ORIGINAL INVESTIGATION

No evidence for enhanced extinction memory consolidationthrough noradrenergic reuptake inhibition—delayed memorytest and reinstatement in human fMRI

Tina B. Lonsdorf & Jan Haaker & Tahmine Fadai &Raffael Kalisch

Received: 27 June 2013 /Accepted: 14 October 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractRationale One promising approach in the current ambition tomaximise treatment benefit for anxiety disorders is the phar-macological enhancement of cognitive–behavioural treatmentefficacy, which can be experimentally modelled by pharma-cological enhancement of extinction learning/consolidation.Noradrenaline (NA) is involved inmemory consolidation, andNAergic innervations are found in brain areas implicated infear conditioning and extinction.Objectives Thus, to enhance extinctionmemory consolidationthrough boosted NAergic signalling, we administered 4 mgreboxetine (RBX) immediately after extinction learning(day 2, 24 h after conditioning on day 1) in a randomised,placebo (PLC)-controlled design. At a delayed memory test(day 8), we probed cued and contextual fear and extinctionmemories before and after a reinstatement manipulation.Results After reinstatement, we find significantly enhancedamygdala and posterior hippocampus activation in the RBXgroup, areas implicated in fear memory expression, while thePLC group exhibited enhanced activation in areas associatedwith extinction memory expression (vmPFC, anterior hippo-campus). No group differences were found in skin conduc-tance responses.

Conclusions Thus, our data do not support our hypothesis thatenhancement of NA signalling may facilitate extinction mem-ory consolidation and provide preliminary evidence that thismight rather enhance fear memories on a neural but notphysiological (skin conductance responses) level.

Keywords Cue conditioning . Context conditioning .

Amygdala . Reinstatement . vmPFC

Introduction

While anxiety disorders are treated with cognitive–behaviouraltherapy (CBT) and pharmacotherapy (e.g. Bandelow et al.2007), there is an ongoing ambition to maximise treatmentbenefit due to considerable non-responder and relapse ratesfor both treatment modalities (van Balkom et al. 1997;Fedoroff and Taylor 2001; Westen and Morrison 2001).One promising approach is the pharmacological enhance-ment of CBT efficacy (D-cycloserine: Davis et al. 2006;Hofmann et al. 2006; Ressler 2004; glucocorticoids:de Quervain et al. 2011).

CBT for anxiety disorders is based on exposure, which canbe experimentally modelled using extinction of acquired fear.During Conditioning , pairings of a neutral conditioned stim-ulus (CS) with an aversive event (US) evokes a conditionedresponse (CR) when the initially neutral stimulus becomes aUS predictor. Subsequent repeated CS presentation withoutthe US leads to a gradual CR weakening (extinction) andcreation of a new memory trace (CS-noUS) that inhibits theinitial fear memory (CS-US) (Bouton 2004; for a review, seeMyers and Davis 2007). After successful extinction, the en-during existence of the CS-US association becomes evidentfrom return of fear phenomena (spontaneous recovery,renewal, and reinstatement) (Bouton 2004).

Electronic supplementary material The online version of this article(doi:10.1007/s00213-013-3338-8) contains supplementary material,which is available to authorized users.

T. B. Lonsdorf (*) : J. Haaker : T. Fadai : R. KalischInstitute for Systems Neuroscience, University Medical CenterHamburg–Eppendorf (UKE), Martinistrasse 52,20246 Hamburg, Germanye-mail: t.lonsdorf@uke.de

R. KalischNeuroimaging Center Mainz (NIC), Focus Program TranslationalNeuroscience, Johannes Gutenberg University Medical Center,Langenbeckstr. 1, 55131 Mainz, Germany

PsychopharmacologyDOI 10.1007/s00213-013-3338-8

Whereas fear conditioning and extinction are establishedmodels for the acquisition and the treatment of anxiety, returnof fear models relapse after successful treatment.

CBT represents a learning process accompanied by alteredgene expression, synaptic plasticity (Kandel 1999) and neuralactivity (Linden 2006). To enhance CBT efficacy, pharmaco-logical agents thus should target core pathways involved inthese learning and memory processes.

Noradrenaline (NA) regulates synaptic plasticity un-derlying (emotional) associative memory processes in-cluding consolidation and reconsolidation (McGaughand Roozendaal 2009) and increases neuronal excitabil-ity and thereby lowers the threshold for LTP-dependentmemory formation (Hu et al. 2007).

Critically, animal studies found that brain areas involved infear conditioning, extinction and their recall are denselyNAergic innervated (Mueller and Cahill 2010), including theamygdala, the hippocampus as well as the (infralimbic) me-dial prefrontal cortex (~human ventromedial prefrontal cortex,vmPFC) in animals (LeDoux 2000; Kim and Jung 2006;Milad and Quirk 2012) and humans (Phelps et al. 2004;Kalisch et al. 2006; Sehlmeyer et al. 2009; Milad et al.2007). These findings provide the theoretical basis forNAergic modulation of conditioning and extinction processes(Mueller and Cahill 2010). In fact, NAergic drugs have beensuggested as potential candidates for adjuncts to exposure-based CBT in anxiety disorders (cf. Mueller and Cahill 2010).

It is established that post-training administration ofNAergic antagonists and agonists in animals impairs or en-hances fear memory consolidation, respectively (Ferry et al.1999; LaLumiere et al. 2005; LaLumiere et al. 2003). Inhumans, NA is involved in consolidation (McGaugh andRoozendaal 2009) and reconsolidation processes (Kindtet al. 2009; Soeter and Kindt 2012; Soeter and Kindt 2011a).In contrast, it is less clear if extinction memory(consolidation), as a model for CBT outcome, can be en-hanced analogously. Animal data suggest that administrationof the selective competitive alpha2-adrenergic receptor antag-onist yohimbine prior to extinction enhances extinction learn-ing (Cain et al. 2004; Morris and Bouton 2007) and reducesreturn of fear at a later test (Janak and Corbit 2011). Further,the necessity of noradrenergic signalling was demonstrated ina study where a blockade of noradrenergic ß-receptors usingpropranolol prior to extinction impaired extinction memoryretrieval on the next day (Mueller et al. 2008). In addition, alsopost-extinction intra-amygdala NA infusions dose dependent-ly enhanced extinction memory (Berlau and McGaugh 2006),while post-training blockade of ß-receptors did not impairextinction (Mueller et al. 2008). In humans, data on the roleof NA in extinction and its consolidation are sparse andcontradictory. In (sub-clinical) claustrophobic patients, phar-macological enhancement of NA transmission via yohimbineduring exposure facilitated short- and long-term fear reduction

(Powers et al. 2009). The same method, however, failed to beeffective in a larger virtual reality treatment study in patientswith fear of flying (Meyerbroeker et al. 2012). Despite ofsuggestive animal work, no human study to date has investi-gated NAergic effects on extinction memory (consolidation)and return of fear.

We therefore enhanced NA signalling during extinctionmemory consolidation using the selective NA reuptake inhib-itor reboxetine (RBX), which enhances NA-levels two tofivefold in the frontal cortex and the hippocampus in animals(Hajós et al. 2004). We employed a multiple-day paradigmconsisting of conditioning (day 1), delayed extinction (day 2)and memory tests before (test 1) and after (test 2) reinstate-ment (day 8).

We hypothesised that administration of RBX during ex-tinction memory consolidation (1) strengthens consolidationof the inhibitory memory trace leading to a dominance ofextinction over fear memory recall at test 1 and (2) a protec-tion from reinstatement of fear at test 2.

Materials and methods

Overview over design

We used a 3-day paradigm (see Fig. 1a) for studyingcontext conditioning (sustained fear) and cue condition-ing (phasic fear) within the same experiment and subject(Grillon et al. 2004a). A similar paradigm has previouslybeen used in our laboratory to disentangle the neuralunderpinnings of cued and contextual fear conditioning(Marschner et al. 2008) as well as the behavioural cor-relates (Haaker et al. 2013) and neural underpinnings ofdelayed extinction recall and reinstatement (LonsdorfT.B., Haaker, J. & Kalisch R., under review). We referto the detailed description below and give a brief over-view in the following.

Three pictures of rooms served as context CSs (“CXT”;duration, 45 s) during each of which one of three discretesymbols (cue CSs, “Cue”; duration, 5 s) were intermittentlyshown twice (see Fig. 1b–d and below for details). Threeconditions (unpredictable, predictable and safe) were realisedthrough different predictability of the electrotactile US whilemaintaining themean number of USs (2) per condition (exceptin the safe). In the unpredictable condition, the cue (UCue) didnot signal the US, making the context (UCXT) the best US-predictor (context conditioning). Cue conditioning in turnshould occur in the predictable condition where the cue(PCue) always co-terminated with the US. In a safe condition,providing control stimuli SCue and SCXT, no US was given.

Conditioning and extinction took place on two consecutivedays (days 1 and 2) in the psychophysiological laboratory andday 8 within the functional MRI (fMRI) environment.

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Participants

Forty-seven male, right-handed participants [age range,25–43 years; mean of 28.6 (SEM, 0.6)] took part in the study,whereof 21 were randomly assigned to the PLC condition and26 to the RBX condition. The study was approved by the localethics committee [Ärztekammer Hamburg (General MedicalCouncil Hamburg)]. Experiments were performed in accor-dance with the ethical standards of the Declaration of

Helsinki. All participants provided written informed consentand received 160 Euro for participation. See also supplemen-tary material for more information.

Five participants had to be excluded due to their wish todiscontinue after day 1, a positive drug test or inappropriatebehaviour in the scanner. This left 42 participants for anal-yses (19 PLC: 25–41 years, mean 29.2±0.9; 23 RBX:25–43 years, mean 28±0.9). Both groups did not differ inage, F (1,40)=1.15, p =0.29.

Fig. 1 Design. Experimental timeline (a) and structure of trials in theunpredictable (b), predictable (c) as well as safe (d) condition. One trialconsisted a context CSs with two superimposed cue CSs occurring duringpre-defined time windows. In between trials a black screen with a white

fixation cross was presented (6–8 s, mean of 7 s) as the inter-trial interval(ITI). Shown is an example of stimulus-condition assignments. Boltdenotes US

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Trial structure

Stimuli were presented in trials that corresponded to thecontinuous presentation of one of the CXTs and lasted for45 s. During a trial, the corresponding cue was presented twicefor 5 s during fixed time windows (with an onset at 13–15 sand 31–35 s after trial onset; Fig. 1b–d). ITI duration was6–8 s with a mean of 7 s. Assignments of the contextual andcue CS to three pairs corresponding to the unpredictable(UCXT, UCue), the predictable (PCXT, PCue) and the safecondition (SCXT, SCue) were consistent across the experi-ment and across days, but varied between participants. Whilestimulus combinations in the unpredictable and predictableconditions were counter-balanced across participants, thestimuli in the safe condition were always the ones shown inFig. 1d. On each day, trials were presented in either of fourpseudo-randomised orders, whereof two started with a pre-dictable and two with an unpredictable condition (balancedacross participants). There were nomore than two consecutivetrials belonging to the same condition. Trials were alwaysgrouped in blocks of nine (three of each condition) that wereseparated by subjective fear ratings (see below).

Day 1

Up to four participants were recorded simultaneously andparticipants were seated in shielded compartments and couldnot see each other during the experiment.

On day 1, recording and stimulation electrodes were at-tached, and US intensity was adjusted to a level of maximumtolerable pain [range, 2.7–32 mA; mean of 8.9 mA (SEM,1.1)]. While calibration was being conducted for one partici-pant, the other participants listened to loud music via head-phones. Participants were asked to rate the painfulness of theUS between 0 (“I feel nothing”) and 10 (“maximally unpleas-ant”) [final rating: range, 3–9; mean of 7.3 (SEM, 0.2)].Subsequently, participants underwent cued and contextualfear conditioning as described above. There were no differ-ences between groups with respect to US intensity and pain-fulness ratings.

During a habituation phase, each of the three trial types waspresented in a shortened exemplary version (CXT presentationfor a total of 7.5 s with cue onset 2.5 s after trial onset) withoutany US. Participants were also familiarised with the fear ratingscales and the use of the keypad. Conditioning consisted of 27trials in 3 blocks (total of 9 trials per condition). In the unpre-dictable condition, one, two or three USs per trial (with amean of2) were randomly administered in fixed time windows (withonsets between 5–7 s, 24–26 s and 39–41 s after trial onset).To avoid that the UCue acquires safety signal properties, twoUSs in total were applied during UCue presentations (1 s aftercue onset, never during the first 2 twoUCue presentations) and toavoid that the onset of theUCXTacquires safety signal properties

a US occurred 2 s after UCXT onset once (never in the firstblock). These three “special cases” (two UCue and one UCXTonset) were omitted from skin conductance response (SCR)scoring. In the predictable condition, the PCuewas always pairedwith a US occurring 4.8 s after cue onset (100 % PCue rein-forcement). Thus, in both conditions, the same total number ofUSs was administered. In the safe condition, no US ever oc-curred. Participants were not informed about the conditioningcontingencies or the learning element beforehand.

On day 1, recording and stimulation electrodes were at-tached and US intensity was adjusted to a level of maximumtolerable pain [range, 2.7–32 mA; mean of 8.9 mA (SEM,1.1)]. There were no differences between groups with respectto US intensity, F(1,40)=1.03, p =0.32 (PLC, 10.1±2 mA;RBX, 7.9±1.2 mA). Participants were asked to rate the pain-fulness of the US between 0 (“I feel nothing”) and 10(“maximally unpleasant”) [final rating: range, 3–9; mean of7.3 (SEM, 0.2)]. Final ratings did not differ between groups,F(1,38)=1.52, p =0.23 (PLC, 7.2±0.3; RBX, 7.4±0.2).

At the end of the experiment, CS-US contingency aware-ness was assessed using a semi-structured interview(BECHARA et al. 1995), based on which participants wereclassified as aware and unaware. In both drug groups, threeparticipants were classified as unaware, while all others wereclassified as aware or partly aware (i.e. being able to correctlyidentify major parts of the conditioning contingencies in thedifferent experimental conditions) of the conditioningcontingencies.

Participants were not instructed about conditioning contin-gencies or their changes on any day.

Day 2

Approximately 24 h after conditioning, participants returnedfor an extinction session (day 2). Stimulation and recordingelectrodes were attached as the day before, without renewedUS intensity calibration. Again, up to four participants wererecorded simultaneously and participants were seated inshielded compartments and could not see each other duringthe experiment.

During the experiment, 18 trials were presented in 2 blocks(total of 6 trials per condition). No US was administered.Participants were not informed beforehand about any changein CS-US contingencies as compared to the previous day.

To target extinction memory consolidation, participantsingested either a pill containing 4 mg (Onur et al. 2009)RBX [Edronax®, tmax 2h, t1/2 approximately 13 h (Dostertet al. 1997)] or a placebo pill (pure capsule filling material:mannitol with Aerosil®) in a double-blind manner immedi-ately after extinction. Beforehand, a third person had random-ly assigned subjects to the groups receiving RBX or placebo.This person never obtained any experimental data nor had anycontact with the participants. Participants were in a fasting

Psychopharmacology

condition for at least 2.5 h prior to drug administration andremained at the laboratory for at least 1 h after drug intake,while blood pressure and pulse frequency was intermittedlymonitored. In addition, participants were interviewed for pos-sible adverse side effects. No serious adverse events werereported. In the PLC group, six participants reported adverseside effects, while four participants in the RBX group reportedside effects.

Day 8

One week after conditioning (day 8; 6–8 days after day 1),participants returned for memory recall tests conducted indi-vidually inside the MR scanner. Stimulation and recordingelectrodes were attached, and no additional US calibrationwas performed. A recall test (test 1) consisted of 18 unrein-forced trials in 2 blocks (total of 6 trials per condition) and wasfollowed by the presentation of a grey screen. Five secondsafter onset of the grey screen, three unsignaled reinstatement-USs were administered (interval 5 s). Two minutes after thelast US, a reinstatement test (test 2, corresponding to test 1)was conducted. Day 8 took place individually inside the MRscanner. Each test phase (tests 1 and 2) was identical to theextinction session (as on day 2) with respect to the number oftrials. The separation of test 1 (to capture recall) and test 2(to capture reinstatement effects) is important to unequivocal-ly separate reinstatement processes from possible confound-ing effects (e.g. recall effects, spontaneous recovery, newcontextual environment). Further, while behavioural data of-ten quantify reinstatement using one or two single trials, this isnot sufficient in fMRI, where larger numbers of repetitions areneeded to acquire sufficient signal/noise ratios.

Skin conductance responses

Skin conductance was measured via self-adhesive Ag/AgClelectrodes placed on the palmar side of the left hand on thedistal and proximal hypothenar. Skin conductance response(SCR) amplitudes (in μS) were scored as the largest responseoccurring 0.9–4.0 s post-stimulus onset. For the CXTs, onlythe onset of a given trial was scored (Marschner et al. 2008), assubsequent CXT phases are confounded US reactions. SCRswere averaged over blocks of three (CXTs) or six (Cues) trials,resulting in three blocks on day 1, two blocks on day 2 andtwo blocks per phase on day 8 (as in Fig. 2).

On days 1 and 2, data were recorded with a BIOPACMP-100 amplifier (BIOPAC Systems Inc, Goleta, CA,USA) with AcqKnowledge 4 software. On day 8 insidethe scanner, a CED2502-SA skin conductance unit withSpike 2 software (Cambridge Electronic Design,Cambridge, UK) was used. Note that this precludes com-parison of days 1 and 2 with day 8 data.

Data were down-sampled to 10 Hz, and phasic skin con-ductance responses (SCRs) to the onsets of context or cue CSswere manually scored offline using a custom-made computerprogram.

Separately for the three experimental days, logarithms werecomputed for all values, to normalise the distribution(Venables and Christie 1980), and these log values wererange-corrected (SCR/SCRmax CR[day]) to account for inter-individual variability (Lykken and Venables 1971). SCR mea-surements that showed recording artefacts or excessive base-line activity were discarded and treated as missing data.

Due to technical difficulties, SCR data from a limitednumber of participants had insufficient data quality and werethus excluded (day-wise) from the analyses [RBX:N(day 1)=3,N(day 2)=1, N(day 8)=7; PLC: N(day 2)=2, N(day 8)=7].Note that high numbers of excluded subjects on day 8 are due tothe technical challenges posed by the combined acquisition ofpsychophysiological and fMRI data.

Subjective fear ratings

At the beginning of each experimental phase and after everytrial block, participants were asked to rate each CS withrespect to the fear/stress/tension that was elicited when theylast saw it. Thus, the first rating on day 2 (prior to extinctionlearning) refers to the last presentation during day 1 and soforth. Ratings were performed on a computerised visual ana-log scale [0 (none)–100 (maximal)].

Ratings were performed using the keyboard (days 1 and 2) ora button-response box (day 8) with the right hand. Selected ratingvalues had to be confirmed by a key press and were otherwisetreated as missing data. Participants were excluded from theanalyses (day-wise) if less than one third of all data points werevalid [not missing; PLC: N(day 1)=2,N(day 2)=2, N(day 3)=2; RBX: none]. Baseline ratings prior to the first experimentalphase (conditioning, day 1) were not included in the analyses.

Data analysis (behavioural data)

Behavioural data were analysed separately for the three experi-mental days as well as the two phases on day 8 (tests 1 and 2),using SPSS 18 for Windows. For fear ratings, mixed-modelANOVAs with stimulus (3) as the within-subject variable wereapplied, while for SCR's stimulus (3)×time (block)mixed-modelANOVAs for days 1 and 2were calculated. Drug groupwas usedas between-subject variable in all analyses. In contrast to fearratings where only few data points throughout the experimentalsessions exist, the factor block was included for SCR's analysesto provide a more fine-grained picture of the learning curves. Forday 8, the ANOVAs testingmemory expression before (test 1) aswell as after (test 2) reinstatement were restricted to stimuluseffects (3) in the first blocks of each test, to account for onlineextinction. A potential enhanced fear memory expression

Psychopharmacology

after versus before reinstatement (test 2> test 1) wasassessed using stimulus (3) × time (2) mixed-modelANOVAs on the last block before and the first block afterreinstatement. Again, drug group was used as betweensubject variables in all analyses.

ANOVAs were followed by contrasts of interest defined apriori. To assess effects of conditioning, we calculated con-trasts showing whether participants successfully discriminatedbetween truly US-predictive stimuli (UCXT, PCue) and thecorresponding non-predictive stimuli (here in particularSCXT and SCue), that is, UCXT>SCXT (for context condi-tioning) and PCue>SCue (for cue conditioning). Note alsothat comparing only stimuli of the same kind (cues with cuesand contexts with contexts) avoids problems related to un-equal number of repetitions for cues and contexts (cues beingpresented twice as frequently as contexts) as well as to differ-ent scaling of event- and block-type regressors (cue andcontext responses) in the imaging data analysis describedbelow (which actually prohibits cue-to-context comparisons).An α -level of p <0.05 was considered significant, andGreenhouse–Geisser correction was applied if necessary.

fMRI data analysis (day 8)

Data were obtained with a 3-T MR scanner (MAGNETOMtrio, Siemens Germany) using a 32-channel head coil (seeSupplementary methods and materials for acquisition details).

Pre-processing [SPM8 (www.fil.ion.ucl.ac.uk/spm) onMatlabR2009b (The MathWorks, Natick, MA, USA)] involvedrealignment, unwarping and normalisation to a sample-specifictemplate, using DARTEL (Ashburner 2007).

Based on the idea of sustained versus phasic fear responses(Grillon et al. 2004a; Davis et al. 2010), a general linear model(for details, see Friston et al. 2006) with a total of 28 regressors

was set up for statistical first-level (single-subject) analysis:one regressor per CXT type (UCXT, PCXT, and SCXT),which modelled the entire trial/CXT presentation of 45 s asa continuous block using a “box car” function, one regressorper cue type (UCue, PCue and SCue), which modelled eachcue onset as an event using a “stick” or delta function. Inaddition, these categorical regressors were parametricallymodulated with a linear decreasing function to capture re-sponse decreases over trials. Both the categorical and theparametric regressors were built separately for tests 1 and 2.Additional nuisance regressors were included to factor outexperimental effects of no interest: Event-related regres-sors modelled the onset of each ITI, each rating blockas well as each reinstatement-US; a block-type regressormodelled the 2-min rest period after the reinstatement-USs. All regressors were convolved with a canonicalhemodynamic response function. Due to the differentscaling of block- and event-type regressors, second-level random-effects analyses of group effects were per-formed separately for CXTs and cues and for tests 1and 2. These analyses used SPM's “full factorial” modeland focused on group comparisons with respect to thecontrasts of interest for context CSs (UCXT>SCXT)and cue CSs (PCue>SCue).

Correction for multiple comparison at an α -level ofp <0.05 was restricted to pre-defined regions of interest(ROIs) and used small volume correction (SVC) based onGaussian random field theory [family-wise error rate method(Friston et al. 2006)].

We expected RBX to enhance extinction memory consol-idation. This wouldmanifest as enhanced activation in regionsassociated with extinction recall at test and/or reduced activa-tion in regions associated with fear memory as compared tothe PLC group. Thus, ROI were defined for regions

Fig. 2 Behavioral data. Fearratings to cue and contextual CSs(a , c RBX; e , g PLC) and SCRsto cue and contextual CSs(b , d RBX; f , h PLC) duringconditioning (day 1), extinction(day 2) as well as tests 1 and 2(day 8). PCue , UCue , SCue cueCSs in the predictable,unpredictable and safe conditions,respectively. PCXT, UCXT,SCXT context CSs in thepredictable, unpredictable andsafe conditions, respesctively.Data show mean±SEM. loglogarithmised, rc range-corrected.Bolt denotes reinstatement-USs

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implicated in extinction recall (vmPFC, the anterior hippo-campus) and fear recall (posterior hippocampus, amygdala,dmPFC; (Sehlmeyer et al. 2009; Mechias et al. 2010; Etkinet al. 2011). ROI centre coordinates were determined byaveraging peak-effect coordinates reported in prior studies offear and extinction expression (see Supplementary Table S1),provided the reported effects had survived appropriate correc-tion for multiple comparisons. Because no such data wereavailable for the amygdala, a probabilistic anatomical maskwas used (http://www.cma.mgh.harvard.edu; threshold, 0.7;Desikan et al. 2006). As in earlier work (Kalisch et al. 2009),subcortical ROIs were spheres of 6 mm radius around thecorresponding unilateral centre coordinate. For cortical ROIs(vmPFC and dmPFC), the x -coordinate was set to 0 and theresulting midline-centred coordinates were used as the centreof a box of dimensions 20×16×16 mm that equally coveredboth hemispheres (see Raczka et al. 2010; Paret et al. 2011).

Results

No significant main or interaction effects involving treatmentgroup were observed on any day in any experimental phase infear ratings or SCRs.

Fear ratings

Irrespective of treatment group, conditioning effects wereindicated by significant main effects for stimulus in fearratings to both cue and context CSs. These were maintainedthroughout extinction and the memory tests before (test 1) andafter reinstatement (test 2) (all p <0.004; see Table 1 forstatistical details and Fig. 2). While during conditioning andextinction for both cue and contextual CSs discrimination wasobserved between all three stimulus types (predictable, unpre-dictable and safe), there was no discrimination between thepredictable and the unpredictable condition at day 8 (tests 1and 2). Both, however, elicited significantly higher ratingsthan the safe condition. The reinstatement test (test 2>test1)yielded no evidence for return of fear (no main or interactioneffects involving the factor time).

Skin conductance responses

Irrespective of treatment group, conditioning effects onday 1 were indicated by a significant main effect of stimu-lus in SCRs to cue and, at trend level, to context CSs(p <0.001 and p =0.07, respectively; see Table 1 for statis-tical details and Fig. 2). While responses in the predictablecondition differed significantly from the safe condition forboth cue and contextual CSs, discrimination between thepredictable and the unpredictable condition was observedonly for cue CSs (PCue>UCue). For contextual CSs,

UCXT differed from SCXT at trend level, while UCXTand PCXT elicited comparable SCRs.

These conditioning effects were, however, not maintainedthroughout extinction and the memory tests before (test 1) andafter reinstatement (test 2), with the exception of a trend-likestimulus effect during test 1 (all other ps<0.17; see Table 1).Unlike in ratings, in SCRs, the reinstatement test (test 2>test1)yielded evidence for (generalised) return of fear to cue andcontextual CSs, as indicated by a main effect of time(see Table 1). There was no interaction effect with stimulus,indicative of generally enhanced SCRs to all cue CSs as aresult of the reinstatement manipulation.

Imaging data (day 8)

Before reinstatement (test 1)

Cue CSs For cue CSs before reinstatement (test 1), the PLCgroup showed significantly higher activation than the RBXgroup in the categorical contrast PCue>SCue within asubgenual cingulate area of the vmPFC ROI (Fig. 3a) as wellas in the left (Fig. 3b) and, at trend level, in the right posteriorhippocampus ROIs (see Table 2 for statistical details). WhilevmPFC and left posterior hippocampus activation were spe-cific for the cue previously predictive of the US as comparedto the control stimulus (PCue>SCue contrast), significantactivation in the right posterior hippocampus was also ob-served when contrasting the stimulus that never in itself pre-dicted the US but was presented in a US-predictive context tothe control stimulus (UCue>SCue contrast) (see Table 2).

In the RBX group (>PLC), a linearly decreasing activationpattern was observed in the right amygdala in the contrastPCue>SCue (Table 2 and Fig. 3c). This stood in contrast tothe categorical right amygdala activation in the PLC group(>RBX) observed at a more lenient exploratory threshold[x ,y,z=20,0,−26; k=21, Z=3.01, p =0.001(uc)], indicating dif-ferent temporal profiles of amygdala activation in the groups.

Context CSs For contextual CSs, significantly higher categor-ical activation was observed in the RBX than in the PLCgroup in the contrast UCXT>SCXT in the right posteriorhippocampus ROI (Table 2 and Fig. 3d).

After reinstatement (test 2)

Cue CSs After reinstatement (Test 2), a linearly decreasingactivation pattern was observed in the PLC group (>RBX) inthe PCue>SCue contrast in the right anterior hippocampusROI (Table 2 and Fig. 4a) as well as, at trend level, in thevmPFC ROI (Table 2). The right anterior hippocampus acti-vation decrease was, however, not specific for the previouslyUS-predictive cue compared to the control stimulus (PCue>SCue contrast), as it was also observed for the contrast

Psychopharmacology

involving the non-reinforced cue that was previously present-ed in the reinforced context (UCue>SCue) (Table 2).Mirroring our behavioural findings (SCRs), this is indicativeof generalised reinstatement effects, as observed by otherspreviously in various dependent measures (Dirikx et al.2004; e.g. Dirikx et al. 2007, 2009; Gazendam and Kindt2012; Kull et al. 2012; Sokol and Lovibond 2012; Kindt andSoeter 2013).

In the RBX group (>PLC), higher categorical activationwas observed within the left amygdala ROI for the contrastPCue>SCue (Fig. 4c). There was also a stronger linear de-crease to PCue than SCue in this group in the posteriorhippocampus (Fig. 4d; see Table 2 for details).

Context CSs The PLC group (>RBX) showed a trend-likemore decreasing activation in the contrast UCXT>SCXT inthe vmPFC ROI (Table 2 and Fig. 4b).

Discussion

The present data do not support the hypothesis that post-treatment administration of drugs targeting the NA system(in particular SNRIs) may be good candidates to pharmaco-logically enhance CBTefficacy via consolidation processes inanxiety disorders. Contrary to our expectations, NA signallingenhancement during extinction memory consolidation did notpreclude or dampen the neural correlates of fear memoryexpression before and after reinstatement. Instead, althoughthere were no treatment group differences at the behaviourallevel, the observed hemodynamic response pattern mightpreliminary point towards a facilitation of return of fear inthe RBX-treated group.

In the framework of fear and extinction, return of fear is thepredominant retrieval of the original excitatory fear memorytrace over the inhibitory extinction memory trace after suc-cessful extinction (Bouton 2004; Vervliet et al. 2013). Inaccordance with a proposed role of the amygdala in CS-USassociation retrieval (Fanselow and Gale 2003; LeDoux2000), previous human studies have implicated the amygdalain returning fear (Kalisch et al. 2006; Agren et al. 2012), whilesubgenual cingulate/vmPFC activation has been observed inextinction recall in humans (e.g. Phelps et al. 2004; Kalischet al. 2006; Milad et al. 2007) and animals (Morgan andLeDoux 1995; Milad and Quirk 2002, 2012).

In our study, compared to the RBX group, the PLC groupshowed higher categorical activation of the subgenual part ofthe vmPFC for cue CSs in concert with categorical posteriorhippocampus activation, an area previously associated withreturning fear in humans (Kalisch et al. 2006, 2009). This maybe interpreted as co-expression of extinction and fear memoryremainders during test 1. Contrary to our hypotheses, we did

Table 1 Behavioral data: statistics

Measure Phase df F p value Eta2 Contrastsa

Cue Ratings C 2,76 48.00 <0.001 0.55 1a

E 2,76 18.77 <0.001 0.33 1b

T1 2,66 8.84 0.001 0.21 2a

T2 2,70 10.06 <0.001 0.22 2b

T2>T1 1,33 <1 0.63

SCR C 2,74 17.10 <0.001 0.32 5

E 2,74 1.23 0.30

T1 2,46 1.84 0.20

T2 2,46 1.39 0.26

T2>T1 1,23 4.99 0.036 0.18 –

Context Ratings C 2,76 73.61 <0.001 0.66 1c

E 2,76 36.55 <0.001 0.49 1d

T1 2,64 14.53 <0.001 0.31 2c

T2 2,72 8.58 0.003 0.19 2d

T2>T1 1,35 <1 0.32

SCR C 2,74 2.93 0.07 0.07 2e

E 2,74 1.82 0.17

T1 2,46 2.84 0.07 0.11 4

T2 2,46 <1 0.83

T2>T1 1,23 7.15 0.014 0.34 –

Main effects of stimulus (predictable, unpredictable, and safe) for bothcue and contextual CSs during conditioning (C , day 1), extinction(E , day 2) and the memory tests on day 8 before (test 1, T1) and afterreinstatement (test 2, T2). Main effects of time are given to index changesfrom T1 to T2 (T2>T1). Interactions are not shown. There were noeffects of treatment group. For analyses of conditioning and extinctioneffects for both CS types, main effects of time were always significant(all ps<0.001) and are not reported in detail here (with the exception ofthe reinstatement test T2>T1). Similarly, stimulus×time interactionswere never significant (all ps>0.09) and are not reported in detail herea (1) All CSs differ significantly from each other: (a) PCue–UCue [F(1,38)=9.85, p=0.003, Eta2 =0.21]; PCue–SCue [F(1,38)=63.77, p<0.001, Eta2 =0.63]; UCue–SCue [F(1,38)=44.39, p<0.001, Eta2 =0.54]. (b) PCue–UCue[F(1,38)=6.91, p =0.012, Eta2 =0.15]; PCue–SCue [F(1,38)=22.95,p <0.001, Eta2 =0.38]; UCue–SCue [F(1,38)=18.98, p <0.001, Eta2 =0.33]. (c) PCXT–UCXT [F(1,38)=4.84, p =0.034, Eta2 =0.11]; PCXT–SCXT [F (1,38)=79.23, p <0.001, Eta2 =0.68]; UCXT–SCXT[F(1,38)=90.04, p <0.001, Eta2 =0.70]. (d) PCXT–UCXT [F(1,38)=4.84, p =0.034, Eta2 =0.11]; PCXT–SCXT [F(1,38)=41.80, p <0.001,Eta2 =0.52]; UCXT–SCXT [F(1,37)=46.84, p <0.001, Eta2 =0.55]. (2)PCue and UCue or PCXT and UCXT do not differ significantly butboth differ from SCue significantly or tendentially: (a) PCue–UCue[F (1,33)<1]; PCue–SCue [F (1,33)=10.37, p =0.003, Eta2 =0.24];UCue–SCue [F (1,33)=12.54, p =0.001, Eta2 =0.28]. (b) PCue–UCue[F (1,35)=2.17, p =0.17]; PCue–SCue [F (1,35)=13.23, p =0.001,Eta2 =0.27]; UCue–SCue [F (1,35)=10.61, p =0.002, Eta2 =0.23].(c) PCXT–UCXT [F (1,32)<1]; PCXT–SCXT [F (1,32)=15.11,p <0.001, Eta2 =0.32]; UCXT–SCXT [F (1,32)=16.37, p <0.001,Eta2 = 0.34]. (d) PCXT–UCXT [F (1,36) < 1]; PCXT–SCXT[F (1,36)=10.27, p =0.003, Eta2 =0.22]; UCXT–SCXT [F (1,36)=8.89, p =0.0015, Eta2 =0.204]. (e) PCXT–UCXT [F (1,37)<1];PCXT–SCXT [F (1,37)=4.82, p =0.034, Eta2 =0.12]; UCXT–SCXT[F (1,37)=3.41, p =0.073, Eta2 =0.08]. (4) PCXT differs significantlyfrom SCXT [F (1,23)=6.23, p =0.02, Eta2 =0.22], UCXT differstendentially from SCXT [F (1,23)=3.78, p =0.06, Eta2 =0.14] andPCXT and UCXT do not differ [F (1,23)<1]. (5) PCue differs signif-icantly from UCue [F (1,37)=24.26, p <0.001, Eta2 =0.40] and SCue[F(1,37)=25.58, p<0.001, Eta2 =0.41] but both do not differ [F(1,23)<1]

Psychopharmacology

not observe enhanced activation in the RBX group in areaspreviously implicated in the recall of extinction memory(e.g. vmPFC, anterior hippocampus).

Together, findings for test 1 do not provide clear results tosupport our initial hypothesis of enhanced extinction memoryrecall following NAergic enhancement during extinctionmemory consolidation.

During test 2, however, categorical amygdala activationwas found to be higher in the RBX (vs. PLC) group to cueCSs. Amygdala involvement in reinstatement has been previ-ously observed in rodents (Laurent and Westbrook 2010; Linet al. 2011) and humans (Lonsdorf, T.B., Haaker, J. &Kalisch,R., under review). Thus, our findings might be cautiouslyinterpreted as reinstated fear memory expression in the RBXgroup (vs. PLC). In support of this preliminary group differ-ences, linearly decreasing activation in regions implicated inextinction memory expression (vmPFC, anterior hippocam-pus) was observed in the PLC group (>RBX). Of note, in thePLC group, vmPFC activation was present both before (test 1)and after reinstatement (test 2).

In sum, these findings do not speak in favour of our initialhypotheses. However, despite of hemodynamic differencesbetween the treatment groups, no group differences in SCRsor fear ratings were found. SCRs, in particular within thefMRI environment, tend to be a very noisy measurementand similarly to our findings; others did not observe an impactof a2- or ß-adrenergic challenge on SCRs and subjective

ratings, while fear potentiated startle (FPS) responses seemedmore sensitive to NAmanipulations (Kindt et al. 2009; Soeterand Kindt 2011b). Thus, future studies should consider the useof FPS. Dissociations between different behavioural measure-ments with respect to NAergic manipulations have previouslybeen discussed in the context of multiple memory systems andemotional (FPS) versus cognitive (SCRs) fear representations(Soeter and Kindt 2012). An alternative explanation may beprovided by Pavlov's “extinction beyond the zero” (Lattal andWood 2013), meaning that, even when behavioural responseshave reached a floor level, changes in molecular and neuronalprocesses proceed. This implies that behavioural tests alonemay not always be sufficient and highlight the value of mo-lecular or imaging studies. In fact, extinction seemed to bequite efficient in reducing psychophysiological responding(SCRs) in our paradigm, whichmay also preclude drug effectson SCRs on day 8. As clinical populations are characterisedby a deficit in extinction learning (Lissek et al. 2005), theresults might turn out differently in patient populations.

In addition, it has to be acknowledged that our paradigmwas not optimised for the quantification of contextual fear inSCRs. That is, SCR onsets are usually measured 0.9–4 s post-stimulus onset (Fowles et al. 1981). Due to US administrationat cue CS offset (in the predictable condition), measurement ofSCRs to the onset of the second and third CXT presentationalone would be confounded with SCRs to the US or itsomission. Further, there was only one US administration

Fig. 3 Brain activation pattern during test 1 (before reinstatement). Forcued CSs (PCue>SCue), higher activation in the PLC as compared to theRBX group was observed in the vmPFC ROI (categorical regressor) (a)as well as in the left posterior hippocampus ROI (categorical) (b). Higheractivation in the RBX as compared to the PLC group was observed in theright amygdala ROI (parametric regressor) (c). Note that linearly decreas-ing contrast parameter estimates in the bar graphs are indicated bynegative values , whereas positive values indicate increasing responses.

For contextual CSs (UXCT>SCXT), higher activation was observed inthe RBX as compared to the PLC group in the right posterior hippocam-pus ROI (categorical) (d). Bar graphs represent average contrast esti-mates for the contrasts PCue>SCue and UCXT>SCXT. Red bordersindicate the location of the exact ROI, whereas white borders serveillustrative purposes only. For illustrative purposes images arethresholded at p<0.01(uc.). *p<0.05 (SVC)

Psychopharmacology

during the first 2 s of any UCXT presentation, and conse-quently, CXT onset might have acquired safety signal proper-ties despite of our attempt to reduce this likelihood by admin-istering one US shortly after UCXT onset. Again, as

mentioned above, FPS would be a good addition to SCRs inour paradigm allowing for more flexibility in time, as probeadministration is in principle possible at any time. Whilemeasurement of FPS in the fMRI environment is not trivial,

Table 2 Imaging results: activa-tion inside the ROIs for thememory tests before (test 1) andafter (test 2) reinstatement forboth cued and contextual fear inthe categorical (cat.) and linearlydecreasing parametric (para.)regressors

r right hemisphere, l left hemi-sphere, uc uncorrected, SVCsmall volume correcteda Trend-wise significance

Contrast Area k(SVC) p(SVC) p(uc) Z(SVC) x y z

Test 1 (before reinstatement)

PLC>RBX

PCue>SCue (cat.) vmPFC 21 0.014 <0.001 3.84 −8 22 −4post. hipp. (l) 9 0.029 0.001 3.13 −36 −36 −10post. hipp. (r) 5 0.078a 0.003 3.72 38 −36 −10

UCue>SCue (cat.) post. hipp. (r) 20 0.006 <0.001 3.76 40 −36 −12RBX>PLC

PCue>SCue (para.) Amygdala (r) 3 0.029 <0.001 3.33 20 −12 −12UCXT>SCXT (cat.) post. hipp. (r) 7 0.037 0.002 2.94 40 −34 −10

Test 2 (after reinstatement)

PLC>RBX

PCue>SCue (para.) ant. hipp. (r) 11 0.021 0.001 3.16 32 −18 −22vmPFC 33 0.094a <0.001 3.11 10 32 −4

UCue>SCue (para.) ant. hipp. (r) 26 0.040 0.002 2.92 30 −20 −20UCXT>SCXT (para.) vmPFC 66 0.074a 0.001 3.17 −6 44 −12

15 0.089a 0.001 3.10 4 48 −20RBX>PLC

PCue>SCue (cat.) Amygdala (l) 12 0.024 0.001 3.27 −28 0 −22PCue>SCue (para.) post. hipp. (l) 5 0.025 <0.001 3.11 −38 −28 −10

Fig. 4 Brain activation pattern during test 2 (after reinstatement). Forcued CSs (PCue>SCue), higher activation in the PLC as compared to theRBX group was observed in the right anterior hippocampus (parametricregressor) (a). Note that linearly decreasing contrast parameter estimatesin the bar graphs are indicated by negative values , whereas positivevalues indicate increasing responses. For contextual CSs (UXCT>SCXT) higher activation in the PLC as compared to the RBX groupwas observed in the vmPFC ROI (parametric regressor) (b). Higher

activation for cued CSs (PCue>SCue) was observed in the RBX ascompared to the PLC group in the left amygdala ROI (categoricalregressor) (c) and the left posterior hippocampus ROI (categorical regres-sor) (d). Bar graphs represent average contrast estimates for the categor-ical and parametric contrasts PCue>SCue and UCXT>SCXT. Red bor-ders indicate the location of the exact ROI, whereas white borders serveillustrative purposes only. For illustrative purposes, images arethresholded at p<0.01(uc.). *p<0.05(SVC)

Psychopharmacology

we have employed a similar behavioural paradigm outside thescanner measuring both SCRs and FPS (Haaker et al. 2013)showing that FPS may in fact better capture context condi-tioning than SCRs in this particular paradigm.

As NA has been shown to modulate associative memorieswith emotional, excitatory content (Krugers et al. 2011) (vanStegeren 2008), it might be possible that inhibitory memorytraces (i.e. extinction memories) may not be equally susceptibleto augmentation by NA in humans, suggesting further explora-tion of this possibility in future studies. Another explanation isthat extinction training might have induced formation of aninhibitory extinction memory trace as well as reactivated theoriginal fear memory trace. Hence, RBX might have hamperedextinction memory expression at day 8 through selective facili-tation of consolidation of the retrieved excitatory fear memory onday 2, whichwould be in accordancewith an involvement of NAsignalling in reconsolidation processes (Kindt et al. 2009; Soeterand Kindt 2012) as well as fear memory consolidation (Soeterand Kindt 2011b) in humans. Both extinction andreconsolidation share common molecular mechanisms (Papeand Pare 2010). Nevertheless, memory labilisation and subse-quent reconsolidation are expected after short CS (re-)exposuretriggers, whereas a long CS exposure should induce extinction.Thus, in our case, the repeated unreinforced CS presentationshould rather have induced extinction. The distinction betweenextinction and reconsolidation has recently been challenged, andit has also been shown that both processes can be triggered inparallel (Lattal and Wood 2013). Therefore, a reconsolidation-based explanation of our results cannot be definitively excludedat present.

Our findings line up with previous human data on NAergicmanipulations of fear memory enhancement, reconsolidation andpharmacological enhancement of CBT. NAergic transmissionenhancement before conditioning through the a2-adrenergic an-tagonist yohimbine strengthened fear memories as indicated bydelayed extinction learning 48 h later and facilitated return of fear(reinstatement and reacquisition; Soeter and Kindt 2011b). Incontrast, no effect of yohimbine on inhibitory memories duringexposure-based CBT was observed (Meyerbroeker et al. 2012;Powers et al. 2009). While NA enhancement facilitated fearmemories, blockade of ß-adrenergic receptors using propranololdisrupted reconsolidation of conditioned fear (Kindt et al. 2009;Sevenster et al. 2013). Clinically, propranolol administration afew hours after a trauma reduced PTSD symptoms severalmonthlater in an early study (Pitman et al. 2002), while newer results donot support this (Hoge et al. 2012).

In sum, while NAergic manipulations generally affectedfear memory (consolidation) in humans, NA does so far notseem to have comparable effects on the consolidation ofextinction memory in humans, while in animals, NA hasbeen shown to enhance extinction learning (Cain et al. 2004;Morris and Bouton 2007; Mueller et al. 2008; Janak andCorbit 2011).

A specific role of NA in enhancing excitatory memorytraces would be in line with its role in arousal-dependentenhancement of emotional memories (McGaugh 2000),which, from an evolutionary perspective, is an adaptive func-tion aiding preparation of appropriate future coping behaviour.

A third possible explanation for RBX-induced fear memo-ry enhancement may be the existence of different critical timewindows for the consolidation of the reactivated (putatively tobe reconsolidated) fear memory and the newly acquired ex-tinction memory. RBX takes approximately 2 h to reach peakconcentrations (tmax), and we administered RBX post-extinction training to preclude drug effects on the extinctionlearning process. Thus, drug effects on molecular events fol-lowing the extinction session may have been affected as afunction of time. In addition, we employed a single-dose ofRBX (4 mg) in healthy male participants only. It can thereforenot be excluded that other study populations (females, patientssuffering from an anxiety disorder) or other drug doses orchronic drug treatment may yield different results.

While the exact molecular mechanisms mediating our ob-served drug-induced differences in hemodynamic responsepatterns 1 week later cannot be elucidated with our methods,it is noteworthy that differences weremost pronounced for cueCSs and marginal for contextual CSs. Prior human studiesfocussing on cue CSs have found modulatory effects ofNAergic challenges (Soeter and Kindt 2011b; Soeter andKindt 2012), while a previous study assessing both condition-ing modalities found an effect of propranolol on the retentionof contextual, but not cued fear (Grillon et al. 2004b).However, Grillon and colleagues assessed context condition-ing by comparing physiological activity prior to cue condi-tioning with physiological activity prior to a recall test for cueCSs (i.e. when returning to the conditioning context). Thus,different implementations and assessments of contextual fearmay explain these discrepant findings.

It has also been suggested that the modulatory effect of NAdepends on the predictable presentation of CSs (Mason 1983;Mueller and Cahill 2010; Goddard et al. 2010). In support ofthis, group differences in hemodynamic responses were mostpronounced for the stimulus that during conditioning was themost reliable predictor of US administration, namely, thePCue (i.e., cue CS in the predictable condition, 100 % rein-forcement). Similarly, also the results of Grillon et al. ( 2004b)may be reinterpreted with respect to predictability rather thanthe mode of condition, as NAergic effects were observedwhen participants were re-exposed to the context previouslypredictive of cue-associated US.

There are some limitations to the study that deserve to bementioned. First, fMRI acquisition was restricted to day 8, whereeffects of drug administration, which occurred after extinctionlearning, might be detectable. While this is cost efficient, theabsence of fMRI data for the acquisition and extinction sessionpreclude comparisons of activation pattern across the

Psychopharmacology

experimental sessions. Second, fMRI acquisition was restrictedto the day (day 8) at which the potential effects of post-extinctiondrug administration were expected. This precludes comparisonsof activation pattern across the experimental sessions and makesit possible that the change from the behavioural laboratory ondays 1 and 2 to the fMRI environment on day 8 may haveinduced undesirable contextual effects (e.g. new learning oruncertainty). Third, as mentioned above, due to different repeti-tions of cue and contextual CSs as well as imaging analysisconstraints, we were unable to directly compare cue and contex-tual CS. As this precludes to directly assess the extent to whichparticipants are using cue versus contextual information to guidetheir responding, future studies should try to use the samenumbers of repetitions. Fourth, in fact, extinction seemed to bequite efficient in reducing psychophysiological responding(SCRs) in our paradigm, and we cannot exclude that drug effectsmight have been different with smaller amounts of extinction onday 2. In addition, as mentioned previously, RBX might ofcourse act differently in patient populations.

The results of our experimental human fMRI study might,despite of no difference in behavioural measurements betweenthe drug groups, yield preliminary evidence that may call forcaution when translated to the clinical setting. RBX adminis-tration, post-CBT in particular, may interfere with consolida-tion of what was learned during exposure therapy by enhanc-ing return of fear.

In sum, our study does not suggest that 4 mg RBX admin-istered after extinction learning may enhance extinction mem-ory consolidation. Of note, and in light of the above-mentioned animal work, our data however do not precludethat RBX administered prior to extinction learning might beefficient, suggesting future studies to focus on this.

Acknowledgements We thank Moritz Berker for help with SCR scor-ing, Dr. Alexandra Thanellou for help with study design and piloting,Dr. Mathias Gamer for providing the software for SCR scoring as well asTimo Krämer, Kathrin Müller and Katrin Wendt for help with fMRI dataacquisition.

Funding and conflict of Interest The authors declare no conflict ofinterest. This work was supported by the State of Hamburg excellenceinitiative (neurodapt! consortium) and the Deutsche Forschungsgemeinschaft(DFG grants KA 1623/3-1 and KA 1623/4-1).

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