158Met (Val-allele carriers vs. Met-allele homozygotes) asbetween-subject variable were conducted for the conditioning and there-conditioning phases separately. To test for differences in conditionedand external inhibition, repeated measures ANOVAs were conductedwith inhibition (AX+vs. AB vs. AC) as a within-subject variable andwith 5-HTTLPR (s-allele carriers vs. l-allele homozygotes) or COMTVal158Met (Val-allele carriers vs. Met-allele homozygotes) as between-subject variable. We introduced order of test phases as a covariate. Totest the hypotheses that the combination of a 5-HTTLPR s-allele andCOMT met-homozygosity results in an enhanced deficit in conditionedinhibition (cf. Lonsdorf et al., 2009b), an additional repeated measuresANOVA was conducted with conditioned inhibition (AX+ vs. AB) as awithin-subject variable, COMT Val158Met as a between-subject variableand order of test phases as a covariate in 5-HTTLPR s-allele carriers only.All analyses were performed using IBM SPSS Statistics 22 and a .05 levelof significance. Greenhouse–Geisser correctionwas used in case of violat-ed sphericity. Nominal degrees of freedomare reported alongwith partialη2 values as an estimate of effect size.

3. Results

3.1. Fear acquisition

As expected, we observed a pronounced main effect of condition onstartle blink responses elicited during conditioning, F(2,188) = 109.17,p b .001, and partial η2 = .54. Startle blink responses were significantlypotentiated when elicited during the presentation of AX+ as well asduring BX− compared to startle responses evoked during the ITIs;AX+ vs. ITI: F(1,95) = 174.14, p b .001, partial η2 = .65; and BX− vs.ITI: F(1,95) = 35.88, p b .001, partial η2 = .27. Importantly, startle po-tentiation during AX+ presentations was significantly stronger thanpotentiation observed during BX− presentations; AX+ vs. BX−:F(1,95) = 91.77, p b .001, partial η2 = .49. Startle potentiation duringAX+ as well as the discrimination of AX+ vs. BX−was not influencedby the 5-HTTLPR genotype (AX+ vs. ITI and AX+ vs. BX− × 5-HTTLPRinteraction: both Fs b 1) during the conditioning phase. In contrast,during re-conditioning 5-HTTLPR s-allele carriers (AX+ N BX−:t(51) = 6.00, p b .001) showed a significantly stronger startle

2 To investigate conditioned and external inhibition processes, only the first four trialsof the AB and AC tests were averaged to test for the immediate transfer of safety (cf.Jovanovic et al., 2005), that is, to minimize the influence of learning effects due to thenon-reinforcement of stimulus A (extinction learning).

Please cite this article as: Wendt, J., et al., Genetic influences on the acqdx.doi.org/10.1016/j.ijpsycho.2014.10.007

potentiation to the AX+ relative to the BX− condition than did homo-zygous l-allele carriers (AX+ N BX−: t(43) = 1.87, p = .07); AX+ vs.BX−× 5-HTTLPR interaction: F(1,94) = 6.15, p b .05, partial η2 = .06;see Fig. 2. As expected, fear learning was not affected by the COMTVal158Met genotype (conditioning and re-conditioning: both Fs b 1).

Skin conductance response magnitudes were also increased in re-sponse to AX+ compared to BX− trials during conditioning; AX+ vs.BX−: F(1,102) = 79.47, p b .001, partial η2 = .44. Replicating previousresults, this effect was neither influenced by 5-HTTLPR nor by COMTVal158Met genotypes (both Fs b 1). During re-conditioning, however,the difference between SCR magnitudes to AX+ and BX− remainedsignificant only for COMT Val-allele carriers; AX+ vs. BX− × COMT:F(1,101) = 12.48, p b .01, partial η2 = .11 (means (SDs) during re-conditioning for Val-allele carriers: AX+ = .19 (.20), BX− = .06(.08); for homozygous Met-allele carriers: AX+ = .10 (.13), BX− =.10 (.11)).

3.2. Fear inhibition

To test for fear inhibition, the potentiation scores (difference fromITI) of the first four test phase trials (AB and AC) were compared tothe mean AX+ potentiation scores of the last four conditioning andthe four re-conditioning trials. A significant AX+ vs. AB vs. AC × COMTinteraction (F(2,184)= 3.66, p b .05, partial η2= .04) indicated a differ-ential influence of COMT Val158Met genotype on conditioned and exter-nal inhibition. Post-hoc tests revealed that only COMT Val-allele carriersshowed a significant inhibition of startle responses during AB comparedto AX+ trials (t(61) = 2.05, p b .05) and more pronounced inhibitionduring AB compared to AC trials (t(61) = 2.08, p b .05). In contrast,homozygous COMT Met-allele carriers showed a pronounced startlepotentiation during AB presentations despite the presence of a safetysignal (AB N ITI: t(33) = 6.63, p b .001) which was significantly largerthan startle potentiation in COMTVal-allele carriers during AB presenta-tions (t(94) = 2.17, p b .05); see Fig. 3, left panel.3

As supported by a significant AB vs. AX+ × COMT interaction in 5-HTTLPR s-allele carriers (F(1,48) = 7.68, p b .01, partial η2 = .14),only 5-HTTLPR s-allele carriers who were also COMT Val-allele carriersshowed a significant inhibition during AB compared to AX+ trials(t(29) = 2.90, p b .01), whereas those s-allele carriers who were alsohomozygotes for the COMT Met-allele did not show any startle inhibi-tion (t(21)=−1.24, n.s.). Supporting this difference in conditioned in-hibition of fear, startle potentiation during AB trials was significantlyhigher in 5-HTTLPR s-allele carriers who were homozygous COMTMet-allele carriers compared to those who were COMT Val-allele car-riers (t(50)= 2.80, p b .01); see Fig. 3, right panel. Exploratory analysisof COMT Val158Met genotype influences on conditioned inhibition ef-fects in homozygous 5-HTTLPR l-allele carriers revealed no significantinteraction (F b 1). Fear inhibition effects were not influenced by theorder of test phases.

Skin conductance responses were also significantly inhibited whenthe safety cue was combined with the excitatory stimulus. SCRs weresignificantly larger to the last four conditioning and the four re-conditioning AX+ onsets than to the first four AB presentations in thetest phase; AX+ vs. AB: F(1,102) = 8.99, p b .01, partial η2 = .08. Thiseffect was not significant for the AX+ vs. AC comparison, F b 1. As indi-cated by a significant interaction (AX+vs. AB× COMT: F(1,101)= 4.65,p b .05, partial η2 = .04), reduced SC responses during AB compared toAX+ trials were found only in COMT Val-allele carriers (AB: t(69) =3.61, p b .01). This interactionwas not a result of differential responding

riers showed augmented startle responses during AB compared to AC trials while the op-posite was true for Val-allele carriers (χ2(1) = 7.81, p b .01). That is, based on the oddsratio, the odds to find the assumed increased inhibitory strength of the conditioned inhib-itor B compared to the external inhibitor C were 3.39 times higher in COMT Val-allele car-riers than in homozygous Met-allele carriers.

uisition and inhibition of fear, Int. J. Psychophysiol. (2014), http://

Fig. 2. Startle blink potentiation (difference from ITI startle responses) to AX+ (black bars) and BX− (white bars) during conditioning (left panel) and re-conditioning (right panel) in5-HTTLPR s-allele and homozygous l-allele carriers. Mean values are displayed with standard errors. Asterisks indicate significant differences. ***p b .001, **p b .01, *p b .05.

5J. Wendt et al. / International Journal of Psychophysiology xxx (2014) xxx–xxx

to AB but rather to a reduced excitatory SCR to AX+ in homozygousCOMT Met-allele carriers during re-conditioning (see above; averageAX+ as well as AB and AC means (SDs) for Val-allele carriers:AX+ = .16 (.17), AB = .10 (.13), AC = .15 (.18); for homozygousMet-allele carriers: AX+ = .10 (.11), AB = .10 (.15), AC = .16 (.16)).SC inhibition in both test trial types was not affected by 5-HTTLPR geno-types (both Fs b 1).

Fig. 3.Mean startle blink potentiation (difference from ITI startle responses) to AX+(blackbarsAB (gray bars) and AC (striped bars) trials. The left panel shows differences in AB and AC test phcarriers only. Mean values are displayed with standard errors. Asterisks indicate significant dif

Please cite this article as: Wendt, J., et al., Genetic influences on the acqdx.doi.org/10.1016/j.ijpsycho.2014.10.007

4. Discussion

Taken together, our results confirm an influence of 5-HTTLPR andCOMT Val158Met polymorphisms on both learning and inhibition offear. During fear conditioning, we found a robust AX+/BX− startleblink potentiation in both 5-HTTLPR s-allele and homozygous l-allelecarriers. During re-conditioning trials, however, this conditioned startle

) during the last four conditioning and the four re-conditioning trials aswell as thefirst fourases in COMT Val158Met genotypes. The right panel shows the AB test in 5-HTTLPR s-alleleferences. ***p b .001, **p b .01; *p b .05.

uisition and inhibition of fear, Int. J. Psychophysiol. (2014), http://

6 J. Wendt et al. / International Journal of Psychophysiology xxx (2014) xxx–xxx

potentiationwasmore pronounced in 5-HTTLPR s-allele carriers indicat-ing a prolonged expression of previously learned fear responses in thosegenotypes. Different from COMT Val-allele carriers, homozygous Met-allele carriers showed a pronounced potentiation of the startle responseduring processing of the fear-conditioned cue despite the presence of asafety signal (AB). As expected, the fear inhibition deficit was specific tothe conditioned inhibitor B and not observed for the external (atten-tion-related) inhibitor C, thus, indicating a deficit in learning inhibitoryassociations in homozygous COMT Met-allele carriers.

4.1. 5-HTTLPR and fear learning

The 5-HTTLPR s-allele has been associated with different anxiety-related personality traits such as neuroticism and harm avoidance(Munafò et al., 2005) and can increase the risk of post-traumatic stressdisorder (PTSD) most probably depending on trauma load (Kilpatricket al., 2007; Koenen et al., 2009; Kolassa et al., 2010a) as well as the se-verity of panic disorder symptoms (Lonsdorf et al., 2009a). Findings of5-HTTLPR-dependent modulation of the amygdala and its prefrontalcoupling during processing of affective relative to neutral information(Dannlowski et al., 2008; Heinz et al., 2005)may provide a link betweensaid polymorphism and fear learning as indexed with startle blinkresponses (Munafò et al., 2008; Todd et al., 2011). However, a currentworking model of molecular processes in the lateral amygdala duringfear acquisition does not include a possible modulation by serotoninsince its role has not been studied in detail yet (Johansen et al., 2011).

In a recent review, Lonsdorf and Kalisch comment that little isknown about the effects of genetic polymorphisms on temporal aspectsof fear acquisition (Lonsdorf and Kalisch, 2011). In our study, both5-HTTLPR genotypes exhibit comparable fear learning during the initialacquisition phase. However, 5-HTTLPR s-allele carriers show significant-ly increased conditioned fear-potentiated startle responses during re-conditioning, thus showing stronger expression of the fear responseover a longer period of time. Thus differences between both groups de-velop later during fear acquisition than in previous experiments(Lonsdorf et al., 2009b). This could be due to differences in stimulusmaterials used: where we used pairs of geometric shapes, Lonsdorfand coworkers used angry faces (Lonsdorf et al., 2009b) as conditionedstimuli. Angry faces are known to activate the amygdala (at least whentheir gaze is directed at the observer: N'Diaye et al., 2009; Sauer et al.,2013) and, therefore, may facilitate the serotonergic influence duringfear acquisition and fear expression. Using geometric shapes as condi-tioned stimuli it might take longer until group differences in the expres-sion of learned fear are revealed.

Replicating previous findings (Lonsdorf et al., 2009b), fear-potentiated startle responses but not conditioned discrimination ofthe skin conductance responses was modulated by the 5-HTTLPR poly-morphism. Thus, our results support the notion that the 5-HTTLPR poly-morphism influences the expression of amygdala driven defensivebehavior during fear – as reflected by startle potentiation – ratherthan the supporting autonomic changes that are not always fear specificbut are also dependent on increased orienting to the reinforced cuedriven by cognitive processes of shock expectancy and contingencyawareness (Hamm and Vaitl, 1996; Soeter and Kindt, 2010). The lackof genotype differences in contingency awareness further strengthensthis notion.

4.2. COMT Val158Met and fear inhibition

COMT Met-allele carriers have been shown to be more frequentamong some PTSD populations (Boscarino et al., 2012; Valente et al.,2011) and homozygous Met-allele carriers seem to exhibit an elevatedrisk for developing PTSD even at low trauma load (Kolassa et al.,2010b). Our results replicate and extend previous findings on reducedfear extinction by COMT Met-allele carriers (Lonsdorf et al., 2009b;Norrholm et al., 2013) which may explain their increased vulnerability

Please cite this article as: Wendt, J., et al., Genetic influences on the acqdx.doi.org/10.1016/j.ijpsycho.2014.10.007

to develop symptoms after experiencing a trauma (cf. Myers andDavis, 2004). Using an conditional discrimination procedure previouslyvalidated in animals (Myers and Davis, 2004) and adapted to humanstartle research (Jovanovic et al., 2005) the current data demonstratethat not only extinction learning but also inhibition of the fear responseby the presence of a learned safety cue is significantly impaired in COMTMet-allele carriers. Thus, reduced extinction of fear observed in Met-allele carriers seems to be indeed a result of impaired building of newinhibitory associations. Animal research suggests that inhibitory learn-ing is mediated by the infralimbic region of the ventromedial prefrontalcortex (vmPFC) which exerts an inhibitory influence on the amygdaladuring extinction of learned fear (Paré et al., 2004; Quirk et al., 2000).Dopamine has been found to suppress inhibitory medial PFC effects onthe amygdala (Rosenkranz and Grace, 2001, 2002). That is, the in-creased dopamine availability in COMT Met-allele carriers (Tunbridgeet al., 2006) might explain their safety learning deficit.

5. Conclusion

Learning theory basedmodels explaining the etiology of anxiety dis-orders proclaim that pathological anxiety is a result of a learned associ-ation between certain cues and a fear response formed during theexperience of a traumatic event (Field, 2006; Mineka and Oehlberg,2008). But neither do all peoplewhohave experienced traumatic eventsdevelop pathological anxiety (Breslau et al., 1991), nor do all patientsrespondwhen treatedwith exposure therapy (Ougrin, 2011). Thus, cur-rent learning theory based etiology models of anxiety disorders have totake into account both the importance of vulnerabilities partly based ongenetic factors and conditioning processes taking place during traumat-ic or stressful life events when explaining the development andmainte-nance of pathological anxiety (Mineka and Zinbarg, 2006; Skelton et al.,2012).

Although the effect sizes of genetic effects are generally small in thepresent study and the effect of 5-HTTLPR and COMT Val158Met polymor-phisms on conditioned inhibition needs to be replicated before drawingextensive conclusions, our results indicate that 5-HTTLPR and COMTVal158Met polymorphism modulate the vulnerability for the develop-ment of anxiety disorders via differentmechanisms: the 5-HTTLPR poly-morphism seems to modulate the stability of fear expression duringcues that have been associated with aversive events and, thus, may af-fect the quality and intensity of the expression of conditioned fearonce consolidated fear memories are activated. The COMT Val158Metpolymorphism seems to modulate a separate mechanism influencinginhibition of fear expression in the presence of learned safety cues(Mineka and Zinbarg, 2006).

Acknowledgements

This work was supported by a grant from the German ResearchSociety (DFG) (WE 2762/5-1). We thank Carmen Hamm and PaulinaTroitzsch for blood sampling, Anja Wiechert for excellent technical as-sistance in the genotyping analyses, CosmaHoffmann for help with par-ticipant recruitment, Svenda Berg andWiebke Grieser for help with thediagnostic interviews, and Henriette Hacker and Laura Troike for helpwith data acquisition.

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