INTPSY-10870; No of Pages 7 International Journal of Psychophysiology xxx (2014) xxx–xxx

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International Journal of Psychophysiology journal homepage: www.elsevier.com/locate/ijpsycho

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 b

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

a r t i c l e

i n f o

Article history: Received 21 July 2014 Received in revised form 16 September 2014 Accepted 10 October 2014 Available online xxxx Keywords: Fear learning Fear inhibition 5-HTTLPR COMT Val158Met Startle blink response

a b s t r a c t As a variant of the Pavlovian fear conditioning paradigm the conditional discrimination design allows for a detailed investigation of fear acquisition and fear inhibition. Measuring fear-potentiated startle responses, we investigated the influence of two genetic polymorphisms (5-HTTLPR and COMT Val158Met) on fear acquisition and fear inhibition which are considered to be critical mechanisms for the etiology and maintenance of anxiety disorders. 5-HTTLPR s-allele carriers showed a more stable potentiation of the startle response during fear acquisition. Homozygous COMT Met-allele carriers, which had demonstrated delayed extinction in previous investigations, show deficient fear inhibition in presence of a learned safety signal. Thus, our results provide further evidence that 5-HTTLPR and COMT Val158Met genotypes influence the vulnerability for the development of anxiety disorders via different mechanisms. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pavlovian fear conditioning involves a procedure in which a biologically neutral stimulus is associated with an aversive unconditioned stimulus (UCS) such as pain or dyspnea. As a consequence, the formerly neutral stimulus (conditioned stimulus; CS) comes to elicit a speciesspecific defensive behavior (e.g., freezing) and supporting physiological adjustments (e.g., increase in autonomic arousal). Fear conditioning has been used as a laboratory tool to study fear and anxiety across species, also uncovering the neural networks that are involved in the acquisition and extinction of fear (Davis et al., 2010; Hamm and Weike, 2005; Milad et al., 2006). The startle reflex and its modulation is probably one of the most promising indicators for the study of defensive behavior across species. In rodents the startle response is reliably potentiated when elicited during a conditioned cue that has previously been associated with a painful aversive UCS (Davis, 1992). Converging evidence now suggests that the amygdala with its many efferent projections represents one of the key structures involved in such fear potentiation of the startle reflex. In humans, the blinking of the eyelid, the most reliable and fastest component of the startle reflex is recorded (Blumenthal et al., 2005). It has been repeatedly demonstrated that the eyeblink component of the startle reflex is also potentiated in humans when elicited during cues that have previously been paired with aversive moderately painful UCSs (Hamm et al., 1993; Hamm and Vaitl, 1996; see Hamm and Weike, ⁎ Corresponding author at: Department of Psychology, University of Greifswald, Franz-Mehring-Strasse 47, 17487 Greifswald, Germany. Tel.: +49 3834 863719. E-mail address: [email protected] (J. Wendt).

2005 for a review). Animal data show that bilateral lesions of the central nucleus of the amygdala block fear-potentiated startle (Hitchcock and Davis, 1986, 1991). Accordingly, fear-conditioned startle potentiation was not completely abolished but significantly impaired in patients with unilateral lesions of the amygdala after resections of the anterior part of the temporal lobe due to medically intractable temporal lobe epilepsy (Weike et al., 2005). Supporting these findings, fear-potentiated startle was completely blocked in a group of four female subjects with Urbach–Wiethe disease from the Northern Cape of South Africa, although half of these subjects had declarative knowledge about the CS– UCS contingencies (Klumpers, 2012). Recent behavioral genetic studies suggest that acquisition but also extinction of fear-potentiated startle is modulated by genetic polymorphisms. Hariri and colleagues (Hariri, 2011; Munafò et al., 2008) showed that a polymorphism in the promoter region of the human serotonin transporter (5-HTT) gene (SLC6A4) is related to amygdala activity evoked by threat cues. Carriers of the short-(s) allele (a 43 bp deletion) of the 5-HTT promoter (5-HTTLPR) show increased amygdala activation during viewing of fearful faces compared to long-(l) allele carriers. Based on these findings, Lonsdorf et al. (2009b) tested fearpotentiated startle in 5-HTTLPR s-allele carriers and l-homozygotes. During acquisition, fear-conditioned startle potentiation was significantly stronger in s-allele carriers than in l/l carriers. The same genetic modulation of fear-potentiated startle was found when participants were instructed that the aversive UCS would follow one of two cues (Klumpers et al., 2012). Depending on certain variations of other genes (neuropeptide S receptor: Glotzbach-Schoon et al., 2013; corticotropin releasing hormone receptor 1: Heitland et al., 2013) 5-HTTLPR

http://dx.doi.org/10.1016/j.ijpsycho.2014.10.007 0167-8760/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

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s-allele carrier also show increased context conditioning. Taken together, these findings indicate that 5-HTTLPR s-allele carriers seem to be more prone to form excitatory CS–UCS associations. The catechol-O-methyltransferase (COMT) gene codes the activity of the enzyme COMT responsible for the O-methylation of catecholamines and contains a number of polymorphisms including Val158Met (Männistö and Kaakkola, 1999). Since carriers of the COMT Met-allele show reduced enzymatic activity, synaptic dopamine availability is higher in Met-carriers (Tunbridge et al., 2006). Homozygous Metallele carriers show reduced extinction of fear potentiated startle (Lonsdorf et al., 2009b) as well as deficient inhibition of the fear response to the unreinforced CS- during safety learning (particularly in PTSD patients; Norrholm et al., 2013). Deficient extinction recall has recently also been found in Met/Met mice (Risbrough et al., 2014). Together, these findings support the hypothesis that homozygous COMT Met-allele carriers show deficits in forming new inhibitory associations after the previously conditioned cue is no longer followed by the aversive UCS. After extinction training, the CS holds two qualitatively different associations, excitatory associations with the UCS per acquisition training and inhibitory associations per extinction training (cf. Myers and Davis, 2004), which are difficult to disentangle. Davis and coworkers therefore used a conditional discrimination paradigm (Jovanovic et al., 2005; Myers and Davis, 2004) which allows for the systematic comparison of excitatory and inhibitory learning by means of two different elements of a compound (A and B) that provide the information whether a third stimulus (X) is paired with an aversive UCS or not (AX +/BX−). In a subsequent summation test (AB) one can test, if the presence of the conditioned safety signal (B) does indeed result in an inhibition of the fear response elicited by the conditioned danger signal (in this case fear responses to AB should be decreased relative to those to AX+). Here, our goal is to investigate the genetic modulation of the acquisition and inhibition of the fear response as indexed by fear-potentiated startle as a measure of defensive behavior and skin conductance as a measure of autonomic arousal. Based on previous findings, we investigated the influence of the 5-HTTLPR and the COMT Val158Met polymorphisms on fear learning and fear inhibition. We expected that 5-HTTLPR s-allele carriers would show increased fear-potentiated startle (AX + N BX −). As described above, homozygous COMT Met-allele carriers were expected to show reduced inhibitory learning which was operationalized as reduced inhibition of startle potentiation in response to AB compared to AX+ trials. To test the hypothesis that the presumed lack of response inhibition in homozygous COMT Met-allele carriers is indeed due to a specific deficit in learning inhibitory CS–UCS associations, we additionally introduced a novel stimulus C conducting a second test phase (AC). According to Pavlov (1927), stimulus C is referred to as an external inhibitor whose inhibiting strength is characterized as an attention-related phenomenon (Myers and Davis, 2004; Pavlov, 1927) and should be generally less pronounced than that of the conditioned inhibitor B (Jovanovic et al., 2005). That is, homozygous COMT Met-allele carriers were expected to show an inhibition deficit in AB but not in AC trials. 2. Method 2.1. Participants Students of the University of Greifswald aged between 18 and 40 years were recruited via notice boards or internet advertisement. Exclusion criteria were mental or neurological disorders, regular intake of medication affecting the central nervous system, color blindness, loss of hearing, and being of non-Caucasion descent. Overall, 412 participants filled out questionnaires and donated a blood sample. Based on their genotypes, 114 individuals whose current and lifetime mental health was verified by way of the Structured Clinical Interview for DSM-IV (SKID; Wittchen et al., 1997) participated in the laboratory

experiment. For three participants, the experiment was terminated due to personal or technical reasons. Five participants were excluded from data analysis because they reported to be unaware of CS–UCS contingencies after finishing the experiment1 (see procedure). Thus, the final sample comprised 106 participants. Of these, 57 subjects were homo- or heterozygous carriers of the 5-HTTLPR s-allele and 35 were homozygous COMT Met158Met carriers (see Table 1 for more detailed information about the genotype groups). Subjects received course credit or up to 30 euros depending on the extent of their participation. Before participation, all subjects signed a written informed consent for the study, which was approved by the Ethics Committee of the University of Greifswald. 2.2. Genotyping DNA extraction from whole blood as well as typing of polymorphisms was performed at the Department of Functional Genomics of the University Medicine Greifswald. For typing of the 5-HTTLPR short/ long promoter polymorphism, PCR was performed essentially as described by Lonsdorf and coworkers (Lonsdorf et al., 2009b) using the primer pair described in this study (forward primer: 5'-TGAATGCCAG CACCTAACCCCTAA-3′; reverse primer: 5′-GAATACTGGTAGGGTGCAAG GAGA-3′) with some minor modifications. In detail, the touchdown PCR was performed using a final MgCl2 concentration of 3 mM, 0.75 mM dNTPs, 20–50 ng template DNA per assay, a 10× Reaction Buffer composed in equal parts by 10× KCl-containing Taq Buffer and 10× (NH4)2SO4-containing Taq Buffer (Thermo Scientific), and 1 U Taq Polymerase (Thermo Scientific). Subsequently, the obtained PCR products (short allele: 336 bp; long allele: 379 bp) were size-separated by electrophoresis using 2.5% agarose-TBE gels, visualized by ethidium bromide staining, and documented using a GeneFlash gel documentation system (Syngene). Typing of the tri-allelic 5-HTTLPR polymorphism was performed exactly as described earlier (Lonsdorf et al., 2009a). COMT Val158Met (rs4680) SNP genotypes were determined using an appropriate TaqMan® assay (C_25746809) and an ABI HT7900 PCR System (both Applied Biosystems). 2.3. Materials, design, and procedure Visual cues were four differently colored geometric shapes (blue pentagon, pink triangle, yellow trapezoid, and black star) which were presented alone or in combination (see below) for 7 s on a screen 2 m in front of the participant. The UCS was a 10 ms train of 500 Hz single electrical 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 UCS was adjusted for each participant individually to a level that was described as “highly unpleasant, but not painful”. 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; COMT Val158Met: F = 1.26, n.s.). The acoustic startle probe was 95 dB(A) white noise presented binaurally via headphones (MDR-CD 170; Sony, Cologne, Germany) for 50 ms. During acquisition, geometric shapes were presented in pairs which were either followed by the UCS (AX +, 100% reinforcement) or not (BX−). The order of reinforced and unreinforced trials was balanced between participants. During the test phases, A and B were presented together (to test for conditioned inhibition) or A was paired with a novel stimulus C (to test for external inhibition). X was always the black star, and the assignment of A, B, and C to one of the remaining geometric shapes was balanced between participants. Acoustic startle probes were presented 5.5 or 6.5 s after every cue onset (see Fig. 1A). The inter-trial intervals (ITIs) varied between 11 and 23 s. 1 Only participants who were aware about CS–UCS contingencies were included in the analysis since deficits in fear inhibition have been found before in unaware subjects using the conditional discrimination paradigm (Jovanovic et al., 2006).

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

J. Wendt et al. / International Journal of Psychophysiology xxx (2014) xxx–xxx Table 1 Mean age and sex distribution in 5-HTTLPR and COMT Val158Met genotype groups. Genotype group 5-HTTLPR s-allele carriers l-allele homozygotes COMT Val158Met Met-allele homozygotes Val-allele carriers

N

Mean age in years (SD)

Sex: male/female

57 49

23.2 (2.9) 24.0 (3.3)

31/26 24/25

35 71

23.1 (2.6) 23.9 (3.3)

19/16 36/35

Upon arrival in the laboratory, each participant signed an informed consent form and was asked to give a urine sample to conduct a rapid drug test (Drug Control Integra Cup; HediMed). Then sensors for physiological data recording and the electrode for electrical stimulation were attached. In an initial habituation phase, six startle probes were presented without any visual foreground stimuli, then visual cues A, B, and X were presented four times each without any UCSs; startle probes were administered 5.5 or 6.5 s after picture onset and additionally six times during ITIs. Then the UCS intensity was adjusted within five trials. The following procedure was segmented into four parts (see Fig. 1B): (1) during conditioning 12 AX+ presentations co-terminated with the UCS, while 12 BX − presentations were never paired with the UCS. The order of conditioned stimuli presentation was pseudorandomized with the constraint of no more than two consecutive presentations of the same shape pair; (2) during the first test phase, A was presented eight times in a compound with B (for half of the participants) or with a novel stimulus C (for the other half of the participants) without any UCS; (3) during re-conditioning four AX+ presentations again co-terminated with the UCS, while four BX − presentations were never paired with the UCS; and (4) during the second test phase, A was presented in compound with either B or C (whichever

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was not presented in the first test phase). The order of AB and AC test phases was balanced between participants. For ethical reasons, the final test phase was followed by an extinction phase in which AX as well as A, B, and X were presented several times without reinforcement. After finishing the experiment, participants were asked several questions to determine their awareness of CS–UCS contingencies. The interview started with a free recall question (“Did you know when you were going to receive an electro-tactile stimulus?”). Then multiple choice questions about the association of the UCS with each of the geometric shapes were asked which applied only to those experimental phases in which electro-tactile stimuli were presented (e.g. “The presentation of an electro-tactile stimulus followed the pentagon (1) mostly yes, (2) mostly no, and (3) not possible to answer.”). Participants were classified as aware if they either answered the free recall question correctly (91%) or were able to correctly identify the AX+ throughout the further interview (in total 95.5%; cf. Weike et al., 2007). There were no differences in awareness between genotype groups. To minimize a possible moderating effect of hormone levels on the association of genotype and conditioned inhibition of fear, women participated in the experiment only between the 2nd and 4th day of their menstruation cycle to ensure low levels of female sexual hormones and their releasing factors (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/AgCl miniature surface electrodes (4 mm diameter; Grass Products) filled with electrolyte (Marquette Hellige) were attached beneath the left eye. A Coulbourn S75-01 bioamplifier served to amplify the raw EMG with a 30 Hz high-pass filter and a 400 Hz Kemo-VBF8-03 low-pass filter. Digital sampling was set to 1000 Hz from 100 ms prior to until 400 ms after onset of the startle probe. The EMG signal was filtered through 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) EDA was recorded from the hypothenar eminence of the palmar surface of the participant's right hand. A Coulbourn S71-22 skin conductance coupler provided a constant 0.5 V across two Ag/AgCl standard electrodes (8 mm diameter; Gelimed) filled with a 0.05 M sodium chloride electrolyte medium. The analog signal was sampled continuously at a rate of 10 Hz. 2.5. Data reduction and response definition

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

2.5.1. Startle blink magnitudes The magnitude of the startle eyeblink was scored offline using a computer algorithm (Globisch et al., 1993) that automatically identified latency of blink onset and peak amplitude in microvolt. Only blinks starting 20–120 ms after probe onset and peaking within 150 ms were scored. No detectable blinks were scored as zero responses. Five participants with excessive baseline activity or recording artifacts (N 20%) were excluded from the data analysis. For the remaining participants, trials with excessive 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 between genotype groups (5-HTTLPR and COMT Val158Met: both Fs b 1). Blink magnitudes were standardized for each participant using a z-score transformation. The standardized responses of each participant were then converted to T-scores [50 + (z × 10)]. To test for fear acquisition, AX+ and BX− mean scores were calculated for the conditioning phase as well as for the re-conditioning phase. To test for fear inhibition, an average AX+ score covering the last four trials of conditioning and the four re-conditioning trials were compared with the first four trials of the AB

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

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and AC test phases.2 Potentiation scores were calculated as difference scores from ITI startle responses. With five additional participants excluded due to technical problems (e.g., detachment of electrodes) the final sample 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 increase between 0.9 and 4.0 s after stimulus onset (first interval response) using a computer program (Globisch et al., 1993). A logarithmic transformation was applied to normalize the distribution (Venables and Christie, 1980) and the log values were range corrected by dividing each individual score by the participant's maximum response to reduce inter-individual variability unrelated to the task (Lykken and Venables, 1971). Mean SCR scores were calculated as described for startle blink magnitudes. Three participants were excluded from SC response analysis due to technical problems (final sample: N = 103). 2.6. Data analysis To test for genotype group differences during acquisition, repeated measures analyses of variance (ANOVAs) with condition (AX+ vs. BX− vs. ITI for startle responses, AX+ vs. BX− for SCRs) as a within-subject variable and 5-HTTLPR (s-allele carriers vs. l-allele homozygotes) or COMT Val158Met (Val-allele carriers vs. Met-allele homozygotes) as between-subject variable were conducted for the conditioning and the re-conditioning phases separately. To test for differences in conditioned and external inhibition, repeated measures ANOVAs were conducted with inhibition (AX+vs. AB vs. AC) as a within-subject variable and with 5-HTTLPR (s-allele carriers vs. l-allele homozygotes) or COMT Val158Met (Val-allele carriers vs. Met-allele homozygotes) as betweensubject variable. We introduced order of test phases as a covariate. To test the hypotheses that the combination of a 5-HTTLPR s-allele and COMT met-homozygosity results in an enhanced deficit in conditioned inhibition (cf. Lonsdorf et al., 2009b), an additional repeated measures ANOVA was conducted with conditioned inhibition (AX+ vs. AB) as a within-subject variable, COMT Val158Met as a between-subject variable and 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 level of significance. Greenhouse–Geisser correction was used in case of violated sphericity. Nominal degrees of freedom are reported along with partial η2 values as an estimate of effect size. 3. Results 3.1. Fear acquisition As expected, we observed a pronounced main effect of condition on startle blink responses elicited during conditioning, F(2,188) = 109.17, p b .001, and partial η2 = .54. Startle blink responses were significantly potentiated when elicited during the presentation of AX + as well as during 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 potentiation during AX + presentations was significantly stronger than potentiation observed during BX − presentations; AX+ vs. BX−: F(1,95) = 91.77, p b .001, partial η2 = .49. Startle potentiation during AX+ as well as the discrimination of AX+ vs. BX− was not influenced by the 5-HTTLPR genotype (AX+ vs. ITI and AX+ vs. BX− × 5-HTTLPR interaction: 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 trials of 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 the non-reinforcement of stimulus A (extinction learning).

potentiation to the AX+ relative to the BX − condition than did homozygous 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 COMT Val158Met genotype (conditioning and re-conditioning: both Fs b 1). Skin conductance response magnitudes were also increased in response to AX+ compared to BX− trials during conditioning; AX+ vs. BX−: F(1,102) = 79.47, p b .001, partial η2 = .44. Replicating previous results, this effect was neither influenced by 5-HTTLPR nor by COMT Val158Met genotypes (both Fs b 1). During re-conditioning, however, the difference between SCR magnitudes to AX + and BX − remained significant only for COMT Val-allele carriers; AX+ vs. BX− × COMT: F(1,101) = 12.48, p b .01, partial η2 = .11 (means (SDs) during reconditioning 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 from ITI) of the first four test phase trials (AB and AC) were compared to the mean AX + potentiation scores of the last four conditioning and the four re-conditioning trials. A significant AX+ vs. AB vs. AC × COMT interaction (F(2,184) = 3.66, p b .05, partial η2 = .04) indicated a differential influence of COMT Val158Met genotype on conditioned and external inhibition. Post-hoc tests revealed that only COMT Val-allele carriers showed a significant inhibition of startle responses during AB compared to AX+ trials (t(61) = 2.05, p b .05) and more pronounced inhibition during AB compared to AC trials (t(61) = 2.08, p b .05). In contrast, homozygous COMT Met-allele carriers showed a pronounced startle potentiation during AB presentations despite the presence of a safety signal (AB N ITI: t(33) = 6.63, p b .001) which was significantly larger than startle potentiation in COMT Val-allele carriers during AB presentations (t(94) = 2.17, p b .05); see Fig. 3, left panel.3 As supported by a significant AB vs. AX+ × COMT interaction in 5HTTLPR 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 carriers showed a significant inhibition during AB compared to AX + trials (t(29) = 2.90, p b .01), whereas those s-allele carriers who were also homozygotes for the COMT Met-allele did not show any startle inhibition (t(21) = −1.24, n.s.). Supporting this difference in conditioned inhibition of fear, startle potentiation during AB trials was significantly higher in 5-HTTLPR s-allele carriers who were homozygous COMT Met-allele carriers compared to those who were COMT Val-allele carriers (t(50) = 2.80, p b .01); see Fig. 3, right panel. Exploratory analysis of COMT Val158Met genotype influences on conditioned inhibition effects in homozygous 5-HTTLPR l-allele carriers revealed no significant interaction (F b 1). Fear inhibition effects were not influenced by the order of test phases. Skin conductance responses were also significantly inhibited when the safety cue was combined with the excitatory stimulus. SCRs were significantly larger to the last four conditioning and the four reconditioning AX+ onsets than to the first four AB presentations in the test phase; AX+ vs. AB: F(1,102) = 8.99, p b .01, partial η2 = .08. This effect was not significant for the AX+ vs. AC comparison, F b 1. As indicated 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 to AX+ trials were found only in COMT Val-allele carriers (AB: t(69) = 3.61, p b .01). This interaction was not a result of differential responding 3 Additional categorical analysis revealed that more homozygous COMT Met-allele carriers showed augmented startle responses during AB compared to AC trials while the opposite was true for Val-allele carriers (χ2(1) = 7.81, p b .01). That is, based on the odds ratio, the odds to find the assumed increased inhibitory strength of the conditioned inhibitor B compared to the external inhibitor C were 3.39 times higher in COMT Val-allele carriers than in homozygous Met-allele carriers.

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

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

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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) in 5-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.

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

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

Fig. 3. Mean startle blink potentiation (difference from ITI startle responses) to AX+ (black bars) during the last four conditioning and the four re-conditioning trials as well as the first four AB (gray bars) and AC (striped bars) trials. The left panel shows differences in AB and AC test phases in COMT Val158Met genotypes. The right panel shows the AB test in 5-HTTLPR s-allele carriers only. Mean values are displayed with standard errors. Asterisks indicate significant differences. ***p b .001, **p b .01; *p b .05.

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

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potentiation was more pronounced in 5-HTTLPR s-allele carriers indicating a prolonged expression of previously learned fear responses in those genotypes. Different from COMT Val-allele carriers, homozygous Metallele carriers showed a pronounced potentiation of the startle response during processing of the fear-conditioned cue despite the presence of a safety signal (AB). As expected, the fear inhibition deficit was specific to the conditioned inhibitor B and not observed for the external (attention-related) inhibitor C, thus, indicating a deficit in learning inhibitory associations in homozygous COMT Met-allele carriers. 4.1. 5-HTTLPR and fear learning The 5-HTTLPR s-allele has been associated with different anxietyrelated personality traits such as neuroticism and harm avoidance (Munafò et al., 2005) and can increase the risk of post-traumatic stress disorder (PTSD) most probably depending on trauma load (Kilpatrick et al., 2007; Koenen et al., 2009; Kolassa et al., 2010a) as well as the severity of panic disorder symptoms (Lonsdorf et al., 2009a). Findings of 5-HTTLPR-dependent modulation of the amygdala and its prefrontal coupling during processing of affective relative to neutral information (Dannlowski et al., 2008; Heinz et al., 2005) may provide a link between said polymorphism and fear learning as indexed with startle blink responses (Munafò et al., 2008; Todd et al., 2011). However, a current working model of molecular processes in the lateral amygdala during fear acquisition does not include a possible modulation by serotonin since its role has not been studied in detail yet (Johansen et al., 2011). In a recent review, Lonsdorf and Kalisch comment that little is known about the effects of genetic polymorphisms on temporal aspects of fear acquisition (Lonsdorf and Kalisch, 2011). In our study, both 5-HTTLPR genotypes exhibit comparable fear learning during the initial acquisition phase. However, 5-HTTLPR s-allele carriers show significantly increased conditioned fear-potentiated startle responses during reconditioning, thus showing stronger expression of the fear response over a longer period of time. Thus differences between both groups develop later during fear acquisition than in previous experiments (Lonsdorf et al., 2009b). This could be due to differences in stimulus materials used: where we used pairs of geometric shapes, Lonsdorf and coworkers used angry faces (Lonsdorf et al., 2009b) as conditioned stimuli. Angry faces are known to activate the amygdala (at least when their gaze is directed at the observer: N'Diaye et al., 2009; Sauer et al., 2013) and, therefore, may facilitate the serotonergic influence during fear acquisition and fear expression. Using geometric shapes as conditioned stimuli it might take longer until group differences in the expression of learned fear are revealed. Replicating previous findings (Lonsdorf et al., 2009b), fearpotentiated startle responses but not conditioned discrimination of the skin conductance responses was modulated by the 5-HTTLPR polymorphism. Thus, our results support the notion that the 5-HTTLPR polymorphism influences the expression of amygdala driven defensive behavior during fear – as reflected by startle potentiation – rather than the supporting autonomic changes that are not always fear specific but are also dependent on increased orienting to the reinforced cue driven by cognitive processes of shock expectancy and contingency awareness (Hamm and Vaitl, 1996; Soeter and Kindt, 2010). The lack of genotype differences in contingency awareness further strengthens this notion. 4.2. COMT Val158Met and fear inhibition COMT Met-allele carriers have been shown to be more frequent among some PTSD populations (Boscarino et al., 2012; Valente et al., 2011) and homozygous Met-allele carriers seem to exhibit an elevated risk for developing PTSD even at low trauma load (Kolassa et al., 2010b). Our results replicate and extend previous findings on reduced fear extinction by COMT Met-allele carriers (Lonsdorf et al., 2009b; Norrholm et al., 2013) which may explain their increased vulnerability

to develop symptoms after experiencing a trauma (cf. Myers and Davis, 2004). Using an conditional discrimination procedure previously validated in animals (Myers and Davis, 2004) and adapted to human startle research (Jovanovic et al., 2005) the current data demonstrate that not only extinction learning but also inhibition of the fear response by the presence of a learned safety cue is significantly impaired in COMT Met-allele carriers. Thus, reduced extinction of fear observed in Metallele carriers seems to be indeed a result of impaired building of new inhibitory associations. Animal research suggests that inhibitory learning is mediated by the infralimbic region of the ventromedial prefrontal cortex (vmPFC) which exerts an inhibitory influence on the amygdala during extinction of learned fear (Paré et al., 2004; Quirk et al., 2000). Dopamine has been found to suppress inhibitory medial PFC effects on the amygdala (Rosenkranz and Grace, 2001, 2002). That is, the increased dopamine availability in COMT Met-allele carriers (Tunbridge et al., 2006) might explain their safety learning deficit. 5. Conclusion Learning theory based models explaining the etiology of anxiety disorders proclaim that pathological anxiety is a result of a learned association between certain cues and a fear response formed during the experience of a traumatic event (Field, 2006; Mineka and Oehlberg, 2008). But neither do all people who have experienced traumatic events develop pathological anxiety (Breslau et al., 1991), nor do all patients respond when treated with exposure therapy (Ougrin, 2011). Thus, current learning theory based etiology models of anxiety disorders have to take into account both the importance of vulnerabilities partly based on genetic factors and conditioning processes taking place during traumatic or stressful life events when explaining the development and maintenance of pathological anxiety (Mineka and Zinbarg, 2006; Skelton et al., 2012). Although the effect sizes of genetic effects are generally small in the present study and the effect of 5-HTTLPR and COMT Val158Met polymorphisms on conditioned inhibition needs to be replicated before drawing extensive conclusions, our results indicate that 5-HTTLPR and COMT Val158Met polymorphism modulate the vulnerability for the development of anxiety disorders via different mechanisms: the 5-HTTLPR polymorphism seems to modulate the stability of fear expression during cues that have been associated with aversive events and, thus, may affect the quality and intensity of the expression of conditioned fear once consolidated fear memories are activated. The COMT Val158Met polymorphism seems to modulate a separate mechanism influencing inhibition 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 Research Society (DFG) (WE 2762/5-1). We thank Carmen Hamm and Paulina Troitzsch for blood sampling, Anja Wiechert for excellent technical assistance in the genotyping analyses, Cosma Hoffmann for help with participant recruitment, Svenda Berg and Wiebke Grieser for help with the diagnostic interviews, and Henriette Hacker and Laura Troike for help with data acquisition. References Blumenthal, T.D., Cuthbert, B.N., Filion, D.L., Hackley, S., Lipp, O.V., van Boxtel, A., 2005. Committee report: guidelines for human startle eyeblink electromyographic studies. Psychophysiology 42, 1–15. http://dx.doi.org/10.1111/j.1469-8986.2005.00271.x. Boscarino, J.A., Erlich, P.M., Hoffman, S.N., Zhang, X., 2012. Higher FKBP5, COMT, CHRNA5, and CRHR1 allele burdens are associated with PTSD and interact with trauma exposure: implications for neuropsychiatric research and treatment. Neuropsychiatr. Dis. Treat. http://dx.doi.org/10.2147/NDT.S29508. Breslau, N., Davis, G.C., Andreski, P., Peterson, E., 1991. Traumatic events and posttraumatic stress disorder in an urban population of young adults. Arch. Gen. Psychiatry 48, 216–222. http://dx.doi.org/10.1001/archpsyc.1991.01810270028003.

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007

J. Wendt et al. / International Journal of Psychophysiology xxx (2014) xxx–xxx Dannlowski, U., Ohrmann, P., Bauer, J., Deckert, J., Hohoff, C., Kugel, H., Suslow, T., 2008. 5HTTLPR biases amygdala activity in response to masked facial expressions in major depression. Neuropsychopharmacology 33, 418–424. http://dx.doi.org/10.1038/sj. npp.1301411. Davis, M., 1992. The role of the amygdala in fear-potentiated startle: implications for animal models of anxiety. Trends Pharmacol. Sci. 13, 35–41. Davis, M., Walker, D.L., Miles, L., Grillon, C., 2010. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology 35 (1), 105–135. http://dx.doi.org/10.1038/npp.2009.109. Field, A.P., 2006. Is conditioning a useful framework for understanding the development and treatment of phobias? Clin. Psychol. Rev. 26 (7), 857–875. http://dx.doi.org/10. 1016/j.cpr.2005.05.010. Globisch, J., Hamm, A., Schneider, R., Vaitl, D., 1993. A computer program for scoring reflex eyeblink and electrodermal responses written in Pascal. Psychophysiology 39, S30. Glotzbach-Schoon, E., Andreatta, M., Reif, A., Ewald, H., Tröger, C., Baumann, C., Pauli, P., 2013. Contextual fear conditioning in virtual reality is affected by 5HTTLPR and NPSR1 polymorphisms: effects on fear-potentiated startle. Front. Behav. Neurosci. 7, 31. http://dx.doi.org/10.3389/fnbeh.2013.00031 (April). Hamm, A.O., Vaitl, D., 1996. Affective learning: awareness and aversion. Psychophysiology http://dx.doi.org/10.1111/j.1469-8986.1996.tb02366.x. Hamm, A.O., Weike, A.I., 2005. The neuropsychology of fear learning and fear regulation. Int. J. Psychophysiol. 57 (1), 5–14. http://dx.doi.org/10.1016/j.ijpsycho.2005.01.006. Hamm, A.O., Greenwald, M.K., Bradley, M.M., Lang, P.J., 1993. Emotional learning, hedonic change, and the startle probe. J. Abnorm. Psychol. 102 (3), 453–465 (Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8408958). Hariri, A.R., 2011. The what, where, and when of catechol-O-methyltransferase. Biol. Psychiatry 70 (3), 214–215. http://dx.doi.org/10.1016/j.biopsych.2011.06.002. Heinz, A., Braus, D.F., Smolka, M.N., Wrase, J., Puls, I., Hermann, D., Büchel, C., 2005. Amygdala-prefrontal coupling depends on a genetic variation of the serotonin transporter. Nat. Neurosci. 8 (1), 20–21. http://dx.doi.org/10.1038/nn1366. Heitland, I., Groenink, L., Bijlsma, E.Y., Oosting, R.S., Baas, J.M.P., 2013. Human fear acquisition deficits in relation to genetic variants of the corticotropin releasing hormone receptor 1 and the serotonin transporter. PLoS One 8 (5), e63772. http://dx.doi.org/ 10.1371/journal.pone.0063772. Hitchcock, J.M., Davis, M., 1986. Lesions of the amygdala, but not of the cerebellum or red nucleus, block conditioned fear as measured with the potentiated startle paradigm. Behav. Neurosci. 100, 11–22. http://dx.doi.org/10.1037/0735-7044.100.1.11. Hitchcock, J.M., Davis, M., 1991. Efferent pathway of the amygdala involved in conditioned fear as measured with the fear-potentiated startle paradigm. Behav. Neurosci. 105, 826–842. http://dx.doi.org/10.1037/0735-7044.105.6.826. Johansen, J.P., Cain, C.K., Ostroff, L.E., LeDoux, J.E., 2011. Molecular mechanisms of fear learning and memory. Cell 147 (3), 509–524. http://dx.doi.org/10.1016/j.cell.2011. 10.009. Jovanovic, T., Keyes, M., Fiallos, A., Myers, K.M., Davis, M., Duncan, E.J., 2005. Fear potentiation and fear inhibition in a human fear-potentiated startle paradigm. Biol. Psychiatry 57 (12), 1559–1564. http://dx.doi.org/10.1016/j.biopsych.2005.02.025. Jovanovic, T., Norrholm, S.D., Keyes, M., Fiallos, A., Jovanovic, S., Myers, K.M., Duncan, E.J., 2006. Contingency awareness and fear inhibition in a human fear-potentiated startle paradigm. Behav. Neurosci. 120, 995–1004. http://dx.doi.org/10.1037/0735-7044. 120.5.995. Kilpatrick, D.G., Koenen, K.C., Ruggiero, K.J., Acierno, R., Galea, S., Resnick, H.S., Gelernter, J., 2007. The serotonin transporter genotype and social support and moderation of posttraumatic stress disorder and depression in hurricane-exposed adults. Am. J. Psychiatry 164, 1693–1699. http://dx.doi.org/10.1176/appi.ajp.2007.06122007. Klumpers, F., 2012. Fear not — Neurobiological Mechanisms of Fear and Anxiety. Ipskamp Drukkers B.V., the Netherlands. Klumpers, F., Heitland, I., Oosting, R.S., Kenemans, J.L., Baas, J.M.P., 2012. Genetic variation in serotonin transporter function affects human fear expression indexed by fear-potentiated startle. Biol. Psychol. 89, 277–282. http://dx.doi.org/10.1016/j. biopsycho.2011.10.018. Koenen, K.C., Aiello, A.E., Bakshis, E., Amstadter, A.B., Ruggiero, K.J., Acierno, R., Galea, S., 2009. Modification of the association between serotonin transporter genotype and risk of posttraumatic stress disorder in adults by county-level social environment. Am. J. Epidemiol. 169, 704–711. http://dx.doi.org/10.1093/aje/kwn397. Kolassa, I.-T., Ertl, V., Eckart, C., Glöckner, F., Kolassa, S., Papassotiropoulos, A., Elbert, T., 2010a. Association study of trauma load and SLC6A4 promoter polymorphism in posttraumatic stress disorder: evidence from survivors of the Rwandan genocide. J. Clin. Psychiatry 71, 543–547. http://dx.doi.org/10.4088/JCP.08m04787blu. Kolassa, I.-T., Kolassa, S., Ertl, V., Papassotiropoulos, A., De Quervain, D.J.-F., 2010b. The risk of posttraumatic stress disorder after trauma depends on traumatic load and the catechol-O-methyltransferase Val(158)Met polymorphism. Biol. Psychiatry 67 (4), 304–308. http://dx.doi.org/10.1016/j.biopsych.2009.10.009. Lonsdorf, T.B., Kalisch, R., 2011. A review on experimental and clinical genetic associations studies on fear conditioning, extinction and cognitive-behavioral treatment. Transl. Psychiatry 1 (9), e41. http://dx.doi.org/10.1038/tp.2011.36. Lonsdorf, T.B., Rück, C., Bergström, J., Andersson, G., Ohman, A., Schalling, M., Lindefors, N., 2009a. The symptomatic profile of panic disorder is shaped by the 5-HTTLPR polymorphism. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 33 (8), 1479–1483. http://dx.doi.org/10.1016/j.pnpbp.2009.08.004. Lonsdorf, T.B., Weike, A.I., Nikamo, P., Schalling, M., Hamm, A.O., Ohman, A., 2009b. Genetic gating of human fear learning and extinction: possible implications for geneenvironment interaction in anxiety disorder. Psychol. Sci. 20, 198–206. http://dx. doi.org/10.1111/j.1467-9280.2009.02280.x.

7

Lykken, D.T., Venables, P.H., 1971. Direct measurement of skin conductance: a proposal for standardization. Psychophysiology 8, 656–672. http://dx.doi.org/10.1111/j.14698986.1971.tb00501.x. Männistö, P.T., Kaakkola, S., 1999. Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors. Pharmacol. Rev. 51 (4), 593–628 (Retrieved from http://www.ncbi.nlm.nih. gov/pubmed/10581325). Milad, M.R., Rauch, S.L., Pitman, R.K., Quirk, G.J., 2006. Fear extinction in rats: implications for human brain imaging and anxiety disorders. Biol. Psychol. 73, 61–71. http://dx. doi.org/10.1016/j.biopsycho.2006.01.008. Mineka, S., Oehlberg, K., 2008. The relevance of recent developments in classical conditioning to understanding the etiology and maintenance of anxiety disorders. Acta Psychol. 127 (3), 567–580. http://dx.doi.org/10.1016/j.actpsy.2007.11.007. Mineka, S., Zinbarg, R., 2006. A contemporary learning theory perspective on the etiology of anxiety disorders: it's not what you thought it was. Am. Psychol. 61 (1), 10–26. http://dx.doi.org/10.1037/0003-066X.61.1.10. Montag, C., Buckholtz, J.W., Hartmann, P., Merz, M., Burk, C., Hennig, J., Reuter, M., 2008. COMT genetic variation affects fear processing: psychophysiological evidence. Behav. Neurosci. 122, 901–909. http://dx.doi.org/10.1037/0735-7044.122.4.901. Munafò, M.R., Clark, T., Flint, J., 2005. Promise and pitfalls in the meta-analysis of genetic association studies: a response to Sen and Schinka. Mol. Psychiatry http://dx.doi.org/ 10.1038/sj.mp.4001706. Munafò, M.R., Brown, S.M., Hariri, A.R., 2008. Serotonin transporter (5-HTTLPR) genotype and amygdala activation: a meta-analysis. Biol. Psychiatry 63 (9), 852–857. http://dx. doi.org/10.1016/j.biopsych.2007.08.016. Myers, K.M., Davis, M., 2004. AX+, BX− Discrimination Learning in the Fear-Potentiated Startle Paradigm: Possible Relevance to Inhibitory Fear Learning in Extinction, (Ci), pp. 464–475 http://dx.doi.org/10.1101/lm.74704.limited. N'Diaye, K., Sander, D., Vuilleumier, P., 2009. Self-relevance processing in the human amygdala: gaze direction, facial expression, and emotion intensity. Emotion 9 (6), 798–806. http://dx.doi.org/10.1037/a0017845. Norrholm, S.D., Jovanovic, T., Smith, A.K., Binder, E., Klengel, T., Conneely, K., Ressler, K.J., 2013. Differential genetic and epigenetic regulation of catechol-O-methyltransferase is associated with impaired fear inhibition in posttraumatic stress disorder. Front. Behav. Neurosci. 7, 30. http://dx.doi.org/10.3389/fnbeh.2013.00030. Ougrin, D., 2011. Efficacy of exposure versus cognitive therapy in anxiety disorders: systematic review and meta-analysis. BMC Psychiatry 11 (1), 200. http://dx.doi.org/10. 1186/1471-244X-11-200. Paré, D., Quirk, G.J., LeDoux, J.E., 2004. New vistas on amygdala networks in conditioned fear. J. Neurophysiol. 92 (1), 1–9. http://dx.doi.org/10.1152/jn.00153.2004. Pavlov, I.P., 1927. Conditioned Reflexes vol. 17. Oxford University Press, p. 448. Quirk, G.J., Russo, G.K., Barron, J.L., Lebron, K., 2000. The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J. Neurosci. 20, 6225–6231 (20/16/6225 [pii]). Risbrough, V., Ji, B., Hauger, R., Zhou, X., 2014. Generation and characterization of humanized mice carrying COMT158 Met/Val alleles. Neuropsychopharmacology 39, 1823–1832. Rosenkranz, J.A., Grace, A.A., 2001. Dopamine attenuates prefrontal cortical suppression of sensory inputs to the basolateral amygdala of rats. J. Neurosci. 21, 4090–4103 (21/11/ 4090 [pii]). Rosenkranz, J.A., Grace, A.A., 2002. Cellular mechanisms of infralimbic and prelimbic prefrontal cortical inhibition and dopaminergic modulation of basolateral amygdala neurons in vivo. J. Neurosci. 22, 324–337 (22/1/324 [pii]). Sauer, A., Mothes-Lasch, M., Miltner, W.H.R., Straube, T., 2013. Effects of gaze direction, head orientation and valence of facial expression on amygdala activity. Soc. Cogn. Affect. Neurosci. 1–7 http://dx.doi.org/10.1093/scan/nst100. Skelton, K., Ressler, K.J., Norrholm, S.D., Jovanovic, T., Bradley-Davino, B., 2012. PTSD and gene variants: new pathways and new thinking. Neuropharmacology 62 (2), 628–637. http://dx.doi.org/10.1016/j.neuropharm.2011.02.013. Soeter, M., Kindt, M., 2010. Dissociating response systems: erasing fear from memory. Neurobiol. Learn. Mem. 94, 30–41. http://dx.doi.org/10.1016/j.nlm.2010.03.004. Todd, R.M., Palombo, D.J., Levine, B., Anderson, A.K., 2011. Genetic differences in emotionally enhanced memory. Neuropsychologia 49 (4), 734–744. http://dx.doi.org/10. 1016/j.neuropsychologia.2010.11.010. Tunbridge, E.M., Harrison, P.J., Weinberger, D.R., 2006. Catechol-O-methyltransferase, cognition, and psychosis: Val158Met and beyond. Biol. Psychiatry 60 (2), 141–151. http://dx.doi.org/10.1016/j.biopsych.2005.10.024. Valente, N.L.M., Vallada, H., Cordeiro, Q., Bressan, R.A., Andreoli, S.B., Mari, J.J., Mello, M.F., 2011. Catechol-O-methyltransferase (COMT) val158met polymorphism as a risk factor for PTSD after urban violence. J. Mol. Neurosci. 43 (3), 516–523. http://dx.doi.org/ 10.1007/s12031-010-9474-2. Venables, P.H., Christie, M.J., 1980. Electrodermal activity. In: Martin, I., Venables, P.H. (Eds.), Techniques in Psychophysiology. Wiley, New York, pp. 4–67. Weike, A.I., Hamm, A.O., Schupp, H.T., Runge, U., Schroeder, H.W.S., Kessler, C., 2005. Fear conditioning following unilateral temporal lobectomy: dissociation of conditioned startle potentiation and autonomic learning. J. Neurosci. 25 (48), 11117–11124. http://dx.doi.org/10.1523/JNEUROSCI. 2032-05.2005. Weike, A.I., Schupp, H.T., Hamm, A.O., 2007. Fear acquisition requires awareness in trace but not delay conditioning. Psychophysiology 44 (1), 170–180. http://dx.doi.org/10. 1111/j.1469-8986.2006.00469.x. Wittchen, H.-U., Zaudig, M., Fydrich, T., 1997. Strukturiertes Klinisches Interview für DSM-IV [Structured Clinical Interview for DSM-IV]. Hogrefe, Göttingen http://dx. doi.org/10.1026//0084-5345.28.1.68.

Please cite this article as: Wendt, J., et al., Genetic influences on the acquisition and inhibition of fear, Int. J. Psychophysiol. (2014), http:// dx.doi.org/10.1016/j.ijpsycho.2014.10.007