Scholarly article on topic 'No evidence for blocking the return of fear by disrupting reconsolidation prior to extinction learning'

No evidence for blocking the return of fear by disrupting reconsolidation prior to extinction learning Academic research paper on "Clinical medicine"

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{Extinction / Fear / Reconsolidation / Amygdala / "Fear conditioning" / COMT}

Abstract of research paper on Clinical medicine, author of scientific article — Tim Klucken, Onno Kruse, Jan Schweckendiek, Yvonne Kuepper, Erik M. Mueller, et al.

Abstract Fear extinction is a central model for the treatment of anxiety disorders. Initial research has reported that the single presentation of a conditioned stimulus prior to extinction learning can permanently block the return of fear. However, only few studies have explored this issue and could not always replicate the findings. The present study examined human fear extinction using a four-day design. On the first day, two neutral stimuli were paired with electrical stimulation (UCS), while a third stimulus (CS−) was not. Twenty-four hours later, one conditioned stimulus (CS+rem) and the CS− were reminded once, 10 min before extinction learning, while the other conditioned stimulus (CS+non-rem) was not presented prior to extinction learning. All stimuli were presented during extinction learning and during two re-extinction sessions (24 h and 6-months after extinction learning) without reinforcement. Blood oxygen level-dependent (BOLD) responses and skin conductance responses (SCRs) to both CS+ and the CS− were explored during acquisition, extinction, and in both re-extinction sessions. Regarding SCRs, the results showed that a single presentation of a conditioned stimulus did not block the return of fear during re-extinction: Fear recovery during re-extinction (24 h and 6-months after extinction learning) was observed for both CS+ compared with the CS− with no difference between CS+rem and CS+non-rem. Regarding BOLD-responses, no significant differences between CS+rem and CS+non-rem were found in region of interest (ROI)-analyses (amygdala, ventromedial prefrontal cortex) during extinction learning and both re-extinction sessions. Whole-brain analyses showed increased BOLD-responses to the CS+non-rem as compared to the CS+rem in several regions (e.g., middle frontal gyrus) during extinction learning and re-extinction (24 h after extinction learning). The present findings suggest that the effect of preventing the return of fear by disrupting reconsolidation seems to be a more labile phenomenon than previously assumed. Possible boundary conditions and implications are discussed.

Academic research paper on topic "No evidence for blocking the return of fear by disrupting reconsolidation prior to extinction learning"

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No evidence for blocking the return of fear by disrupting reconsolidation prior to extinction learning

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Tim Klucken a,b,*> Onno Kruse a,b,Jan Schweckendiek a,b, Yvonne Kuepper Erik M. Mueller d, Juergen Hennig c and Rudolf Stark a,b

a Department of Psychotherapy and Systems Neuroscience, Justus Liebig University Giessen, Germany b Bender Institute of Neuroimaging, Justus Liebig University Giessen, Germany c Personality Psychology and Individual Differences, Justus Liebig University Giessen, Germany d Department of Clinical Psychology, Justus Liebig University Giessen, Germany

ARTICLE INFO

ABSTRACT

Article history: Received 3 June 2015 Revised 4 October 2015 Accepted 16 March 2016 Reviewed 10 July 2015 Action editor Peter Kirsch Published online 24 March 2016

Keywords: Extinction Fear

Reconsolidation Amygdala Fear conditioning COMT

Fear extinction is a central model for the treatment of anxiety disorders. Initial research has reported that the single presentation of a conditioned stimulus prior to extinction learning can permanently block the return of fear. However, only few studies have explored this issue and could not always replicate the findings.

The present study examined human fear extinction using a four-day design. On the first day, two neutral stimuli were paired with electrical stimulation (UCS), while a third stimulus (CS-) was not. Twenty-four hours later, one conditioned stimulus (CS+rem) and

the CS- were reminded once, 10 min before extinction learning, while the other conditioned stimulus (CS+non-rem) was not presented prior to extinction learning. All stimuli were presented during extinction learning and during two re-extinction sessions (24 h and 6-months after extinction learning) without reinforcement. Blood oxygen level-dependent (BOLD) responses and skin conductance responses (SCRs) to both CS+ and the CS- were explored during acquisition, extinction, and in both re-extinction sessions.

Regarding SCRs, the results showed that a single presentation of a conditioned stimulus did not block the return of fear during re-extinction: Fear recovery during re-extinction (24 h and 6-months after extinction learning) was observed for both CS+ compared with the CS- with no difference between CS+rem and CS+non-rem. Regarding BOLD-responses, no significant differences between CS+rem and CS+non-rem were found in region of interest (ROI)-analyses (amygdala, ventromedial prefrontal cortex) during extinction learning and both re-extinction sessions. Whole-brain analyses showed increased BOLD-responses to the CS+non-rem as compared to the CS+rem in several regions (e.g., middle frontal gyrus) during extinction learning and re-extinction (24 h after extinction learning).

* Corresponding author. Department of Psychotherapy and Systems Neuroscience, Justus Liebig University Giessen, Otto-Behaghel-Str. 10 F, 35394 Giessen, Germany.

E-mail address: tim.klucken@psychol.uni-giessen.de (T. Klucken). http://dx.doi.org/10.1016/j.cortex.2016.03.015

0010-9452/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

The present findings suggest that the effect of preventing the return of fear by disrupting reconsolidation seems to be a more labile phenomenon than previously assumed. Possible boundary conditions and implications are discussed.

© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Fear conditioning and extinction are well-established models for the development, maintenance, and treatment of anxiety disorders (Goode & Maren, 2014; Milad & Quirk, 2012; Milad, Rosenbaum, & Simon, 2014; Vervliet, Craske, & Hermans, 2013). While fear associations can be rapidly acquired and persist over time, the extinction memory is susceptible to disruptions and cannot permanently block the initial fear memory (Myers & Davis, 2007). Consequently, treatments of psychiatric disorders based on extinction learning (e.g., exposure therapy) often produce effective short-term fear reductions, but relapses are not uncommon (Choy, Fyer, & Lipsitz, 2007; Lipsitz, Mannuzza, Klein, Ross, & Fyer, 1999; Sharma, Thennarasu, & Janardhan Reddy, 2014). Therefore, the identification of specific factors that may decrease the return of fear and the number of relapses are of high clinical interest.

Differential fear conditioning paradigms typically consist of different phases (fear acquisition, extinction learning, and reextinction). During fear acquisition, one or two neutral stimuli (CS+) are initially paired with electrical stimulation (UCS), while another stimulus (CS-) is not. After a few trials, the CS+ elicits conditioned responses (CRs) such as increased skin conductance responses (SCRs), startle amplitude, or subjective ratings (Hamm & Weike, 2005; Lang, Davis, & Ohman, 2000). After that, the CS+ and CS- are repeatedly presented without the UCS (extinction learning), which finally results in a decrease of CRs in subjective and physiological responses (Milad & Quirk, 2012; Myers & Davis, 2007; Quirk & Mueller, 2008). During this time, the extinction memory is mainly modulated by the amygdala (Quirk & Mueller, 2008). After that, the CS+ and the CS- are again presented without reinforcement (re-extinction), e.g., 24 h after extinction learning. The return of fear could be observed under a variety of conditions such as spontaneous recovery, reinstatement, and renewal (Bouton, 2002; Myers & Davis, 2002, 2007). Spontaneous recovery can be described as the reappearance of previously extinguished CRs after a delay following extinction learning without any further learning sessions due to the mere passage of time. Reinstatement refers to the reoccurrence of CRs after extinction learning through the presentation of an unpredictable UCS. Finally, renewal refers to the reactivation of CRs if a subsequent test session is conducted in a different context than the extinction phase. Many methods have been developed to analyze the reoccurrence of CRs of fear during re-extinction. While some studies compared the responses towards the CS+ and the CS-during the first half or the first trial of re-extinction, others authors calculated different "fear-recovery indices" (e.g., first re-extinction trial minus last extinction learning trial; Schiller, Kanen, LeDoux, Monfils, & Phelps, 2013; Schiller et al., 2010).

Recently, animal and human studies have demonstrated that the re-occurrence of conditioned fear during reextinction can be prevented by different techniques, which presumably alter the initial fear memory (Agren, 2014; Agren, Furmark, Eriksson, & Fredrikson, 2012; Johnson & Casey, 2015; Kindt, Soeter, & Vervliet, 2009; Liu et al., 2014; Nader, Schafe, & Le Doux, 2000; Schiller et al., 2010, 2013; Warren et al., 2014). A frequently used technique in human studies is the presentation of a previously conditioned stimulus (CS+rem) 10 min prior to extinction learning without reinforcement, while the other conditioned stimulus (CS+non.rem), also previously paired with the UCS during fear acquisition, is not presented prior to extinction learning (Schiller et al., 2010, 2013). It has been suggested that this single presentation of the CS+rem reactivates the original CS+/UCS memory, which enables a new "CS+/no-UCS" association during extinction learning to be permanently incorporated (Agren, 2014; Schiller et al., 2010). Influential studies have demonstrated successful blocking of the return of fear to the CS+rem as compared to the CS+non.rem during re-extinction when using this procedure (Schiller et al., 2010, 2013). Regarding the underlying neural correlates, a previous study found increased activity in the ventromedial prefrontal cortex (vmPFC) and altered effective connectivity to the CS+non.rem compared to the CS+rem during extinction (Schiller et al., 2013). Regarding re-extinction, increased amygdala responses to the CS+non_rem were found compared to the CS+rem, which has been assumed as an indicator for fear responses (Agren, 2014; Schiller et al., 2013).

However, other studies using similar paradigms could not replicate these promising findings (Golkar, Bellander, Olsson, & Ohman, 2012; Kindt & Soeter, 2013; Soeter & Kindt, 2011). They showed a return of fear to both CS+ and could not find any differences between the CS+rem and the CS+non_rem. In a recent review, Agren (2014) hypothesized that these contrary results might be due to specific subgroups in which the blocking is effective. It was argued that the Val158Met-poly-morphism in Catechol-O-Methyl-Transferase (COMT) is of special interest, because recent studies have been able to show an association of the COMT Val158Met-polymorphism with fear acquisition and extinction learning (Agren, Furmark et al., 2012; Lonsdorf et al., 2009; Wendt et al., 2014). For instance, Lonsdorf and colleagues showed deficits in extinction learning as well as a poorer treatment outcome in extinction-based therapy in Met/Met individuals (Lonsdorf et al., 2009, 2010, 2011). However, no study has investigated the association between the COMT Val158Met-polymorphism and delayed extinction recall or return of fear.

Based on the above-mentioned findings, the present study aimed to investigate the following: First, we investigated po-

tential SCR differences between CS+rem and CS+non-rem durm

extinction learning and re-extinction. Second, we explored the underlying neural correlates of processing the CS+rem compared with the CS+non_rem. As a supplement, we also explored the potential association between the COMT Val158-Met-polymorphism, fear acquisition, extinction learning, and the return of fear. SCRs and neural activity (blood oxygen level-dependent - BOLD signal change) were measured during all phases (fear acquisition, extinction learning, and reextinction). We hypothesized augmented SCRs to the CS+non_rem compared to the CS+rem during re-extinction. Regarding the neural correlates, increased vmPFC activity was expected in the contrast CS+non.rem versus CS+rem during extinction learning and increased amygdala responses to the

non-rem

compared to the CS+rem during re-extinction.

Material and methods

2.1. Participants

One hundred and thirty six Caucasian subjects (75 females; MAge = 23.93; SDAge = 4.15) participated in this study. All were native German speakers, right-handed, and had normal or corrected-to-normal vision. Subjects reporting current or past mental illnesses, chronic diseases, or a consumption of psy-chotropic drugs were excluded. All subjects gave written informed consent and received 50 V for their participation. We restricted our sample to individuals showing reliable SCRs (>.02 ms to one UCS-presentation) and increased SCRs to the CS+ as compared to the CS- during fear acquisition (mean differential SCRs >.02 ms). This criterion reduced the sample size to 70 (23 Met/Met, 28 Met/Val, 19 Val/Val) participants. The exclusion ratio is comparable to that of other studies (Agren, Engman et al., 2012; Agren, Furmark et al., 2012; Schiller et al., 2013). As a supplement, we also conducted an additional analysis on the entire sample (without any SCR exclusion criteria). The additional analysis yielded comparable results.

Eight subjects could not be recruited again for the fourth day after six months, leading to a sample size of 62 subjects for this day (19 Met/Met, 26 Met/Val, 17 Val/Val). There was no significant deviation from Hardy-Weinberg equilibrium [c2(i) < .1.60; p > .2]. Subjects were also interviewed using a self-developed standardized interview for stressful life events and for psychiatric disorders in the previous months on the first experimental and on the last experimental day.

Several power analyses were conducted to estimate the sample size necessary to adequately address the comparison between CS+rem and CS+,

non-rem

. A conservative threshold of

95% sensitivity (1-beta = .95) and a significance level of a = .05 were set for the power analyses based on the results by Schiller et al. (2010). The required sample size was below 23. The study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional ethics committee.

2.2. Fear acquisition and extinction paradigm

The fear acquisition and extinction protocol consisted of four subsequent phases (day 1: fear acquisition; day 2: extinction

learning; day 3: 24 h re-extinction; day 4: 6-month reextinction). SCRs and BOLD-responses were measured during all phases.

On the first day (fear acquisition), 16 trials of each CS+ (CS+rem; CS+non.rem; blue and yellow squares) or 16 trials of the CS- were presented. Each trial started with the presentation of a fixation cross with a variable duration (0-2.5 sec) to achieve a stimulus-onset-asynchrony. After that, a CS (CS+rem; CS+non.rem; CS-) was presented for 8 sec. The UCS (duration: 100 msec) was delivered 7.9 sec after the CS+ onset and co-terminated with the CS+ offset, while the CS- was never associated with the UCS. In contrast to a 38% partial reinforcement rate, which was used by Schiller et al. (2010), we used a 50% reinforcement rate (cf. Golkar et al., 2012). A fixation cross was presented during the inter-trial-interval (ITI). The duration of the ITI depended on the stimulus-onset-asynchrony (5.5-8 sec), because the length of a trial was always 16 sec. A black computer screen was presented at any other time of the experiment. The instructions were to pay attention to the computer screen and try to figure out the relationship between the CS and the UCS.

On the second day (extinction learning), the CS+rem and the CS- were presented once in a counterbalanced order without reinforcement 10 min before the extinction learning started, while the other CS+ (CS+non_rem) was not reminded (Schiller et al., 2010). Subjects had to watch a movie about landscapes ("Colours of Earth - Faszination Natur 2") during the 10-min break. Extinction learning consisted of 11 presentations of the CS+non_rem and 10 presentations of the CS+rem and the CS- to ensure an equal number of presentations during extinction learning, because the CS+non.rem had not been reminded before.

On day three (24 h after extinction learning) and day four (6-months after extinction learning), a reinstatement procedure was conducted inside the scanner to reactivate the fear memory before the re-extinction session started. Accordingly, the time that elapsed between the last extinction learning trial and the first reinstatement trial was around 24 h for day three and around 6-months for day four. During reinstatement, five unsignalled UCS (US to US interval: 13.5-18.5 sec) were applied, while a black computer screen was presented. Around 2 min after the last UCS application of the reinstatement, the re-extinction sessions (10 trials of each CS) started without reinforcement. A pseudorandomized stimulus order was used on each experimental day, which ensured the presentation of all CS with equal frequency in the first and the second half, with no more than two presentations in succession and an equal frequency of all CS for the first trial. In addition, the UCS was presented equally often in the first and in the second half of day one.

The UCS intensity was set individually using a gradually increasing procedure to achieve an "unpleasant but not painful" level of sensation. No UCS-recalibration was made on day 2,3, and 4, to ensure the same number of UCS applications for each subject. A custom-made impulse-generator (833 Hz; 5 mA) provided transcutaneous electrical stimulation (UCS) through two Ag/AgCl electrodes (1 mm2 surface), which was triggered via an optic fiber cable. Electrodes were fixed to the middle of the left shin.

2.3. Skin conductance measuring

SCRs were sampled using Ag/AgCl electrodes filled with isotonic (.05 M NaCl) electrolyte medium placed at the nondominant left hand. An SCR was defined as the highest phasic response following stimulus onset. Therefore, the largest difference between a minimum, which had to occur within 1-8 sec after onset of the CS, and the subsequent maximum was extracted using Ledalab 3.4.4 (Benedek & Kaernbach, 2010). SCRs were ln (mS + 1), corrected for violation of normal distribution of the data. Outlier responses (SCRs ± 3 standard deviations) were excluded.

Mean SCRs to CS+ and CS- were analyzed via analysis of variance (ANOVA) with the factors CS-type (CS+rem, CS+non. rem, CS-) x number of trials (e.g., 16 in fear acquisition), followed by Bonferroni-corrected post-hoc tests, using SPSS 22 (SPSS Inc. Chicago, Illinois, USA). Furthermore, the fear recovery indexes (SCR of first re-extinction trial minus SCR of last extinction learning trial) of the CS+rem with the CS+non. rem were compared to analyze potential differences between both CS+. In addition, we also investigated the first trial separately (SCR of the first trial of the CS+rem minus SCR of the first trial of the CS+non.rem), because Schiller et al. (2013) found the greatest differences between both CS+ in this trial. Finally, in line with other studies (Golkar et al., 2012; Schiller et al., 2010, 2013), we also conducted these analyses with the first interval response only (1-4 sec after CS onset) but results were comparable.

2.4. Magnetic resonance imaging

Subjects were scanned using a 1.5 T whole-body tomograph (Siemens Symphony with a quantum gradient system) with a standard head coil. 160 T1-weighted images (MPRage, 1 mm slice thickness) were acquired in sagittal orientation. Functional imaging consisted of 420 images in a T2*-weighted echo-planar imaging (EPI) sequence. For functional images, a T2*-weighted gradient EPI sequence was used with 25 slices covering the whole brain (slice thickness = 5 mm; 1 mm gap; descending slice procedure; TR = 2.5 sec; TE = 55 msec; flip angle = 90°; field of view = 192 x 192 mm; matrix size = 64 x 64). The orientation of the axial slices was paralleled to the orbitofrontal cortex-bone transition in order to minimize susceptibility artefacts in prefrontal areas. Prior to all statistical analyses, data were preprocessed as described previously (Klucken, Kruse et al., 2015).

The functional data were analyzed using a random-effects general linear model. For each day, separate first level models were calculated. In line with Schiller et al. (2013), separate boxcar regressors (CS duration) for each CS were calculated for each model of extinction learning and both re-extinction sessions (24 h and 6-months after extinction learning) and were divided into an early (first half) and a late (second half) phase, because Schiller et al. (2013) showed the greatest differences in the early, but not the late phase during extinction learning and re-extinction. We also conducted an alternative model using a stick function, but results were comparable.

On the first day, regressors for the UCS and the non-UCS (time window corresponding to the UCS after the CS-) were also introduced in the model (maximum dependency between

CS+ and UCS < .20). The six movement parameters estimated in the preprocessing realignment step were entered into all first level models. The voxel-based time series was filtered with a high pass filter (time constant = 128 sec).

On the group level, a 3 (CS+rem, CS+non.rem, CS-) x 2 (early phase, late phase) ANOVA was computed in SPM8 to explore main effects of CS-type, phase, and CS-type by phase interaction effects. The threshold for whole-brain analyses (for extinction learning and re-extinction) was set to p < .001 and k > 10 to detect potential differences with a liberal criterion. We focused our region of interest (ROI) analyses on the amygdala and the vmPFC, because (1) both of these structures are crucially involved in fear acquisition, extinction learning, and re-extinction; and (2) previous studies found significant differences between CS+rem and CS+

non-rem

in these two

structures only (Agren, Engman et al., 2012; Schiller et al., 2013). ROI analyses were performed with a threshold of p < .05 (family-wise-error; FWE-corrected) and k > 5. The amygdala mask was taken from the "Harvard Oxford cortical and subcortical structural atlases" provided by the Harvard Center for Morphometric Analysis. The vmPFC mask was created with MARINA (Walter et al., 2003) and has been used in previous studies (e.g., Hermann, Keck, & Stark, 2014; Klucken, Schweckendiek et al., 2015; Klucken, Schweckendiek, Merz, Vaitl, & Stark, 2013).

An additional analysis was conducted exploring differences between the CS+rem and the CS+non.rem within the first trial during re-extinction. This procedure was chosen because the comparison of the first trial of the CS+rem and the CS+non_ rem is of special interest, due to previous findings that showed differences between both CS+ in the first trial (parallel to the SCR-analyses of re-extinction; cf. Schiller et al., 2013). Therefore, we specified a new first level model for each subject including all trials as separate regressors (i.e., CS+rem_trial_1,

CS+rem_trial_2,..., CS+non-rem_trial_1, CS+non-rem_trial_2,...).

This results in 3 x 10 conditions (10 trials for the CS+rem, CS+non_rem, and for the CS-). Next, contrasts were created for each comparison of interest (e.g., CS+rem_trial_1 - CS+non_ rem_trial_1) and analyzed on the second level with the same whole-brain and ROI-analyses parameters as described above.

Results

Fear acquisition (day 1)

We will only briefly describe the fear acquisition results to ensure that CS+rem and CS+non.rem did not differ, because the primary aim of the present study was to explore extinction learning and re-extinction. In addition, we did not expect significant differences, because an inclusion criterion for this study was successful conditioning on the first day.

3.1.1. SCRs

The ANOVA revealed a main effect of CS-type [F(2, 68) = 55.34; p < .001], trial [Fp, 69) = 31.88; p < .001], as well as a CS-type x trial [F(2, 68) = 13.29; p < .001] interaction effect. The post-hoc tests showed increased SCRs to the CS+rem and to the CS+non.rem as compared to the CS- (Fig. 1), but no significant differences between the two CS+.

Fig. 1 - Mean skin conductance responses (ln (1 + ms)) for the CS+rem, CS+non-rem, and the CS— for each trial for the first day (fear conditioning) and the second day (extinction learning). *indicates significantly (p < .05) increased responses as compared to the CS— during extinction learning.

3.1.2. Hemodynamic responses

We found a main effect of CS-type, showing increased amygdala responses to the CS+rem (x/y/z = 30/-4/-26; z = 4.07; p = .002) and to the CS+non.rem (x/y/z = -30/-4/-26; z = 3.74; p = .006) as compared to the CS-. We did not find increased left (z = 2.15; pFWE-corr = .24) or right (z = 2.34; pfwe-corr = .18) amygdala activations in the contrast

CS+rem CS+non-rem

or vice versa (pFWE-corr > .50).

3.2. Extinction learning (day 2)

3.2.1. SCRs

The main research question was whether differences between the CS+rem and the CS+:

non-rem

would occur during extinction learning and during re-extinction. The ANOVA revealed a main effect of CS-type [F(2, 68) = 10.14; p < .001] and trial [F(9, 61) = 42.32; p < .001], showing increased SCRs to the CS+rem and to the CS+non.rem as compared to the CS-, but no differences between both CS+ (p > .20). In addition, we focused on the first trial of the CS+rem and the CS+non_rem, because Schiller et al.

(2013) found the greatest differences for the first trial. We found increased SCRs to both CS+ as compared to the CS- (all p < .05), but no differences between the CS+rem and the CS+non_rem (p = .969; effect size: r = .002; Fig. 1).

3.2.2. Hemodynamic responses

Because the aim of the study was to explore potential differences between the CS+rem and the CS+non_rem, we conducted whole-brain analyses as well as ROI-analyses and computed

the COntrasts CS+rem — CS+non-rem and CS+non-rem — CS+rem.

In addition, because previous studies also found differences in the early half of extinction learning (Schiller et al., 2013), we also analyzed BOLD-responses during the early phase of extinction learning.

Regarding ROI-analyses, we did not find significant BOLD-responses during the whole extinction phase for the contrast CS+rem - CS+non.rem in the amygdala ("peak voxel": x/y/z = 27/2/-26; z = 1.56; pFWE-corr = .558) or in the vmPFC (x/y/ z = -12/62/-2; z = 1.34; pFWE-corr = .909). In addition, no significant activation was found for the opposite contrast (CS+non-rem - CS+rem) in the amygdala (x/y/z = -12/-7/-17; z = 1.45; pFWE-corr = .585) or in the vmPFC (x/y/z = -9/29/-23;

z = 2.34; pFWE-corr = .449). Moreover, ROI-analyses showed no increased activation during the early phase of extinction learning for the contrast CS+rem - CS+non_rem in the amygdala (x/y/z = 18/-10/-11; z = 1.99; pFWE-corr = .355), the vmPFC (x/y/ z = -9/29/-23; z = 2.34; pFWE-corr = .449), and for the opposite contrast (amygdala: x/y/z = -24/-7/-20; z = 1.96; pFWE-corr = .350); vmPFC (x/y/z = 30/-7/-20; z = 1.19; pFWE-corr = .698). Finally, ROI-analyses did not show significant differences in the contrasts CS+rem - CS- and CS+non.rem - CS-.

Whole-brain results did not show increased activations to the CS+rem compared with the CS+non_rem (or vice versa) during the whole extinction phase (all puncorrected > .001), but increased BOLD-responses in several regions in the contrast CS+non_rem - CS+rem in the early half of extinction learning and increased activations of the CS+rem and the CS+non_rem as compared with the CS- (Table 1).

3.3. Re-extinction (day 3 and day 4)

3.3.1. SCRs

Day 3 (24-h after extinction learning). The ANOVA showed a significant main effect of CS-type [F(2, 68) = 6.35 p < .01] and trial [F(9, 61) = 16.01; p < .001], with increased SCRs in the first trial to both CS+, which diminished over time. We also calculated the recovery index and found increased recovery for the CS+rem and the CS+non_rem (all p < .05). Notably, no significant differences could be found between the CS+rem and the CS+non.rem in the first trial (p = .269; effect size: r = .07) or for the recovery index (p = .46; effect size: r = .04).

Day 4 (6-months after extinction learning). The ANOVA revealed a significant main effect of time [F(9, 53) = 14.03; p < .001] and a CS-type x time trend [F(18,44) = 1.65; p = .084]. In addition, increased SCRs to both CS+ as compared to the CS-could also be found in the first re-extinction trial as well as an increased recovery index to both CS+ (all p < .05). Again, no significant differences were observed between the CS+rem and the CS+non_rem in the first trial (p = .230; effect size: r = .068), or for the recovery index (p = .461; effect size: r = .08). In addition, we also investigated the differences between CS+rem and CS+non.rem including all subjects, but results were comparable (see supplement for details).

Table 1 - Localization and statistics for whole-brain results for day 2 (extinction learning).

Contrast Structure Side k x y z zmax P

Whole phase CS^rem CS^non-rem CS^non-rem CS^rem No significant differences No significant differences

CS+rem CS Occipital cortex L 663 -3 -88 1 6.25 <.001

Supramarginal gyrus L 44 -60 -31 25 3.90 <.001

Insula L 32 -45 8 -7 3.90 <.001

Middle temporal gyrus R 21 45 -67 7 3.60 <.001

Middle temporal gyrus L 12 -48 -64 7 3.46 <.001

Insula R 24 48 5 1 3.45 <.001

CS+non-rem CS Occipital cortex L 719 -3 -91 1 5.85 <.001

Insula R 10 36 20 -8 3.33 <.001

Early phase CS+rem CS+non-rem No significant differences

CS+non-rem CS+rem Temporal gyrus L 22 66 -31 -14 3.96 <.001

Middle frontal gyrus L 11 -42 23 52 3.69 <.001

Orbitofrontal cortex R 13 3 32 -11 3.62 <.001

CS+rem CS Occipital cortex R 390 12 -85 7 4.90 <.001

Insula R 191 33 23 7 4.65 <.001

Insula L 220 -36 14 7 4.32 <.001

Middle temporal gyrus R 38 48 -64 10 4.02 <.001

Supramarginal gyrus L 81 -66 -25 28 4.03 <.001

Limbic lobe L 37 -3 14 22 3.79 <.001

Cerebellum R 12 9 -40 -8 3.67 <.001

CS+non-rem CS Occipital cortex L 350 -3 -94 1 4.48 <.001

Orbitofrontal cortex R 127 33 23 -8 4.38 <.001

Supramarginal gyrus L 79 -54 -28 25 4.06 <.001

Insula L 22 -33 26 4 3.64 <.001

Limbic lobe R 15 12 -25 34 3.59 <.001

Middle temporal gyrus R 10 66 -28 -14 3.58 <.001

Inferior temporal gyrus L 16 -42 -40 -26 3.51 <.001

The threshold was p < .001 (uncorrected; whole-brain results according to SPM8). All coordinates are given in MNI space. L: left hemisphere, R: right hemisphere.

3.3.2. Hemodynamic responses

Day 3 (24-h after extinction learning). Regarding ROI-analyses, no increased activation could be found for the contrast CS+rem - CS+non.rem during the whole phase in the amygdala or in the vmPFC (no suprathreshold clusters). Moreover, no increased ROI-activation was found for the opposite contrast (amygdala: x/y/z = -18/-13/-14; z = 2.73; pFWE-corr = .097; vmPFC: x/y/z = -3/-59/-23; z = 3.12; pFWE-corr = .105). In addition, no significant BOLD-responses could be found to the CS+rem compared to the CS+non_rem during the early phase (amygdala: x/y/z = -21/-7/-11; z = .20; pFWE-corr = .837; vmPFC: x/y/z = 9/29/-14; z = 1.30; pFWE-corr = .943). In addition, we did not find any significantly increased ROI-activations for the opposite contrast (amygdala: x/y/z = -24/-4/-23; z = 2.13; pFWE-corr = .304; vmPFC: x/y/z = 0/62/-14; z = 1.91; pfwe-corr = .755). No significant differences were found in ROI-analyses in the contrasts CS+rem - CS- and CS+non_ rem - CS-. Finally, no significant differences between CS+rem - CS+non.rem (or vice versa) were found in all ROI when only comparing activations in the first trial.

Increased whole-brain results were found for the contrast CS+non_rem - CS+rem in the whole phase and in the first trial, but not for the early half of re-extinction, while the opposite contrast showed only significant (uncorrected) hippocampal activations in the first trial (see Table 2).

Day 4 (6-months after extinction learning). ROI-analyses showed no increased activation for the contrast

CS+rem - CS+non.rem during the whole phase (amygdala: x/y/ z = -30/-1/-20; z = 1.02; pFWE-corr = 745; vmPFC: x/y/z = 6/44/ -1; z = 3.20; pFWE-corr = .081). In addition, we did not find any increased ROI-activation for the opposite contrast (amygdala: x/y/z = -18/-7/-20; z = .97; pFWE-corr = .757; vmPFC: x/y/ z = -12/68/-2; z = 1.25; pFWE-corr = .931). Moreover, no significant BOLD-responses to the CS+rem compared to the CS+non. rem during the early phase could be observed in the amygdala (x/y/z = -15/-4/-14; z = .74; pFWE-corr = .804) or in the vmPFC (x/y/z = -9/44/-8; z = 2.83; pFWE-corr = .197). In addition, we did not find any significantly increased ROI-activations for the opposite contrast (amygdala: x/y/z = 12/-7/-17; z = 2.02; pFWE-corr = .352; vmPFC: x/y/z = 15/29/-17; z = 1.58; pFWE-corr = .874). Regarding the first trial, no significant differences between both CS+ were found in ROI-analyses. Finally, no significant

ROI-activations were found in the contrasts CS+r<

CS- and

CS+non_rem - CS-. Whole-brain analyses showed increased BOLD-responses to the CS+rem as compared to the CS+non_rem only in the precuneus in the whole phase and in the occipital cortex, precuneus, and supramarginal gyrus in the first trial and pronounced activation to the CS+rem as compared to the CS- in the occipital cortex (Table 3; Fig. 2).

3.4. COMT Val158Met-polymorphism

A detailed results section and a description of the genotyping and data analyses are included in the supplement. In short, regarding SCRs, neither main effects nor interactions with the

Table 2 - Localization and statistics for whole-brain results for day 3 (re-extinction).

Contrast Structure Side k x Y z zmax p

Whole phase CS^rem CS^non-rem No significant differences

CS^non-rem CS^rem Postcentral gyrus R 25 48 -19 34 3.76 <.001

Precentral gyrus R 17 12 -28 73 3.72 <.001

Middle temporal gyrus L 12 -60 -7 -26 3.67 <.001

No distinct region R 59 21 2 16 3.90 <.001

CS+rem CS Occipital cortex R 260 12 -82 7 4.56 <.001

CS+non-rem CS Occipital cortex R 782 12 -82 10 5.08 <.001

Precuneus R 132 15 -58 22 4.09 <.001

Inferior temporal gyrus R 89 51 -64 -5 4.00 <.001

Early phase CS+rem CS+non-rem CS+non-rem CS+rem No significant differences No significant differences

CS+rem CS Occipital cortex L 283 -6 -82 7 4.96 <.001

CS+non-rem CS Precuneus L 16 -21 -49 4 3.58 <.001

Middle temporal gyrus R 24 48 -67 16 3.55 <.001

First trial CS+rem CS+non-rem Precentral gyrus R 18 27 -19 70 3.69 <.001

Superior parietal gyrus R 23 27 -58 64 3.65 <.001

CS+non-rem CS+rem Hippocampus L 14 -27 -19 -17 3.81 <.001

The threshold was p < .001 (uncorrected; whole-brain results according to SPM8). All coordinates are given in MNI space. L: left hemisphere, R: right hemisphere.

Table 3 - Localization and statistics for whole-brain results for day 4 (re-extinction).

Contrast Structure Side k x y z zmax p

Whole phase CS+rem CS+non-rem CS+non-rem CS+rem Precuneus L 44 -3 -61 No significant differences 19 3.66 <.001

CS+rem CS Occipital cortex L 81 -12 -85 4 4.33 <.001

Occipital cortex R 75 15 -73 13 4.18 <.001

CS+non-rem - CS- Occipital cortex R 33 3 -85 4 3.72 <.001

Early phase CS+rem CS+non-rem CS+non-rem CS+rem No significant differences No significant differences

CS+rem CS Occipital cortex L 31 -9 -85 7 3.53 <.001

Occipital cortex R 21 21 -73 13 3.66 <.001

CS+non-rem - CS- No significant differences

First trial CS+rem CS+non-rem Occipital cortex R 45 36 -82 22 4.96 <.001

Precuneus R 81 2 -70 31 3.91 <.001

Supramarginal gyrus R 17 39 -40 43 3.76 <.001

CS+non-rem CS+rem No significant differences

The threshold was p < .001 (uncorrected; whole-brain results according to SPM8). All coordinates are given in MNI space. L: left hemisphere, R: right hemisphere.

COMT Val158Met-polymorphism were observed during fear acquisition, extinction learning, and re-extinction. BOLD-responses also showed no significant amygdala and vmPFC differences during fear acquisition, extinction learning, and re-extinction with respect to the COMT Val158Met-polymorphism.

4. Discussion

The present study explored the impact of a single CS+ presentation prior to extinction learning on re-extinction. SCRs and BOLD-responses between the reminded and non-reminded CS+ were compared during all phases. The results revealed similarly increased SCRs to the CS+rem and to the CS+nonrem as compared to the CS- during extinction learning and re-extinction in the present study. Consistent with the lack of CS+rem versus CS+non_rem differences in SCRs, ROI-

analyses also showed no significant differences between the both CS+ during extinction learning and re-extinction. Moreover, no association with the COMT Val158Met-poly-morphism was observed.

The present findings could not replicate previous results (Agren, Engman et al., 2012; Agren, Furmark et al., 2012; Schiller et al., 2013; Schiller et al., 2010), which had suggested that a single presentation of a CS+ prior to extinction learning had a substantial impact on re-extinction. Yet, our results support other studies also showing no differences between the CS+rem and the CS+non_rem during re-extinction (Golkar et al., 2012; Kindt & Soeter, 2013; Soeter & Kindt, 2011). In order to explain these inconsistent findings, different boundary conditions are discussed, which might have influenced the results.

First, it could be assumed that differences in the experimental design such as CS duration, ITI, exact reinstatement procedure, UCS-recalibration, etc. might alter the effects

Fig. 2 — Mean skin conductance responses (In (1 + ms)) for the CS+rem, CS+non-rem, extinction (day 3 and day 4). Recovery index (below) for re-extinction for the CS+rel significantly (p < .05) increased responses (return of fear) as compared to the CS—.

and the CS— for each trial during rem and the CS+non_rem. 'indicates

(Agren, 2014; Golkar et al., 2012). For instance, Schiller et al. (2010) and the present study presented the CS+rem and the CS- prior to extinction learning, while other studies presented the CS+rem before extinction learning only. In a recent review, Haaker, Golkar, Hermans, and Lonsdorf (2014) hypothesized that slight differences in the conditioning design could significantly influence the return of fear. Second, the exclusion ratios in the present study (~50%) as well as the fMRI-study (~73%) by Schiller et al. (2013) were higher compared to other extinction studies (e.g., ~32% in Golkar et al., 2012), which may also impact findings. However, the present results were comparable, if the entire sample was investigated (see Supplement). Nevertheless, independent replications are required to investigate this unexpected exclusion ratio in more detail.

Third, it is also possible that different analysis strategies might have led to the heterogeneous findings. A variety of different approaches exist to analyze the return of fear index. While some studies calculated the recovery index based on the first trial of re-extinction and the last extinction learning trial (e.g., Schiller et al., 2013), other studies compared SCRs of the first trial (or the first half) during re-extinction of each CS+ with each other. To account for this, we conducted several analyses to explore differences between both CS+ during the early half only or at a single trial level, but did not find any significant SCR differences between the CS+rem and the CS+non_rem. However, differences in the analysis methods as well as the comparison of CRs over different days could be problematic, because of habituation effects at the end of day 2 and large arousal effects on day 3 and day 4. In addition, it

should be noted that the used procedure does not allow a distinction of the exact mode of return of fear because reinstatement and spontaneous recovery effects are intermixed (Haaker et al., 2014).

Moreover, it is also possible that differences in the ratios of contingency aware and contingency unaware subjects between the studies are responsible for the contrary findings. Previous studies showed that contingency awareness impacts on SCRs and neural activity, which were measured in the present study (Hamm & Weike, 2005; Klucken, Kagerer et al., 2009; Klucken, Tabbert et al., 2009; Mertens et al., 2015; Weike et al., 2005; Weike, Schupp, & Hamm, 2007). However, previous studies as well as the present study did not measure contingency awareness explicitly to prevent a repeated presentation of the CS, which might also have impacted extinction processes. Instead, we instructed subjects to pay attention to potential CS/UCS contingencies, which is in line with previous studies (Schiller et al., 2010, 2013).

Finally, Agren (2014) hypothesized that the initial effects may occur in specific subgroups only, which differ in certain genotypes. For instance, an association between fear acquisition and extinction with genetic variation in the serotonin transporter gene (serotonergic transporter-linked polymorphic region; 5-HTTLPR, rs25531) and the Val158Met-poly-morphism in the COMT was found, which are both closely related to fear learning, extinction, and updating of previously learned contingencies (Agren, Furmark et al., 2012; Crisan et al., 2009; Hermann et al., 2012; Klucken, Alexander et al., 2013; Klucken, Schweckendiek et al., 2015; Klumpers, Heitland, Oosting, Kenemans, & Baas, 2012;

Lonsdorf et al., 2009, 2011; Wendt, et al., 2014). As a supplement, we reanalyzed our results with respect to the COMT Val158Met-polymorphism, but did not find any significant associations with the COMT Val158Met-polymorphism. However, a previous study also found no relationship between the COMT Val158Met-polymorphism during extinction learning with SCRs, but with conditioned startle amplitude, suggesting that COMT Val158Met-polymorphism might be associated with specific response systems (Lonsdorf et al., 2009). A possible explanation for this selective effect is the assumption that conditioned startle amplitudes and SCRs are based, at least partly, on different neural circuits (Hamm & Weike, 2005; Weike et al., 2005). While conditioned startle amplitude is primarily mediated by amygdala activations (Davis & Whalen, 2001; Hamm & Weike, 2005), conditioned SCRs could be observed without an involvement of the amygdala (Weike et al., 2005; Tabbert, Stark, Kirsch, & Vaitl, 2006). Thus, the potential association of COMT Val158Met-polymorphism on extinction learning and re-extinction might be visible only in predominantly amygdala-dependent response systems.

Regarding (uncorrected significant) whole-brain results, we found differences between the CS+rem and the CS+non_rem during extinction learning and re-extinction. For instance, increased BOLD-activations were found to the CS+non.rem compared to the CS+rem in the orbitofrontal cortex, the middle frontal gyrus during the early phase of extinction learning. During extinction learning, it is necessary to adapt previously learned contingencies to new circumstances. The middle frontal gyrus has been linked to contingency awareness during fear acquisition (Carter, O'Doherty, Seymour, Koch, & Dolan, 2006). It seems possible that the process to change the CS/UCS relationship of the CS+non_rem may require more explicit effort than the adaptation to the CS+rem, which may have led to the observed effects in this structure. In addition, increased activations to both CS+ in contrast to the CS- were also found in prefrontal areas, the limbic lobe, the occipital cortex, and further structures during extinction learning and/ or re-extinction (see tables for detailed results). However, it should be pointed out that these whole-brain analyses are partly unexpected and the threshold for whole-brain analyses was set to a liberal criterion (uncorrected p < .001). Therefore, the results should be treated with caution until an independent replication is available.

In conclusion, we would like to point out that the present results do not argue against the phenomenon of blocking the return of fear in general. Various animal and human studies found successful blocking of the return of fear (Agren, 2014; Johnson & Casey, 2015; Kindt et al., 2009; Liu et al., 2014; Nader et al., 2000; Schiller et al., 2010, 2013) and it is also possible that our results might be due to false-negative probability. Nevertheless, the unexpected (null) results may support the view that the reported effects may exist in specific constellations only and may require specific parameters, which are not entirely clear to date (Agren, 2014; Golkar et al., 2012). For instance, Schiller and colleagues also reported data of subjects without successfully blocked return of fear (Schiller et al., 2013). Therefore, future studies may help to give more insight into the impact and boundaries and the opportunity for blocking the return of fear more efficiently.

Nevertheless, the possibility of preventing the return of fear through disrupting reconsolidation is still fascinating and could someday be implemented in treatments for anxiety disorders (Shiban, Brutting, Pauli, & Muhlberger, 2015).

Financial disclosures

All authors report no biomedical financial interests or other potential conflicts of interest.

Acknowledgments

This study was supported by a research grant from the Deutsche Forschungsgemeinschaft German Research Foundation to the first author (Grant number: Klu 2500/1-1).

Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.cortex.2016.03.015.

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