inhibitory repetitive transcranial magnetic stimulation (rtms) of the dorsolateral prefrontal cortex...

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NeuroImage xxx (2014) xxx–xxx

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Inhibitory repetitive transcranial magnetic stimulation (rTMS) of thedorsolateral prefrontal cortex modulates early affective processing

OFPeter Zwanzger a,1, Christian Steinberg b,1, Maimu Alissa Rehbein b, Ann-Kathrin Bröckelmann b,

Christian Dobel b, Maxim Zavorotnyy a, Katharina Domschke a, Markus Junghöfer b,⁎a Department of Psychiatry, Mood and Anxiety Disorders Research Unit, University Hospital Muenster, D-48149 Muenster, Germanyb Institute for Biomagnetism and Biosignalanalysis, University Hospital Muenster, D-48149 Muenster, Germany

⁎ Corresponding author at: Institute for Biomagnetism aof Muenster, D-48149 Muenster, Germany. Fax: +49 251

E-mail address: [email protected] (M. Jung1 Peter Zwanzger and Christian Steinberg contributed e

both be regarded as first author.

http://dx.doi.org/10.1016/j.neuroimage.2014.07.0031053-8119/© 2014 Published by Elsevier Inc.

Please cite this article as: Zwanzger, P., et al.,modulates early affective pro..., NeuroImage

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Article history:Accepted 5 July 2014Available online xxxx

Keywords:AnxietyCortico-limbic controlAmygdalaTMSMEG

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RThe dorsolateral prefrontal cortex (dlPFC) has often been suggested as a keymodulator of emotional stimulus ap-praisal and regulation. Therefore, in clinical trials, it is one of the most frequently targeted regions for non-invasive brain stimulation such as repetitive transcranialmagnetic stimulation (rTMS). In spite of various encour-aging reports that demonstrate beneficial effects of rTMS in anxiety disorders, psychophysiological studies ex-ploring the underlying neural mechanisms are sparse. Here we investigated how inhibitory rTMS influencesearly affective processingwhen applied over the right dlPFC. Before and after rTMS or sham stimulation, subjectsviewed faceswith fearful or neutral expressionswhilewhole-headmagnetoencephalography (MEG)was record-ed. Due to the disrupted functioning of the right dlPFC, visual processing in bilateral parietal, temporal, and occip-ital areas was amplified starting at around 90 ms after stimulus onset. Moreover, increased fear-specificactivation was found in the right TPJ area in a time-interval between 110 and 170 ms. These neurophysiologicaleffects were reflected in slowed reaction times for fearful, but not for neutral faces in a facial expression identi-fication task while there was no such effect on a gender discrimination control task. Our study confirms the spe-cific and important role of the dlPFC in regulation of early emotional attention and encourages future clinicalresearch to use minimal invasive methods such as transcranial magnetic (TMS) or direct current stimulation(tDCS).

nd Biosignalanalysis, University83 56874.höfer).qually to this paper and should

Inhibitory repetitive transcranial magnetic stim(2014), http://dx.doi.org/10.1016/j.neuroimag

© 2014 Published by Elsevier Inc.
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RRIntroduction

Several neuroimaging studies have shown that the processing ofemotionally arousing auditory, visual, and olfactory stimuli is accompa-nied by increased activation within a distributed neural network of theamygdala, prefrontal, parietal and modality-specific sensory cortex re-gions (Bradley et al., 2003; Royet et al., 2000; Sabatinelli et al., 2005;Sander and Scheich, 2001). The various brain regions involved in thisnetwork have been ascribed different roles in emotion processing andregulation. In particular, the prefrontal cortex (PFC) has been suggestedto be a key modulator of executive functions (e.g. the prediction of be-havioral consequences) and to exert emotional appraisal either directlyor indirectly, thus, serving the purpose of cognitive–emotional control(for review see Ochsner and Gross, 2005). Previous studies haveshown that the dorsolateral part of the prefrontal cortex (dlPFC) influ-ences emotional stimulus categorization (Cacioppo et al., 1993;

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Freedman et al., 2003), emotional evaluation, emotional memory(Dolcos et al., 2004), and emotional regulation (Meyer-Lindenberget al., 2005) and thus, is considered to play a major role in top-downemotional control. It has also been discussed whether the amygdala, akey structure of emotional stimulus processing, which is reciprocallyconnected with the ventromedial and the orbitofrontal cortex, isunder continuous top-down control by prefrontal cortical areas (DavisandWhalen, 2001; Quirk and Beer, 2006; de Almeida et al., 2009; for re-view see Domschke and Dannlowski, 2010). Considering the importantrole of the dlPFC in top-down emotional control, it is obvious that thisarea became a target for not only basic emotional research, but alsomore clinical and treatment-oriented investigations.

One way to investigate the neuromodulatory role of the dlPFC is tomanipulate its functionality bymeans of repetitive transcranialmagnet-ic stimulation (rTMS). Over the last 15 years, rTMS has been establishedas an excellent research tool, which is capable to selectively modulatecortical activity (Barker et al., 1986). Based on the principle of electro-magnetic induction, rTMS can exert either inhibitory (b1 Hz) or excit-atory (N10 Hz) effects on neuronal functioning depending on theapplied stimulation frequency. Previous studies investigating dlPFCfunction in healthy controls reported anxiety-modulating effects ofrTMS. For instance, after inhibitory stimulation of the right dlPFC

ulation (rTMS) of the dorsolateral prefrontal cortexe.2014.07.003

http://dx.doi.org/10.1016/j.neuroimage.2014.07.003

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subjects revealed a stronger orienting-response towards angry faces(d'Alfonso et al., 2000). van Honk et al. (2002) could show that low-frequency rTMS over the right PFC reduced vigilant emotionalresponses to fearful faces. Repetitive TMS of the right dlPFC was consis-tently associated with disrupted prefrontal–amygdala connectivity inanother study, which fits to the proposed role of the PFC in the regula-tion of mood and anxiety (Vanderhasselt et al., 2011).

While probably the majority of studies have used hemodynamicfunctional neuroimaging techniques to investigate mechanisms of top-down emotion regulation, the temporal dynamics of those mechanismsare lesswell explored. It is generally accepted that certain regions in thePFC exert top-down influences on the processing of visual information(e.g. Bar et al., 2006) and neurophysiological studies show that these in-fluences might already take effect within the first 100 ms (Rudraufet al., 2008; Bayle and Taylor, 2010; Steinberg et al., 2013). In a very re-cent TMS and electroencephalography (EEG) study, Mattavelli et al.(2013) reported that stimulation of the right medial PFC modulatedthe amplitude of early EEG components such as the P1 and N1. Giventhat the generators of these components are believed to be located inextrastriate occipito-temporal and parietal cortices as well as in partsof the fusiform gyrus (e.g. Di Russo et al., 2001), these results demon-strate that the PFC is able to exert top-down influences on posteriorand even distant brain areas in a rapid fashion. Studies on non-clinically low- and high-anxious individuals show that differences inearly visual processing (P1, N1, P2) of emotional information are morepronounced in high relative to low anxious subjects (e.g. Bar-Haimet al., 2005; Holmes et al., 2008) whereas those differences betweengroups become attenuated (Holmes et al., 2008, but see Rossignolet al., 2005) or are even absent (Walentowska and Wronka, 2012) atlater stages of processing. Similar results have been observed in pa-tients. For instance, there is evidence for abnormalities in early visualprocessing of fear-related material in specific phobia, already at thestage of the P1 (80–130 ms), the N170 (130–220 ms, see e.g. Kolassaand Miltner, 2006; Kolassa et al., 2007) and the early posterior negativ-ity (EPN, 150–280 ms, Mühlberger et al., 2009; Wieser et al., 2010).

Considering that hemodynamic investigations of anxiety patientsshow diminished PFC recruitment during emotion processing (e.g.Bishop et al., 2004; Shin et al., 2005),which is often interpreted as an in-sufficient top-down control on a hyperactive amygdala, it appearstempting to manipulate activity in PFC areas to achieve changes in theneural affective network. Such theory based manipulations of cortical

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Fig. 1

Please cite this article as: Zwanzger, P., et al., Inhibitory repetitive transcramodulates early affective pro..., NeuroImage (2014), http://dx.doi.org/10.1

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functions might be very promising for future approaches in treatingpsychiatric diseases like anxiety disorders. Although rTMS seems tohave anxiety-reducing effects (see Zwanzger et al., 2009), so far onlyfew studies have investigated its use for modulating the processing ofanxiety-related information. Instead, rTMS has been largely deployedas a new treatment method for major depression and consequently,there is only sparse evidence for possible therapeutic effects in anxietyand anxiety disorders.

The study at hand aims at further elucidating the role of the PFC inthe direct and indirect cognitive–emotional control of brain areas impli-cated in anxiety-related processing. It investigateswhat consequences atargeted inhibition of dlPFC function using low-frequency rTMS has onthe time-course of fear-related emotion processing measured bymeans ofmagnetoencephalography (MEG). Building on the assumptionthat the dlPFC exerts an inhibitory control over the emotion network,we expected amplified affective processing already during early pro-cessing stages as a consequence of reduced dlPFC inhibition.

Materials & method

General paradigm

Participants viewed faces with fearful or neutral expressions whilewhole-head MEG was recorded (see Fig. 1). Event related neural re-sponses were measured after but also before brain stimulation to ac-count for possible baseline group differences. A control group withsham stimulation and a between-subject designwere chosen to accountfor possible placebo effects and possibly differing conscious perceptionsof sham and real stimulation.

Participants

Forty healthy right-handed adults (23 women, mean age =26.3 years, age range = 19–38) with normal or corrected to normal vi-sion participated in the experiment. All providedwritten informed con-sent and were paid for participation. The study protocol was approvedby the ethics committee of the Medical Faculty, University of Muensterin accordance with the Declaration of Helsinki. All participants con-firmed to have no recorded history of neurological or psychiatricdisorder.

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nial magnetic stimulation (rTMS) of the dorsolateral prefrontal cortex016/j.neuroimage.2014.07.003


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Stimuli

Photographs of 40 individuals (20male, 20 female) were used in thepresent study, in which the individuals displayed either a fearful or aneutral facial expression. The 80 stimuli (40 fearful, 40 neutral faces)were cropped (including hair) using Adobe Photoshop®, were convert-ed to grayscale, and were placed above a homogeneous gray back-ground. Images were 10.5 cm height (297px @ 72dpi) and the nasionwas aligned to the center of the screen (fixation cross) to prevent addi-tional eye movements. All stimuli were taken from the Karolinska Di-rected Emotional Faces (KDEF) database (Lundqvist et al., 1998).

Transcranial magnetic stimulation

Transcranial magnetic stimulation was performed using aMagVenture MagPro X100 Option stimulator (Magventure, Farum,Denmark). A 70-mm figure-eight shaped coil was used to apply 1 Hzlow-frequency repetitive transcranial magnetic stimulation (rTMS) for30 min over the right dlPFC with a stimulation intensity of 120% relatedto the individual motor threshold. Individual resting motor threshold(RMT) was determined for the left abductor pollicis brevis muscle(APBM) and defined as the minimum stimulus intensity that producesa motor evoked potential in at least 50% of 10 trials. The position ofthe right dlPFC was marked 5 cm anterior in a parasagittal line to thescalp location for optimal APBM stimulation according to the standardprotocol used in the majority of clinical trials investigating the benefitsof prefrontal rTMS in therapeutic interventions. Herwig et al. (2001) re-vealed that, if appliedwith this standard setting, TMS over the left hemi-sphere predominately targets the dlPFC areas BA8 and BA9, in a fewcases also the premotor cortex (BA6, frontal eye field). Themotor cortexrelated TMS positioning should account for the known frontal petaliawhich is qualified by extended right-hemispheric frontal and especiallydlPFC regions (Watkins et al., 2001). We are thus confident that thestimulation in the present study targeted the right-hemispheric BA8and BA9.

For sham stimulation the figure-eight coil was oriented tangentiallyto the skull without touching the scalp. This one-wing 90 degree shamstimulation is unlikely to induce any measurable electrical currentswithin the underlying brain tissue (see Lisanby et al., 2001). It shouldbe noted thatwe used a between-subject design also to account for pos-sible differences in muscular or scalp sensations evoked by sham or realTMS stimulations. Since subjects had no previous rTMS experience andwere informed that the motor-threshold TMS test several days beforewas very different from the sustained rTMS session, subjects in theshamgroup should not have been able to detect any conspicuity. The in-fluence of corresponding sources of errors for between group compari-sons should thus be minimal.

Behavioral tests

Subjects had to complete three tests which assessed possible behav-ioral effects of the rTMS intervention: Before and after MEG measure-ments, participants had to rate each of the 80 facial stimuli withrespect to the perceived hedonic valence and emotional arousal on a9-point rating scale by means of the Self-Assessment-Manikin scales(SAM rating) devised by Bradley and Lang (1994). The valence scaleranged from unpleasant (1) to pleasant (9) and the arousal scale fromcalm (1) to exiting (9). In a second and a third test, participants had toidentify the gender (male vs. female) and the facial expression (fearfulvs. neutral) of consecutively presented faces via button press in a speed-ed forced choice discrimination task. The order of tasks (gender/emo-tion) as well as the assignment of response keys (left/right) wasrandomized across subjects. Presentation of stimuli was fully random-ized in all behavioral tests. Valence and arousal rating data from onesubject had to be rejected because of technical problems. Thus, SAM rat-ings from 39 subjects were analyzed. Valence rating data from two


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further subjects were discarded because of extreme values (more than3 times the interquartile range of the boxplot). All post-hoc tests werecorrected for multiple comparisons using the Bonferroni–Holmprocedure.

Magnetoencephalography (recording and data processing)

Visual evoked magnetic fields were measured using a high-densitywhole-head 275-channel MEG system (Omega 275, CTF, VSMMedtechLtd.) with first-order axial SQUID gradiometers. FourMEG sensors werepermanently disabled during data recording. Continuous MEG data inthe frequency band between 0 and 150 Hz were recorded with a sam-pling rate of 600 Hz. Data preprocessing, artifact rejection and correc-tion, averaging, statistics, and visualization were conducted with theMatlab-based EMEGS software (www.emegs.org; Peyk et al., 2011).Responses were down-sampled to 300 Hz and filtered offline between0.01 and 148 Hz. To optimize the detection of early and transientbrain responses in the higher frequency range, further low-pass filteringwas not applied. Data was averaged from 200ms before to 600ms afterstimulus onset and baseline-adjusted using a 150 ms pre-stimulus in-terval. The method for statistical control of artifacts in high-densityEEG/MEG data was used for single trial data editing and artifact rejec-tion (Junghöfer et al., 2000). This procedure (1) detects individual chan-nel artifacts, (2) detects global artifacts, (3) replaces artifact-contaminated sensors with spline interpolation statistically weightedon the basis of all remaining sensors, and (4) computes the variance ofthe signal across trials to document the stability of the averaged wave-form. The rejection of artifact-contaminated trials and sensor epochs re-lies on the calculation of statistical parameters for the absolutemeasuredmagnetic field amplitudes over time, their standard deviationover time, the maximum of their gradient over time (first temporal de-rivative), and the determination of boundaries for each of these threeparameters. On average, 2.6 (SD: 0.8) out of 271 sensors (0.96%) wereinterpolated and 43.8 (SD: 17.6) out of 640 (6.8%) trials were rejectedby the artifact rejection procedure. The number of interpolated sensorsand the number of rejected trials did not differ across the experimentalconditions.

After averaging, cortical sources of the event related magnetic fieldswere estimated using the L2-Minimum-Norm-Estimates method (L2-MNE; Hämäläinen and Ilmoniemi, 1994). The L2-MNE is an inversemodeling technique applied to reconstruct the topography of the pri-mary current underlying the magnetic field distribution. It allows theestimation of distributed neural network activity without a priori as-sumptions regarding the location and/or number of current sources(Hauk, 2004). In addition, of all possible generator sources only thoseexclusively determined by the measured magnetic fields are consid-ered. A spherical shell with evenly distributed 2 (azimuthal and polardirection, radial dipoles do not generate magnetic fields outside of asphere) × 350 dipoles was used as source model. A source shell radiusof 87% of the individually fitted head radius was chosen, roughly corre-sponding to the gray matter depth. Across, all participants and condi-tions, a Tikhonov regularization parameter k of 0.2 was applied.Topographies of source direction independent neural activities—thevector length of the estimated source activities at each position—werecalculated for each individual participant, condition, and time pointbased on the averaged magnetic field distributions and the individualsensor positions for each participant and run.

Combining high-density MEG with advanced source localizationmethods, analysis was based on the neural generator activities availablefor every test source, time point, and participant.

To explore valence effects independent of brain stimulation, esti-mated neural activation in the pre-TMS/pre-Sham phase was analyzed.Data acquired during this baseline session were submitted to arepeated-measures ANOVA including the within-subject factor Valence(fearful vs. neutral) and the between-subject factor Group (Sham vs.TMS).


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To assess effects of brain stimulation, differences between pre- andpost-TMS/Shamphaseswere submitted to anANOVAwith identical fac-tors. This baseline-adjusted analysis takes possible influences ofpreexisting variations into account and captures possible fluctuationsof brain activity (e.g. habituation effects) throughout the course of thewhole experiment in an accurate manner (see Steinberg et al., 2012).

Non-parametric cluster based permutation tests as suggested byMaris and Oostenveld (2007) were applied. To this end, Monte Carlosimulations of identical analyses based on 1000 random permutationsof the complete data set of participants and experimental conditionswere conducted. Based onfirst level statistical effectswith a significancecriterion of p b .05, spatiotemporal clusters of at least six neighboringtest sources and at least 15 ms intervals were used for the clusterbased statistics (p b .05).

Identified spatiotemporal clusters showing significant modulationsof neural activations were further analyzed using post-hoc t-tests inorder to specify the direction of effects.

Procedure

Prior to the start of the study, subjects were randomly assigned toone of two experimental groups, either the Sham or the TMS group (be-tween-subject design). At the beginning, individual head shapes weredigitized using a Polhemus 3Space® Fasttrack to determine the head co-ordinate system for later calculation of spherical head models. Next,participants were sat in a dimly lit, sound attenuated and magneticallyshielded chamber. To monitor the participant's head position andmovementwithin theMEG scanner, three landmark coils were attachedto the two auditory canals and the nasion. Visual stimulation was pro-vided via a mirror system passing the image onto a translucent projec-tion screen. Viewing distance was 86 cm corresponding to 7° of visualangle given the size of the stimuli (10.5 cm in height). Subjects wereinstructed to passively view the stimulation. Online monitoring of re-corded magnetic fields for the occurrence of alpha waves served as anindex for fatigue. According to this, no subject had to be excludedfrom the study. In the pre-TMS MEG phase (overall 9 min), all faceswere presented in randomized order for 600 ms with an inter-stimulus-interval (ISI) of 1100±300ms. In total, 40 fearful and 40 neu-tral faces (stemming from 40 individuals) were repeated four timescomprising 320 stimulus presentations. Following this, participantshad to complete the SAM rating as well as the emotion and gender dis-crimination tasks. In the SAM rating all 80 stimuli were presented for600 ms and immediately after offset the SAM scales appeared. Re-sponses were made by button presses while moving a green cursor tothe desired position along the scales. In the emotion and gender dis-crimination task, subjects were instructed to respond as fast as possible.Stimuli were presented for 600 ms and as soon as the subjectresponded, the next trial was presented after an ISI of 700 ± 200 ms.After a short break repetitive TMS was delivered at 1 Hz with a total of1800 pulses over a period of 30 min above the right dlPFC. As men-tioned above, sham TMS was applied by turning the coil 90° with onewing oriented to the skull. During real or sham stimulation participantssat in a comfortable chair. Once rTMS or sham stimulation finished, thesecondMEG session followed. The experimental designof this post-TMSMEG session was identical to the pre-TMS session, except differing ran-domizations of the stimuli. Finally, participants had to complete thesame behavioral tasks as at the beginning of the experiment (differentrandomization). Avoiding confusion, the assignment of response keyswas kept constant related to the pre-TMS phase.

In contrast to the rule of thumb that inhibitory TMS effects on motorcortex excitability last for as long as TMS was applied (e.g. Chen et al.,1997), there is some evidence that rTMS effects of inhibitory dlPFC stim-ulations might last for a shorter duration. For instance, Eisenegger et al.(2008) applied 1 Hz low-frequency rTMS to the right dlPFC for 15 minand showed that an increase of the regional cerebral blood flow (rCBF)measured by means of positron emission tomography (PET) lasted for


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at least 9 min before returning to baseline after 13 min. Assuming a lin-ear relationship, effects after 30 min rTMS could be expected to last for atleast 18 min. MEG data, SAM ratings and emotion/gender ratings wereacquired within 5–14, 16–19½, and 20½–27 minute time-intervalsafter rTMS stimulation, respectively (see timeline in Fig. 1). Thus, whileneural activitymeasured byMEG should be fully affected by the stimula-tion, behavioral performance could be influenced to a lesser degree.However, since neural measures were of predominant importance,MEG data were always acquired first. Overall, it took the participantsroughly about 90 min to complete the whole experiment.

Results

MEG results

Baseline valence effectsIn the baseline session prior to rTMS or Sham intervention, strong

main effects of Valence were observed in an interval ranging from 60to 160 ms with greater responses for fearful as compared to neutralfaces. These affect-specific responses occurred in bilateral occipital andtemporal areas (see Fig. 2). More specifically, differential activationwas found in a bilateral occipital (F(1,38) = 32.73; p b .001), a left an-terior temporal (F(1,38) = 24.68; p b .001) and a right mid-temporalcluster (F(1,38) = 16.06; p b .001). Fearful faces evoked stronger neu-ral activation compared to neutral faces in all regions. There were nomain effects of Group or interaction effects of Valence and Group.Thus, the two groups did not differ in overall brain activation and inemotional processing before the intervention.

Effects of rTMSThe spatiotemporal analysis of estimated neural activation revealed

general and valence-specific effects of treatment significant on the clus-ter level in two intervals and two regions.

As shown in Fig. 3, a widely distributed source cluster covering theoccipital, superior temporal, and parietal areas of the left hemisphereshowed a huge main effect of Group in a time-interval ranging from90 to 150 ms. To assess hemispheric specificity of this effect, source ac-tivity at two mirror symmetric source clusters on the left and righthemisphere within this interval were analyzed by a Valence (fearfulvs. neutral) by Hemisphere (left vs. right) by Group (TMS vs. Sham)ANOVA.

This analysis revealed a main effect of Group which was character-ized by a general increase of neural activity in the TMS as compared tothe Sham group (F(1,38) = 5.47; p = .025). This main effect was mod-ulated by a Valence × Group interaction (F(1,38) = 4.44; p = .042) asvalence-specific difference activations (fearful vs. neutral) were relative-ly increased after TMS stimulation (t(19)= 2.32; p= .031), while emo-tion processing in the Sham group did not change (t(19) = −0.78; p=.443). There was also a significant Group by Hemisphere interaction(F(1,38)= 4.71; p= .036) indicating stronger activity in the right com-pared to the left hemisphere in the Sham group (t(19) = 2.47; p =.023) whereas – as consequence of the predominately left hemisphericactivation increase in the TMS group – no such effect was observed inthe TMS (t(19) = −0.86; p = .403) group.

In another interval between 110 and 170 ms, group-dependentemotional modulation was found above right-hemispheric superiortemporal and inferior parietal areas, presumably reflecting neural activ-ity around the temporal parietal junction (TPJ, see Fig. 4). As above,laterality effects were assessed by comparing evoked neural activity intwo mirror symmetric left and right-hemispheric clusters with a Va-lence by Hemisphere by Group ANOVA.

There was a main effect of Group (F(1,38) = 6.32; p = .016)again stemming from relatively increased activity in the TMS ascompared to the Sham group. Furthermore, a significant three-way interaction of Valence, Hemisphere, and Group (F(1,38) =5.43; p b .025) revealed that there was a substantially larger



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right-hemispheric difference between fearful and neutral face pro-cessing in the TMS (t(19) = 2.17; p = .043) compared to theSham group (t(19) = −1.69; p = .107), whereas no such differ-ence occurred in the left hemisphere (all p N .05).

In summary, rTMS over right-hemispheric dlPFC led to an overallamplified face processing between 90 and 150 ms irrespective of stim-ulus valence in bilateral parietal, temporal, and occipital areas. In addi-tion, the TMS group exhibited a treatment-related affect-specific boostin the processing of fearful vs. neutral faces which has not been ob-served in the Sham group. The same kind of effect, an additional in-crease of fear-specific activation, also appeared between 110 and 170ms around the right TPJ area. The global power2 of the difference sourcewaveforms (post–pre session) for the cluster in each hemisphere isshown in Fig. 5.

Behavioral results

SAM ratings

Valence. The repeated-measures ANOVA including the within-subjectfactors Session (pre-TMS vs. post-TMS) and Valence (fearful vs. neutral)and the between-subject factor Group (TMS vs. Sham) yielded a signifi-cantmain effect of Session (F(1,35)= 4.972; p= .033) showing that thevalence ratings in thepost-TMS/post-Sham (mean= 4.25; SD=0.36) ascompared to the pre-TMS/post-Sham phase (mean: 4.13; SD = 0.37),most presumably as consequence of a mere exposure effect (Zajonc,1980), increased towards more positive ratings. Not surprisingly, therewas a significant main effect of facial expression (F(1,35) = 251.98; pb .001), characterized by lower valence ratings for fearful (mean =3.27; SD = 0.51) as compared to neutral (mean = 5.1; SD = 0.46)faces. Therewas neither a significantmain effect of group nor an interac-tion with other factors (all p N .05). The participant's gender did not in-fluence the valence ratings as a main effect of gender or interactions ofgender with the other factors were absent (all ps N .05).

Arousal. The repeated-measures ANOVAwith the within-subject factorsSession (pre-TMS/pre-Sham vs. post-TMS/post-Sham) and Arousal(fearful vs. neutral) and the between-subject factor Group (TMS vs.Sham) revealed results similar to the valence ratings: there was a

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2 The mean squared activity across all estimated sources in the model as an index ofglobal neural activation.


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main effect of session (F(1,37)= 5.393; p= .026) as well as a main ef-fect of facial expression (F(1,37) = 114.78; p b .001) showing thatarousal ratings decreased from pre-TMS/pre-Sham (mean = 3.61; SD= 1.32) to post-TMS/post-Sham (mean = 3.34; SD = 1.39) sessionsand that theywere higher for fearful (mean= 4.68; SD= 1.79) as com-pared to neutral (mean= 2.27; SD= 1.08) faces. Again, there was nei-ther a main effect of Group nor an interaction with other factors (allp N .05). The participant's gender did not influence arousal ratings (allps N .05).

Emotion and gender discrimination task

We measured accuracy (error rates) and reaction times (RTs) foreach participant in the emotion and gender discrimination tasks. Datafrom three subjects had to be excluded from further analysis becauseof technical problems, and hence 37 subjects remained in the data set.Please note, when error rates and even RTs were comparedwithin con-ditions, there were not any significant differences between groups forboth the emotion and the gender tasks. Thus, differences in task difficul-ty are unlikely to account for the effects reported below.

In the emotion discrimination task, two outliers (more than 3 timesthe interquartile range of the boxplot) were detected and excludedfrom further analysis. Repeated-measures ANOVA featuring the factorsSession (pre-TMS/pre-Sham vs. post-TMS/post-Sham), Facial Expres-sion (fearful vs. neutral) and Group (TMS vs. Sham) revealed a main ef-fect of Session (F(1,33) = 6.097; p = .019) characterized by a generaldecrease in reaction time between pre (mean = 828 ms; SD =172ms) and post (mean= 772 ms; SD= 146 ms) sessions. A main ef-fect of Facial Expression (F(1,33) = 42.201; p = .000) showed thatfearful faces (mean= 741ms; SD= 146) were recognizedmuch fasterin comparison to neutral faces (mean= 859ms; SD= 163). There wasalso a Session × Facial Expression interaction (F(1,33) = 10.457; p =.003), stemming from a decrease in RT for neutral faces (t(34) = 3.43;p = .002) from pre to post whereas RTs did not change for fearfulfaces (t(34)= 1.00; p= .322). In spite of the training effect for neutralfaces, RTs for fearful faces (mean=729ms; SD=133)were still signif-icantly (t(34)= −4.83; p b .001) faster than for neutral faces (mean=815 ms; SD = 174), even if only post session values were considered.Most importantly, these effects were modulated by an interaction ofSession, Facial Expression, andGroup (F(1,33)= 6.078; p= .019). Sub-jects in the TMS groupwere relatively slower in rating fearful than neu-tral faces after TMS intervention (t(15) = 3.33; p = .005), whereas



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subjects in the Sham group became generally faster in responding toboth fearful and neutral faces while the difference between RTs for fear-ful and neutral faces (t(18) = .675; p = .508) was not significant (seeFig. 6). The gender of the participants did not influence RTs.

Regarding accuracy, after eliminating three outliers (more than 3times the interquartile range of the boxplot), we found a significantSession × Facial Expression interaction (F(1,32) = 12.992; p b .001).Post-hoc t-tests revealed that error rates for fearful faces decreasedfrom pre-TMS/pre-Sham (mean = 6.91; SD = 7.05) to post-TMS/post-Sham (mean = 3.6; SD = 4.53) session (t(33) = 3.1; p = .004)while errors for neutral faces from pre-TMS/pre-Sham (mean = 2.43;

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Fig. 4


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slightly increased (t(33) = −2.136; p = .04). No main effect or inter-actions with Group were found.

For the analysis of the RT data in the gender discrimination task threesubjects had to be excluded from further analysis (more than 3 times theinterquartile range of the boxplot). In the remaining data set we found amain effect of Session (F(1,32) = 19.5; p b .001) and a main effect ofFace Gender (F(1,32) = 42.851; p b .001). Reaction times were fasterin the post-TMS/post-SHAM (mean = 647 ms; SD = 90) as comparedto the pre-TMS/pre-SHAM (mean = 684 ms; SD = 106) session andfor recognizing male (mean= 630 ms; SD= 89.8) compared to female

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(mean= 701ms; SD= 109.6) faces. There were neither main effects ofGroup or of the participants' sex nor interactions of these factors withFace Gender or Session.

Examining accuracy in the gender discrimination task, a main effectof FaceGenderwas found (F(1,35)= 20.947; p b .001). That is, subjectsmade less errors when seeing a male (mean = 5.54%; SD = 4.93) ascompared to a female face (mean= 1.32%; SD= 1.79). No further sig-nificant effects were found.

It should be noted that the mean RTs calculated across sessions andconditions showed that both the TMS group (Emotion: mean =767 ms, SD = 99 ms; Gender: mean = 654, SD = 64; t(15) = 4.659;p = .000) and the Sham group (Emotion: mean = 827 ms, SD =174 ms; Gender: mean = 694, SD = 139; t(18) = 8.16; p = .000)were unexpectedly faster in discriminating gender compared to emo-tion. Although the corresponding error rates for the emotion rating(TMS: mean = 3.7%, SD = 2.5%; SHAM: mean = 4.5%, SD = 3.4%;t(32) = − .85; p = .401) and the gender rating (TMS: mean = 3.5%,SD = 2.5%; SHAM: mean = 3.4%, SD = 2.2%; t(35) = .10; p = .915)did not differ between groups, these overall RT effects could indicatesome differences in task difficulty which would limit inferences regard-ing the affect specificity of the result.

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Fig. 6


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post measures for emotion and gender ratings did not differ betweengroups. Based on the RT difference values for emotion (Δpost–pre

mean(fearful/neutral)) and gender (Δpost–pre mean(male/female)) rat-ings, both the TMS (Emotion:Δpost–pre= −42.6ms, SD=99.2ms; Gen-der: Δpost–pre = −35.5 ms, SD = 46.2 ms; t(15) = − .343; p = .736),and the SHAM group (Emotion: Δpost–pre = −66,4 ms, SD = 151 ms;Gender: Δpost–pre = −49.7 ms, SD = 67.1 ms; t(18) = − .604; p =.553) showed comparable training effects in the emotion and the genderratings. These results suggest the absence of a floor effect in the gendercondition. Thus, an effect of treatment on the gender rating could havebeen observed if present, which is why differences in task difficulty arerather unlikely to account for the reported results.

Discussion

The present study investigated the impact of inhibitory rTMS of theright dlPFC on early affective processing as measured by differentialMEG activation during the presentation of fearful and neutral faces.Our results showed that dlPFC stimulation was followed by overall in-creased event related neural activity at bilateral parietal and temporal

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3 Please note that the magnetic counterpart the EPNwhich has been initially describedin EEG splits up into an early and a late EPN-M in MEG (Peyk et al., 2008).


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cortex regions starting around 90 ms after stimulus onset. Importantly,rTMS-treatment of the right dlPFC led to not only a global increase ofneural activity, but also stronger differential emotional responses tofearful compared to neutral faces in predominately right-hemispherictemporal visual areas from 110 up to 170 ms and additionally also inthe left-hemispheric occipito-parietal regions from 90 to 150 ms. Itshould be mentioned that the analyzed time-intervals and regionswere partially overlapping, and thus, it seems plausible that the interac-tion found in the early interval partially reflects the interaction found inthe later time-interval. Behavioral tests revealed an adverse effect ofrTMS on judging fearful facial expressions, but not on judging stimulusgender. Overall, the electrophysiological results suggest that diminisheddlPFC functioningboosts preferential processing of emotionally relevantmaterial in cortical affective networks. However, the behavioral datasuggest that the dlPFC dysfunction eventually interferes with task rele-vant behavior if proper affective processing is required to complete thetask.

As described by Corbetta and Shulman (2002), activation oftemporoparietal and inferior frontal areas may serve as a detectionsystem for behaviorally relevant stimuli that is particularly sensitive tosalient or unexpected cues. Therefore, it might be suggested thatrTMS-induced changes in cortical network activity enhance specific at-tentional networks responsible for the detection of unexpected situa-tions. Increased attention towards dangerous situations followingrTMS-induced inhibition of right dorsolateral prefrontal activity is inline with several reports showing behavioral effects of inhibitory trans-cranial magnetic stimulation. According to work by van Honk andSchutter, low-frequency rTMS of the right dlPFC led to altered patternsof anxiety-related behavior (Schutter et al., 2001; van Honk et al.,2002). Similarly, in an experiment focusing on selective attention toangry faces, the same rTMS mode resulted in an increase of selectiveattention towards angry faces after right-hemispheric inhibition(d'Alfonso et al., 2000).

Interestingly, such effects have also been observed in patients. Apartfrom a large body of evidence on antidepressant effects of rTMS over thedlPFC (Burt et al., 2002; Couturier, 2005; Gross et al., 2007;Martin et al.,2003; Slotema et al., 2010), several reports conclude on putative anxio-lytic effects of rTMS which would render it an effective method for thetreatment of anxiety disorders (Zwanzger et al., 2009). The majority ofthese studies were conducted in patients with posttraumatic stress dis-order (PTSD). Here, clinical effects were observed in small case studiesshowing a decrease of PTSD symptomatology after right prefrontaltreatment (Grisaru et al., 1998; McCann et al., 1998). Moreover, benefi-cial effects were reported in patients with panic disorder receivingsuprathreshold low-frequency rTMS over the right prefrontal cortex(Garcia-Toro et al., 2002; Mantovani et al., 2007; Prasko et al., 2007;Zwanzger et al., 2002). Although the total number of studies is limitedcompared to the large set of data in major depression these resultspoint towards anxiety-modulating effects of rTMS in anxiety and anxi-ety disorders and therefore underline the important role of the rightdorsolateral prefrontal cortex in the processing of anxiety-relatedstimuli.

However, the majority of these studies have applied different rTMSprotocols ranging from single-treatment sessions up to daily treatmentfor 2–3 weeks. Since some studies show that a single-treatment sessionof rTMS does not have any effect on anxiety (Zwanzger et al., 2009), itremains unclear, whether a single-treatment session is capable to mod-ulate neuronal activity sufficiently to obtain clinically relevant effects.However, we argue that the effect size of the stimulation depends onthe strength of the stimulus applied andwe suggest that a lack of effectsin experimental panic induction might be due to the strong stimulus ofthe panicogenic agent (cf., Zwanzger et al., 2009). Indeed, studies on at-tentional processing of emotional information reveal that a single ses-sion of rTMS can lead to significant effects. For instance, Leyman et al.(2009) showed that a single session of high-frequency rTMS, using a10 Hz stimulation protocol over the right dlPFC, produced a significant


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impairment in the ability to inhibit negative information in a negativeaffective priming task. Similarly, in a double blind placebo-controlledcrossover study, Vanderhasselt et al. (2009) reported that one sessionof high-frequency rTMS over the left dlPFCwas followed by a significantimprovement in task-switching performance, whereas no effect onmood was observed. Moreover, our findings fit nicely with previous re-ports showing opposite effects when high-frequency rTMS is appliedover the right dlPFC. In a study using fMRI, De Raedt et al. (2010)showed that high-frequency rTMS of the right dlPFC resulted in an im-paired disengagement from angry faces, which was associated with de-creased activity in the right dorsolateral prefrontal and the cingulatecortex, but with increased activity in the amygdala. These results pointtowards an altered attentional control of affective processing followingright prefrontal stimulation.

Our resultsmight also be discussed in the context of hemispheric lat-eralization. According to the so called “valence-asymmetry hypothesis”after which withdrawal-related emotions such as anxiety are predomi-nately processed by the right hemisphere, whereas the left hemisphereis more associated with approach-related emotions, such as positivemood (Davidson and Irwin, 1999). Along with this hypothesis, there isevidence that anxiety is associated with increased right-hemisphericactivity (Heller et al., 1998). In light of this model, our strategy using in-hibitory rTMS to decrease right dlPFC top-down emotional controlmight be interpreted as an approach of generally enhancing the right-hemispheric processing of fear relevant stimuli which at the sametime is accompanied by increased selective attention towards threat. Al-ternatively, our results could also be explained by findings from faceperception studies (e.g. Bentin et al., 1996; Kanwisher et al., 1997;Kloth et al., 2006) showing that there is a right-hemispheric dominancefor processing faces which can be seen for facial emotional expressionsaswell (e.g. Pourtois et al., 2005). Thus, this dominance could have beenlost as consequence of rTMS.

Finally, the results obtained in the current study accordwithfindingsfrom basic and clinical affective research. For instance, ERP componentsin early and midlatency time-intervals such as the occipital P1 (90–130 ms) and the occipito-temporal early posterior negativity (EPN,120–280 ms) have been found to be susceptible to different kinds of vi-sual emotional stimuli. The P1 emotion effect is well documented foremotional facial expressions (Batty and Taylor, 2003; Dolan et al.,2006; Pizzagalli et al., 1999; Pourtois et al., 2005) and is believed toreflect the early encoding of emotional features and/or to be related tothe (later) allocation of attentional resources to the corresponding stim-uli. The EPN emotion effect has been found in response to various visualemotional stimuli such as faces (Schupp et al., 2004), scenes (Junghöferet al., 2001), words (Kissler et al., 2007), and even hand gestures(Flaisch et al., 2009). This affect-specific response has been associatedwith facilitated emotional processing in ventral visual areas and hasbeen interpreted in line with the concept of motivated attention (Langet al., 1997). Research investigating the impact of clinical and subclinicalanxiety on emotional processing showed that there are noticeable dif-ferences between individuals in the processing of affective stimuli de-pending on their actual anxiety level. These differences were found tomainly manifest in early and midlatency ERP components such as thealready mentioned P1 (Kolassa and Miltner, 2006) and the EPN(Mühlberger et al., 2009; Wieser et al., 2010). In the current study, theearly and the midlatency activations seen in occipito-temporal areasare in line with previously described time-intervals and locations ofthe P1 and the (early phase of the) EPN3 components. According tothe literature, the effects shown here may reflect the exaggerated per-ceptual processing of emotionally and motivationally significant cueswhich is associated with an increase of attention towards those stimuli.

Inhibitory rTMS of the right dlPFC led to a general amplification ofbrain activity in bilateral occipito-temporal–parietal cortex areas and



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to increased affect-specific responses at the right TPJ region. Inactivationof the right dlPFC might result in a loss of inhibition which is usuallyimposed on limbic structures and other cortical areas. In other words,releasing the ‘brake’ in the right dlPFC seems to influence affective visualprocessing at early stages and recalls how affective stimuli are proc-essed in anxious people or individuals suffering from anxiety disorders.Ourfindings are important for not only understanding affective process-ing per se, but alsomight be of interest for future, more clinically orient-ed studies. For instance, in PTSD, a loss of (cortical) inhibition (and ahyperactivation of the amygdala) is discussed thatmight be compensat-ed by applying rTMS to the corresponding cortical regions. However, re-garding the role of the dlPFC in affective processing and its contributionto anxiety disorders, more research is needed investigating e.g. theimpact of dlPFC rTMS on the processing of pleasant emotions and theinterplay between inhibitory/excitatory stimulation and left/righthemisphere.

Neurophysiological effects in this study come along with modulatedbehavior in an affect-specific discrimination, but not in an emotion-independent control task. Interestingly, the behavioral effects occurredafter only one treatment session with rTMS and in spite of a relativelylong time lag between brain stimulation and behavioral testing, whichpossibly reduced the impact of the treatment (see procedure above).

The emotion-selective deceleration of reaction times seen here cor-responds with other reports showing interfered visual emotional faceprocessing after TMS intervention. For instance, a significant slowingof reaction times has been reported for fearful compared to happyfaces following TMS over right somatosensory cortex (Pourtois et al.,2004). In another study, TMS applied to the medial prefrontal cortexalso led to prolonged reaction times, when angry facial expressionswere presented to the subjects (Harmer et al., 2001). Of course, TMSstimulation sites vary across studies (including the present one),however, all targeted regions are relevant for the processing of emo-tionally relevant visual stimuli. The prolonged reaction times for identi-fying fearful faces in the current study might be explained in differentways: Inhibitory top-down control usually imposed by the dlPFC onemotion-related areas such asmedial and ventrolateral PFC is importantto appraise the relevance of a stimulus in a given context (e.g. Wessinget al., 2013) and thus prevents the occurrence of disproportionate emo-tional reactions towards affective stimuli. The TMS stimulation appliedhere might have caused a loss of control over emotional responses to-wards fearful faces, which amplified fear-specific reactions, such asfight or flight preparation, during affective stimulus presentations, anddrew attentional resources away from the actual cognitive task (i.e.,emotion categorization). TMS might have also disturbed the process ofemotion categorizing per se which is why the fearful facial emotionalexpressions were more difficult to detect. Indeed, stimulus categoriza-tion is a function which is known to be mediated by the dlPFC(Cacioppo et al., 1993; Freedman et al., 2003). Thus, the impaired con-trol of attention towards and/or the deteriorated categorization of emo-tional stimuli might account for the behavioral results seen in thecurrent study.

Lastly, it should also bementioned that there were not any effects ofrTMS on affective face processing at the site of stimulation, at least forthe analyzed time-intervals. This is in line with findings from otherstudies which did not report rTMS effects at site of stimulation(dlPFC), either, when regional cerebral blood flow or glucose metabo-lism was measured (Kimbrell et al., 2002; Ohnishi et al., 2004; Speeret al., 2003). However, based on the idea that rTMS might have evokedsustained effects which could not have overcome the cluster-level crite-rion, we specifically analyzed the complete time-interval (from 0 to600 ms) for Group × Session and Group × Session × Valence interac-tions. As a result, one region at the site of stimulation as well as anothercorresponding contralateral region revealed an enhanced emotion-unspecific neural activity after rTMS but not after sham stimulation(please see supplementary content) which could be interpreted as aneffect of valence-unspecific neural noise induced by rTMS (e.g. Walsh


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and Cowey, 2000). It remains unclear under which conditions onesided rTMS to the dlPFC evokes enhanced ipsilateral (Eisenegger et al.,2008) or enhanced bilateral (Nahas et al., 2001) neural activity at thesite of stimulation.

Taken together, our data show that inhibitory rTMS of the rightdlPFC is followed by an increased activation in response to potentiallythreatening stimuli in areas related to both visual (emotional) process-ing and visual selective attention. Overall the data confirm a specific andimportant role of the prefrontal cortex in the regulation of emotional at-tention and furthermore underline the idea of the dlPFC being a targetfor future clinical research and therapeutic intervention usingminimal-ly invasive methods such as transcranial magnetic (TMS) or direct cur-rent stimulation (tDCS).

Acknowledgments

This work was supported by a grant of the DeutscheForschungsgemeinschaft SFB TRR-58 subproject C01 to MarkusJunghöfer, Peter Zwanzger and Christo Pantev. We thank A. Wollbrink,Karin Berning, Ute Trompeter, Hildegard Deitermann and Janna vonBeschwitz for technical assistance. We also thank two anonymous re-viewers for their helpful comments and suggestions to improve the qual-ity of this work. The first two authors, Peter Zwanzger and ChristianSteinberg, contributed equally to this work.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.neuroimage.2014.07.003.

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