European Neuropsychopharmacology (2013) 23, 8308410924-977X/$ - see frhttp://dx.doi.org/1

nCorrespondenceMedical Sociology, UUniversity, PauwelssTel.: +49 241 808900

E-mail address: bwww.elsevier.com/locate/euroneuroThe role of 5-HT in response inhibitionand re-engagement

Barbara Druekea,n, Sonja M.A. Schlaegela, Anke Seiferta, Olaf Moellerb,Gerhard Grnderb,c, Siegfried Gauggela, Maren BoeckeraaDepartment of Medical Psychology and Medical Sociology, RWTH Aachen University, GermanybDepartment of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty,RWTH Aachen University, GermanycJlich Aachen Research Alliance JARA, Translational Brain Medicine, Germany

Received 4 December 2012; received in revised form 9 April 2013; accepted 4 May 2013KEYWORDSInhibitory motorcontrol;Stopchangeparadigm;5-HT;Escitalopramont matter & 20130.1016/j.euroneur

to: Departmentniversity Hospitaltrae 30, D-520744; fax: +49 241 80drueke@ukaachenAbstractIn animal and human research, the neurotransmitter serotonin (5-HT) has been implicated ininhibitory control. Using functional magnetic resonance imaging (fMRI), the present studyinvestigated the acute effects of pharmacological modulation of the serotonergic system on brainactivation during response inhibition and re-engagement in healthy human volunteers. In arandomized double-blind placebo-controlled cross-over design 14 men received either a single oraldose of the selective serotonin reuptake inhibitor (SSRI) escitalopram (10 mg) or a placebo. At thetime of the expected plasma peak concentration, participants performed a stopchange task duringfMRI. Escitalopram did not affect behavioural performance, since the main effect did not revealsignificant differences between reaction times of go-, stop- or change-trials. During successfulresponse inhibition, escitalopram, however, was associated with enhanced brain activation in rightprefrontal cortex, right supplementary/pre-motor and bilateral cingulate cortex, and subcorticalregions. During inhibition failures, escitalopram also modulated a broad network of brain regions,including anterior cingulate, right parietal cortex, right orbitofrontal cortex, and areas in righttemporal cortex and subcortical regions. During response re-engagement escitalopram increasedbrain activation in right inferior frontal gyrus and precuneus as well as in left middle temporalgyrus. The results implicate the involvement of 5-HT in neural regulation of response inhibition andre-engagement. This study also provides evidence that 5-HT affects both action restraint andaction cancellation through modulation of activation of brain areas. The results support the viewfor a fronto-striatal circuitry for response inhibition in conjunction with serotonin.& 2013 Elsevier B.V. and ECNP. All rights reserved.Elsevier B.V. and ECNP. All rightso.2013.05.005

of Medical Psychology andAachen, RWTH AachenAachen, Germany.3389004..de (B. Drueke).1. Introduction

The dynamic control of behaviour is a crucial ability inadapting one's behaviour in changing situations. Deficits inor the inability to exert behavioural control can lead toreserved.

www.elsevier.com/locate/euroneurodx.doi.org/10.1016/j.euroneuro.2013.05.005dx.doi.org/10.1016/j.euroneuro.2013.05.005dx.doi.org/10.1016/j.euroneuro.2013.05.005http://crossmark.dyndns.org/dialog/?doi=10.1016/j.euroneuro.2013.05.005&domain=pdfmailto:bdrueke@ukaachen.dedx.doi.org/10.1016/j.euroneuro.2013.05.005

8315-HT in stopping and changing behaviourdifficulties relating to the flexible interaction with theenvironmentas can be seen in patients with several mentalor neurological disorders, such as attention deficit hyper-activity disorder (ADHD), obsessive compulsive disorder(OCD), trichotillomania, and Parkinson's disease (Schacheret al., 2007; Chamberlain et al., 2006; Gauggel et al.,2004). Much recent research has focused on (a) the neuralimplementation of behavioural control as a means of gaininga better understanding of the neuropathology of the afore-mentioned disorders and (b) the behavioural correction andadaption.

Response inhibition is a central aspect of behaviouralcontrol, because it is the first step in behavioural adjust-ment and entails suppression of an already planned or eveninitiated action. Response inhibition, i.e., the ability to stopa motor response after initiation, can be measured by usingthe Stop signal task, a well established paradigm forinvestigating inhibitory functions in laboratory settings(Logan, 1994). A key measure of response inhibition is thestop signal reaction time (SSRT), which is the time it takesto stop a motor response. The underlying theory for theestimation of SSRT is described by the so-called horse racemodel (see Section 2 and Logan, 1994). Neural structuresassociated with response inhibition are the dorsolateral,inferior, superior, orbital, and mesial frontal cortex, supple-mentary and pre-supplementary motor area, anterior cin-gulate gyrus, temporal and parietal lobes, and the basalganglia (Aron et al., 2003, 2004; Aron and Poldrack, 2006;Rubia et al., 2003; Eagle et al., 2008).

Exact localization of response inhibition has, however,been difficult. Nevertheless, evidence is accumulating fromneuroimaging studies with the stop signal task that the rightinferior frontal cortex (IFC) is critical for inhibiting analready initiated motor response, since response inhibitionhas consistently activated this region (Aron et al., 2004;Rubia et al., 2003). In a functional magnetic resonanceimaging (fMRI) study of patients with lesions of the rightfrontal cortex, Aron et al. (2003) showed that damage tothe right inferior frontal gyrus (BA 45) is crucial for responseinhibition. In a near-infrared spectroscopy study, Boeckeret al. (2007) found that stopping a response was associatedwith activation of right-lateralized prefrontal cortex.Recent research on response inhibition has also implicatedthe subthalamic nucleus (STN), which is an inhibitory outputstructure of the basal ganglia (Aron and Poldrack, 2006;Li et al., 2008). Aron and Poldrack (2006) suggested thatresponse execution is inhibited through a hyperdirect path-way via the STN, which again suppresses the basal-gangliathalamocortical output. Evidence for an involvement of thebasal ganglia also comes from a study with patients suffer-ing from Parkinson's disease (Gauggel et al., 2004). Deficitsin response inhibition might be due not only to structuraldamage, but also to deficits in neurotransmitter function-ing. The above-mentioned mental or neurological disorderssuch as ADHD, OCD or Parkinson's disease are not onlyassociated with deficits in response inhibition, but they alsoshow abnormal serotonin (5-HT) functioning, suggestingthat 5-HT is also involved in response inhibition (Del-Benet al., 2005; Rubia et al., 2005; Cools et al., 2008).Furthermore, the prefrontal cortex, which is clearly relatedto response inhibition, is a region that is connected toascending serotonergic projections from dorsal and medianraphe nuclei localized in the brain stem (see Cools et al.,2008).

Evidence for an involvement of 5-HT in response inhibi-tion comes from Clarke et al.'s (2007) animal study, in whichthey found a deficit in performance on a serial discrimina-tion reversal (SDR) task after prefrontal 5-HT depletion.This deficit was not due to a failure to approach a previouslyunrewarded stimulus, but was instead attributed to a failureto inhibit responding to a previously rewarded stimulus. In astudy with rats, Eagle et al. (2008) also found a selectiveimpairment in inhibition performance after 5-HT depletionin waiting, but not in the stop-signal reaction time (SSRT).Rubia et al. (2005) found evidence of a serotonergicmodulation with acute tryptophan depletion (ATD) of rightorbito-inferior PFC as well as superior and medial temporalcortices using a Go-Nogo task in healthy volunteers. Incontrast to the suggested involvement of 5-HT in responseinhibition, the previous research using the stop-signal taskwith the healthy volunteers showed that 5-HT had no effecton performance (Eagle et al., 2008; Robbins, 2007;Chamberlain and Sahakian, 2007). Chamberlain et al.(2006) found an enhanced response inhibition in humansafter intake of atomoxetine, a selective norepinephrinereuptake inhibitor (SNRI), but an unaffected SSRT afterintake of citalopram, a selective serotonin reuptake inhi-bitor (SSRI). As well, Dougherty et al. (2010) were not ableto find response inhibition impulsivity to be affected afterATD depletion in healthy participants performing a GoStopimpulsivity paradigm, while response initiation and conse-quence sensitivity impulsiveness were found to be affected.They suggested different biological processes may underliespecific components of impulsivity.

Despite the growing information about the neural basisof inhibitory motor control, there are still several openquestions. Thus, it is still not known to what extent 5-HT isinvolved in processes of both successful and unsuccessfulinhibition of initiated actions in healthy humans. Previousstudies have indicated little or no involvement of 5-HT inresponse inhibition, but there have been few such studieswith humans. It is even more unclear whether a subsequentprocess, i.e., response re-engagement, is altered throughserotonergic modulation. The StopChange paradigm hasbeen considered as an extension of the Stop-signal task, asboth tasks seem to differ only in the number of requiredmental processes. Of special interest in the StopChangetask is the change process which follows the inhibitionprocess and requires the subsequent re-engagement intoan alternative action. This process is called either responsere-engagement or change process or action reprogramming.In a recent study Verbruggen et al. (2008) found evidencethat all three processes (i.e., go-, stop- and re-engagementprocesses) are involved in the change paradigm and thatthey function mostly independent of each other in serial orclose-to serial order. On neural level, Boecker et al. (2010)found activations for changing a response in inferior parietallobes, bilateral IFC, and medial frontal cortex, includingpre-SMA, frontopolar cortex and insula. These results weresupported by the results from a TMS-study by Neubert et al.(2010) who found pre-SMA and rIFG to have direct influencesan primary motor cortex (M1) (for a detailed review onresponse-reengagement see Boecker et al. (2013)). Little isknown about the pharmacological effects of a serotonergic

B. Drueke et al.832manipulation on response re-engagement so far. Druekeet al. (2010) found in a study with healthy human volunteersno effects of escitalopram on response re-engagement asmeasured with the StopChange task which might be due tothe very low dose of 10 mg escitalopram.

Therefore, the main goal of the present study was tofurther investigate the open questions outlined above. Weinvestigated differential involvement of serotonergic sys-tem on brain activation patterns during response inhibitionand re-engagement in healthy humans, using the selective5-HT reuptake inhibitor (SSRI) escitalopram, which is amongthe most selective reuptake inhibitors available for humanuse at the moment. Because of the inconsistent results inprevious studies, we wanted to verify response inhibitionand re-engagement as precisely as possible using the stopchange paradigm, which is much more precise than the Go/No-Go paradigm. To our knowledge, no previous study hasinvestigated serotonergic modulation by combining pharma-cological and fMRI methodologies with the StopChangeparadigm, especially with a focus on the following processof response re-engagement. On the basis of prior research,we hypothesized that performance in the stopchange taskwould not be affected by 5-HT modulation. Consistent withDel-Ben et al.'s (2005) results we expected, however, thatescitalopram would modulate brain activations in right PFCthat were associated with response inhibition and re-engagement. Finally, we hypothesized that escitalopramwould also alter brain activation especially in anteriorcingulate during failed inhibition.

2. Experimental procedures

2.1. Participants

Healthy male volunteers (n=14) aged between 18 and 39 yearsparticipated in this study. Most were university students fromvarious academic departments. All participants were recruited viaposters and announcements in seminars. The means and standarddeviation for body weight and height respectively were 77.578.7 kg and 18278.3 cm. All subjects were drug-free and non-smokers, and underwent a drug screening before inclusion. Toestablish physical and mental health, participants underwent acareful screening for any hormonal, cardiovascular and neurologicaldiseases, which was based upon a health questionnaire and inter-views. Furthermore, blood samples of the participants were ana-lyzed in order to their verify physical health. In addition, all ofthem underwent a semi-structured clinical interview for mentaldisorders performed by a psychiatrist who is highly specialised onthe diagnosis of mental disorders. Exclusion criteria were a personalor family history of major mental or neurological disorder in first-degree relatives, somatic illness and regular alcohol or illicit drugabuse. For safety during MRI scanning, all participants affirmed thatthey were free of any metal pacemaker and body tattoos. On thedate of the tests, participants were not allowed to smoke, drinkalcohol, or eat chocolate, nuts or bananas. No participant had to beexcluded from the study due to any kind of acute or chronicphysical disease or mental disorder.

Prior to the main experiment, participants were thoroughlyinformed about the objectives and procedure of the study, thesubstances used and possible side-effects of the pharmacologicalintervention. After all information was presented and the subjectshad no further questions they signed the informed consent form thenext day. Participants were allowed to withdraw from the experi-ment at any time.Challenge-tests were performed during two separate sessions(placebo and escitalopram) one week apart. On the day of theexperiment, lunch was taken between 12:00 and 12:30 p.m. After-wards food intake was not allowed until the tests were completed.

The study was approved by the Ethics Committee of the RWTHAachen University and the Federal Institute for Drugs and MedicalDevices (BfArM).

2.2. Design

The study had a randomized, double blind cross-over design withtwo treatments (placebo, 10 mg escitalopram), each of which wasgiven in a single oral dose in counterbalanced order, i.e. half of theparticipants received escitalopram in the first session and placeboin the second one, and vice versa in the other half. A time intervalof exactly one week between each session was chosen as it issufficient for complete washout, since the elimination half life ofescitalopram is 2733 h (Rao, 2007).

2.3. Drugs

Escitalopram is a selective serotonin reuptake inhibitor (SSRI) withhigh affinity and the greatest selectivity tested so far for the human5-HT transporter. Escitalopram is the S-enantiomer of the SSRIcitalopram since pharmacology studies showed that the therapeuticactivity of citalopram resides in the S-enantiomer (Burke et al.,2002). It is indicated for the treatment of major depression andanxiety disorders (Burke et al., 2002). Following oral administra-tion, escitalopram is rapidly absorbed, with maximal plasma con-centrations achieved approximately 34 h after drug intake(Sogaard et al., 2005). It exhibits linear and dose-proportionalpharmacokinetics with mainly hepatic biotransformation. It shows aplasma elimination half-life (t1/2) of 2733 h, which is consistentwith once-daily dosing. Steady state plasma concentrations arereached within approximately one week with once-daily adminis-tration of 1020 mg, the commonly used dose (Aronson and Delgado,2004). Compared to placebo, first improvements of symptoms areseen within 12 weeks of commencing treatment, as verified bydepression rating scales (Burke et al., 2002). Escitalopram isgenerally well tolerated in clinical trials. For our study we usedCipralexs and a matching placebo provided by Lundbeck.

2.4. Experimental procedure

Each testing day lasted 4.5 h and participants were monitored withregards to any side effects. On the day of the experiments (at 1:30p.m.), subjects were seated in a comfortable chair, and bloodpressure and heart rate were measured. Drugs were administered20 min after arrival in identical capsules to maintain the blindnessfor subjects and experimenters. Afterwards, subjects completedseveral questionnaires and practised the stopchange task outsidethe MRI scanner. About 3 h after the drug intake, the participantstarted the stopchange task and at the same time functionalneuroimaging via MRI scanning of the brain was performed, as this isthe time of mean maximal plasma concentration of escitalopram(Sogaard et al., 2005). As the stopchange paradigm consists ofmany trials, the functional scanning was conducted in two subses-sions of which each session lasted 18 min. Half of the trials werepresented in the first subsession and the other half in the secondone and a short break between subsessions was offered. Finally, ananatomic scan was made using a T1 weighted structural scan.

2.5. MRI scanning procedure

Images were acquired at the University Hospital of the RWTHAachen University, using a 1.5-T Philips Gyroscan NT with standard

8335-HT in stopping and changing behaviourhead coil and foam padding to restrict movement. Axial multisliceT2-weighted images were obtained with a gradient-echo planarimaging sequence (TE=50 ms; TR=2800 ms; 64 64 matrix; flipangle=901; 30 slices, 3.4375 3.4375 mm2 in-plane resolution;slice thickness 3.75 mm; no gap, voxel size 3.75 3.75 3.75),covering the entire brain. The scanning session consisted of twosubsessions, each starting with 10 dummy scans that were notrecorded for data analysis to allow tissue to reach steady statemagnetization. During each subsession, 380 volumes were acquired.

Inherent in the stopchange paradigm is the fact that theexperimenter does not know in advance in which of the stop- andchange-trials the participant will successfully inhibit or change theinitiated response and in which he will fail. To ensure an optimizedscanning of the hemodynamic response in all brain slices for all fourcritical stop- and change conditions, namely StopInhibit, StopRe-spond, StopChange and ChangeRespond, the experiment was pro-grammed with an adaptive TR-class tracking algorithm to ensure anoptimum capture of the hemodynamic response for all four condi-tions (for details see Boecker et al. (2010)).

A T1 weighted structural scan was also acquired for each subjectfor co-registration. No abnormalities were found for any of the 14subjects.2.6. Stopchange paradigm

The stopchange task is a computer-based test developed toevaluate response inhibition and response-re-engagement. Thestopchange paradigm is composed of four different trial types:go-trials, stop-trials, change-trials and null events (see Figure 1).The duration of the stimulus trials (go-, stop- and change-trials) was3350 ms whereas the null events lasted either 2800 or 3350 ms.During null-events, a blank screen was presented, and these wereincluded into the experiment according to the randomized event-related design proposed by Burock et al. (1998).

The go-trials required the participants to execute a simple motorresponse following the presentation of two stimuli. They made up70% of the stimulus trials and were part of a simple choice reactiontime task in which participants had to discriminate a black circleand a black triangle on a computer monitor. Depending on which ofFigure 1 Graphical illustration of the trial types used in the event-most important parameters.the two go-stimuli was presented, participants had to respondeither with a left or right key press using index and ring fingers oftheir right hand. On stop-trials (15% of stimulus trials) the choicereaction time task was followed by an occasional and unpredictableauditory stop signal (a 1000 Hz tone of 500 ms duration) after avariable delay (stimulus onset asynchrony, SOA). It signalled theparticipants to inhibit the execution of their response to the choicereaction time task. In another 15% of the stimulus trials a changesignal (a 400 Hz tone of 500 ms duration) was presented after avariable delay. This signal indicated that participants should nowinhibit their response to the choice reaction time task and insteadpress the middle of the three response buttons with their rightmiddle finger.

The stop signal delay (stop-SOA) and the change signaldelay (change-SOA) were set by a staircase-tracking algorithm(Kaernbach, 1991), which adapted to the response rate. The stop-SOA and the change-SOA were adjusted independently in a way thatparticipants reached an inhibition rate of approximately 50% (stopcondition) or a change rate of likewise approximately 50% (changecondition). Setting an accuracy of 50% ensures an equal probabilityfor stopping as well as for not stopping, i.e., the probability ofstopping is neither overestimated nor underestimated. Thestaircase-tracking algorithm worked as follows: initially the stop-SOA and the change-SOA were set at 250 ms. Depending on thesuccess in response inhibition in stop-trials or success in changing inchange-trials, the SOA was increased by 50 ms in the following stop-trial or change-trial. Thus, a greater difficulty for participants toinhibit or change their responses was produced. If, however,participants failed to inhibit or change their response, the SOAwas decreased by 50 ms in the next stop- or change-trial, enhancingthe chance of successful inhibition or changing. This trackingprocedure provides a possibility to estimate inhibition performance,the stop signal reaction time (SSRT), which is relatively stable toany violation of independence between go- and stop-processes.Moreover, the individual adjustment of stop signal delay and changesignal delay maintains a high level of difficulty in inhibition and atthe same time homogeneity across subjects by making the partici-pants work on the edge of their inhibitory capacity.

Four blocks, containing 44 trials each, were performed tointroduce the different types of trials. Participants were instructedrelated stopchange paradigm and the horse-race model with its

B. Drueke et al.834to respond as fast as possible, while keeping a high level ofcorrectness. They were briefed not to slow down and wait for apossible stop or change signal thus delaying their response, but totry hard to withhold the response after hearing a stop signal orchange the response after the corresponding change signal. Toprevent them from developing a strategy participants were toldthat they would not be able to withhold the response at all timessince the computer would adapt to their results aiming for a successrate of 50%.

The participants took two subsessions of the stop change taskwith 308 trials each (196 go-trials, 42 stop-trials, 42 change-trialsand 28 null events) during fMRI scanning. The visual stimuli werepresented via a head-mounted video display designed to meet MRrequirements. If necessary, vision was adjusted to normal by placingcorrecting lenses in the goggles. The auditory stimuli were pre-sented via headphones at an individually adapted volume level,which was able to penetrate the noise of the MRI scanner.

2.7. Behavioural data analysis

Behavioural data were analyzed using the Statistical Package forthe Social Sciences (SPSS) Version 16.

The following parameters of the stopchange paradigm wereanalyzed: GoRT for correctly answered go-trials, ChangeRT forcorrectly answered change-trials, stop-SOA for stopsignal delay andchange-SOA for change-signal delay. On the basis of these parameters,the SSRT for successfully inhibited responses was estimated bysubtracting the delay of the measured reaction time of go-trials(SSRT=mean GoRT minus stop-SOA). CSRTwas calculated accordingly.Also, we determined StopRespondRT for trials of unsuccessfullyinhibited but correctly answered tasks and ChangeRespondRT for trialsof unsuccessful changing but correctly answered tasks.

To verify the working of the tracking algorithm the rate ofsuccessful stopping was determined, and had a range between 40%and 60%. A factorial analysis of variance (ANOVA) for repeatedmeasures with a repeated measurement factor substance (10 mgescitalopram versus placebo) was performed to analyze the depen-dent variables SSRT, CSRT, reaction time on go-trials (GoRT),reaction time on change trials (ChangeRT), StopRespondRT as wellas ChangeRespondRT.

2.8. Imaging data analysis

Functional images acquired from the scanner were analyzed withstatistical parametric mapping software (SPM5; http:/www.fil.ion.ucl.ac.uk/spm/). Images were motion corrected and realigned toeach participants' first image. Data were normalized into standardstereotactical space. Spatial smoothing was performed on thenormalized functional images using a Gaussian kernel of 8 mmFWHM. No participant had to be discarded from further analysisbecause of movement artefacts exceeding a limit of one voxel sizeper axis. First-level analysis was performed on each subject togenerate a design matrix using a random effects model with thefollowing events: Go, StopInhibit, StopRespond, StopChange, ChangeRespond and Null-event. For the go-conditions, events weremodelled at the time of the go-stimulus and for stop- andchange-conditions, events were modelled at the time of thepresentation of the auditory stimulus. For second-level analysis, a fullfactorial ANOVA with factors substance and condition was performed. Contrasts were calculated using whole brain analysis to assessthe effect of escitalopram: StopInhibitescitalopram4StopInhibitplacebo,StopRespondescitalopram4StopRespondplacebo, Goescitalopram4Goplacebo,StopInhibitescitalopram4StopInhibitplacebo, ChangeRTescitalopram4ChangeRTplacebo, ChangeRespondescitalopram4ChangeRespondplacebo.

All statistical maps for response inhibition and go were thre-sholded at po0.005 (uncorrected), except for the contrasts Chan-geRT and ChangeRespond, which were thresholded at po0.01(uncorrected). To correct for multiple comparisons, we estimatedan appropriate cluster size threshold using a Monte Carlo simulationof the brain volume implemented in Matlab (https://www2.bc.edu/slotnics/scripts/cluster_threshold_beta.m) (Slotnick et al., 2003;Forman et al., 1995). A Monte Carlo simulation has the advantage ofsensitivity to small effect sizes, while it still corrects for multiplecomparisons across the whole brain volume. The simulation indicated that for an assumed individual voxel type I error of po0.005,uncorrected, a cluster size of 8 contiguous resampled voxels wasrequired to correct for multiple comparisons across the whole brainat po0.05 (based on 10,000 iterations for our 64 64 32 functional image matrix with a full-width at half-maximum kernel of8 mm). We complemented this analysis with exploratory comparisons for response re-engagement (ChangeRTescitalopram4ChangeRTplacebo, ChangeRespondescitalopram4ChangeRespondplacebo) with athreshold of po0.01 (uncorrected) in order to have a first idea of aserotonergic manipulation on response reengagement. Anothercomplex contrast was calculated because of its repeated presencein the literature: StopInhibit minus GoEscitalopram4StopInhibit minusGoPlacebo and, vice versa, StopInhibit minus Goplacebo4StopInhibitminus Goescitalopram. Statistical maps were thresholded at po0.05(uncorrected) with cluster sizes of 5 or more voxels for this contrast.

3. Results

3.1. Behavioural results

On the behavioural level the standard parameters of thestopchange paradigm were determined: mean go-RT for correctly answered go-trials;

mean change-RT for correctly answered change-trials;

mean Stop-Respond-RT, i.e., the response time in stop-trials for which the participant did not stop; mean change-Respond-RT, i.e., the response speed inchange-trials in which the participant did not change; mean stop-SOA;

mean change-SOA;

SSRT, i.e., the time needed to inhibit an already initiatedmotor response in stop-trials; CSRT, i.e., the time needed to inhibit an already initiatedresponse in change-trials.

Means7s.d. for GoRT were 4657168 ms for placebo and400764 ms for escitalopram. Means7s.d. for SSRT are230754 ms for placebo and 247745 ms for escitalopram.For ChangeRT means7s.d. are 638786 ms with placebo and653794 ms with escitalopram. StopRespond revealed 381735 ms with escitalopram and 4327149 ms with placebo.

Analysis of variance revealed no significant differencesfor the main effect of substance for GoRT (F=2.2, df=1,p=0.16), SSRT (F=1.1, df=1, p=0.32), ChangeRT (F=0.8,df=1, p=0.36) and StopRespond (F=1.8, df=1, p=0.19),which means that results with escitalopram did not differsignificantly from placebo.

4. fMRI results

4.1. Response inhibition (StopInhibit)

Results of brain activation during response inhibition arerepresented in Table 1. Successful response inhibition for

http:/www.fil.ion.ucl.ac.uk/spm/http:/www.fil.ion.ucl.ac.uk/spm/https://www2.bc.edu/~slotnics/scripts/cluster_threshold_beta.mhttps://www2.bc.edu/~slotnics/scripts/cluster_threshold_beta.m

Table 1 Brain activations during successful and failed response inhibition.

Region BA Hemisphere x y z Z-score

1. Successful response inhibition(a) Escitalopram4placeboCingulate gyrus 31 Left 20 57 25 3.48Parietal lobe precuneus 7 Left 16 68 29 3.08Medial frontal gyrus 10/11 Right 8 42 12 3.43Middle temporal gyrus 21 Left 48 8 13 3.32Anterior cingulate 25 Left 4 7 10 3.24Superior/middle frontal gyrus 8 Right 24 14 40 3.15Cingulate gyrus 24 Right 16 1 48 3.14

(b) Placebo4escitalopramSuperior frontal gyrus 8 Right 8 38 53 3.02Superior frontal gyrus 8 Left 4 42 53 2.95

2. Failed response inhibition(a) Escitalopram4placeboPostcentral gyrus 3 Right 51 17 56 4.28Parahippocampal gyrus 35 Left 24 24 22 3.86Cingulate gyrus 24/32 Right 16 17 25 3.81Middle temporal gyrus 21 Right 48 2 34 3.78Middle/superior temporal gyrus 19/22 Left 32 58 10 3.73Caudate head Left 8 19 4 3.68Precentral gyrus 4 Right 20 21 49 3.57Superior temporal gyrus 21/38 Right 48 3 10 3.36Medial frontal gyrus 25 Right 12 7 14 3.11Lentiform nucleus, Putamen Right 16 7 7 2.72Putamen Right 16 7 7 2.72Middle temporal gyrus 21 Left 51 5 13 3.09Fusiforme gyrus 37 Left 48 51 11 2.87

Statistical maps were thresholded at po0.05 corrected across the whole brain. No increased activation in the placebo versusescitalopram was found for failed response inhibition.

8355-HT in stopping and changing behaviourStopInhibitescitalopram4StopInhibitplacebo was associated withsignificantly increased activation (po0.05, corrected acrossthe whole brain) in right orbito frontal cortex (OFC) (BA 10/11, see Figure 2), as well as right middle frontal gyrus (BA 8)and right cingulate (BA 24). Left lateralized activation wasseen in cingulate (BA 31), anterior cingulate (BA 25),precuneus (BA 7) and auditory cortex (BA 21). Althoughactivation was bilaterally located, the focus was right-lateralized. In the contrary condition, response inhibitionfor StopInhibit placebo4StopInhibit escitalopram activationswere found bilaterally in the superior frontal gyrus (BA 8).

4.2. Failed response inhibition (StopRespond)

Results of brain activation during failed response inhibitionare represented in Table 1. StopRespond escitalopram4StopRespond placebo significantly activated (po0.05 cor-rected across the whole brain) right somatosensory cortex(BA 3), right primary motor cortex (BA 4), right cingulate(BA 24/32), right middle and superior temporal gyrus (BA21, BA 38), right medial frontal gyrus (BA 25) as well as rightputamen. Left-hemispherical activation was observed in theparahippocampal gyrus, caudate head and in the temporalcortex (BA 37). Left auditory cortex (BA 19, BA 22) was alsoactivated. Brain activations are demonstrated in Figure 3.There were no activations found for the reverse conditionplacebo4escitalopram.

4.3. Response re-engagement (ChangeRT)

ChangeRT-trials for ChangeRTescitalopram4ChangeRTplaceboshow enhanced activations in left middle temporal gyrus(BA 21), right inferior frontal gyrus (BA 45/13), right insula(BA 13) and right precuneus (BA 7). The reverse conditionChangeRTplacebo4ChangeRTescitalopram revealed only activa-tion in left middle frontal gyrus (BA 11).

4.4. Failed response re-engagement(ChangeRespond)

ChangeRespond trials for the ChangeRespondescitalopram4ChangeRespondplacebo only show enhanced activations in rightinsula (BA13). The reverse condition ChangeRespondplacebo4ChangeRespondescitalopram revealed activation in right fusiformgyrus (BA 37), right inferior frontal gyrus (BA 47), right and leftparahippocampal gyrus (BA 19/35), left hippocampus, rightsuperior frontal gyrus (BA 8), right and left precentral gyrus

Figure 2 Enhanced brain activation in BA 10/11 during response inhibition with escitalopram.

Figure 3 Brain activation during failed inhibition for escitalo-pram4placebo (po0.05 corrected across the whole brain).

B. Drueke et al.836(BA 4/6/44), right insula (BA 13/44), left precuneus (BA 7/19)as well as left putamen, substantia nigra and lateral globuspallidum.

4.5. Go-trials

Brain activations for go-trials are listed in Table 2. Enhancedactivation in go-trials for Goescitalopram4Goplacebo was right-lateralized (po0.05 corrected across the whole brain):insula (BA 13), anterior and posterior cingulate (BA 30,BA 32), superior temporal gyrus (BA 38, BA 13), postcentralgyrus (BA 1) and in the middle frontal gyrus (BA 9). Left-lateralized activations were located in the subthalamicnucleus and middle temporal gyrus (BA 21). Brain activa-tions during go-trials for escitalopram4placebo are shownin Figure 3. Go-trials for Goplacebo4Goescitalopram were alsoassociated with a right-lateralized network of brain regionsin the middle frontal gyrus (BA 10, BA 11, BA 6), medialfrontal cortex including supplementary motor cortex andprecentral gyrus (BA 6), superior frontal gyrus (BA 8, BA 10),caudate head, precuneus (BA 7) and middle temporal gyrus(BA 21). Left medial and middle frontal cortex (BA 6) as wellas superior frontal gyrus (BA 10) also showed activation.

4.6. Response inhibition minus Go

Brain activations for StopInhibit minus Goescitalopram4StopInhibit minus Goplacebo are listed in Table 2 and

Table 2 Brain activations for the contrasts StopInhibit minus Go.

Region BA Hemisphere x y z Z-score

1. StopInhibit minus Go(a) Escitalopram4placeboParietal lobe precuneus 7 Left 20 68 48 4.06Superior parietal lobe 7 Right 28 63 51 3.23Insula 13 Left 40 8 14 3.22Claustrum 13 Left 28 9 18 3.10Posterior cingulate 29 Right 12 42 17 2.99Thalamus Right 8 35 9 2.74Thalamus Right 28 31 2 2.98Lentiform nucleus Putamen Left 24 7 19 2.98Caudate Left 20 10 26 2.75Insula 13 Left 32 6 26 2.66Middle frontal gyrus 6 Right 24 6 41 2.87Frontal lobe sub-gyral 6 Right 20 1 55 2.5Midbrain red nucleus Right 4 27 5 2.73Claustrum Left 24 20 14 2.72

(b) Placebo4escitalopramCingulate gyrus 31 Left 20 30 31 3.08Thalamus right 8 4 4 2.93

Statistical maps were thresholded at po0.01 uncorrected.

Figure 4 Enhanced brain activations by escitalopram com-pared to placebo for the contrast StopInhibit minus Go(po0.01, uncorrected).

8375-HT in stopping and changing behaviourpresented in Figure 4. The contrast StopInhibit minusGoescitalopram4StopInhibit minus Goplacebo (po0.05 uncor-rected) was associated with significant activation in bilat-eral parietal lobe (BA 7), left hemispheric insula (BA 13),claustrum (BA 13), lentiform nucleus and caudate. Activa-tion was also found in right-lateralized posterior cingulate(BA 29), thalamus, middle frontal gyrus (BA 6) as well as rednucleus. The contrast StopInhibit minus Goplacebo4StopInhi-bit minus Goescitalopram showed activations in right thalamusand left cingulate (BA 31).5. Discussion

The present study investigated the acute effects of phar-macological modulation of 5-HT on brain activation duringresponse inhibition and re-engagement in healthy humanvolunteers. 14 men performed a stopchange task after oraladministration of the SSRI escitalopram. Escitalopram didnot affect inhibitory performance, but was associated withalterations in brain activation patterns compared to pla-cebo. Escitalopram enhanced brain activation in rightprefrontal cortex, including right OFC, in right supplemen-tary/premotor and bilateral cingulate cortex, as well as insubcortical regions during response inhibition. In the condi-tion of failed stopping, escitalopram activated a widespreadnetwork of brain regions, including anterior cingulate, rightparietal cortex, right OFC, areas in right temporal cortexand subcortical regions. Response re-engagement led toactivation in right inferior frontal gyrus and precuneus andleft middle temporal gyrus. During the go-process, escita-lopram increased activation in numerous regions such asright anterior and posterior cingulate and in right prefron-tal, parietal and temporal cortex as well as in subcorticalareas.

The lack of effect of escitalopram on task performance isin line with various studies that found no effect of 5-HTmodulation on response inhibition using either a stop signal

B. Drueke et al.838task (Bari et al., 2009; Clark et al., 2005; Chamberlainet al., 2006) or a go/no-go task (Del-Ben et al., 2005; Everset al., 2006, Rubia et al., 2005). The stop signal task andgo/no-go tasks differ in design of paradigm and, as a result,in determination of inhibitory performance. However, thelack of effects of 5-HT on task performance seems to besteady across paradigms. These studies also used differentmethods of intervention in the serotonergic system. Thefact that none succeeded to demonstrate an effect oninhibitory performance implies that modulation of theserotonergic system has no effect on response inhibition,at least not as reflected in task performance. This is alsosupported by the results from Drueke et al. (2010), whoinvestigated 36 healthy human participants with the StopChange paradigm and found neither an effect on stoppingnor on changing behaviour after a serotonergic modulationwith 10 mg escitalopram.

The focus of interest was the role of escitalopram. Forreasons of control, we also reviewed fMRI data of responseinhibition in the placebo condition. During the stop-process,we found activation that has been related consistently toresponse inhibition, including right lateral IFC, right DLPFC,the right supplementary motor area and temporal regions.Multiple fMRI studies (Aron et al., 2003; Aron and Poldrack,2006; Rubia et al., 2003) also showed activation in theregions mentioned above as relevant. As we were able toreproduce these crucial stopping areas, we assume that ourstudy measured response inhibition and that alterations inbrain activations were induced by escitalopram.

During the change process, we also found activationrelated to response re-engagement, including the inferiorand medial frontal cortex.5.1. Response inhibition

Our findings of enhanced brain activations in right prefron-tal cortex, including right OFC, with escitalopram duringresponse inhibition are consistent with the results of pre-vious research (Del-Ben et al., 2005; Rubia et al., 2005).The OFC has been implicated in mediation of inhibitorycontrol (Rubia et al., 2005). Eagle et al. (2008) alsosuggested that the OFC is involved in the stopping processby mediating stopping via an orbitofrontaldorsomedialstria-tum-pathway. However, there are also differences to theresults of Del Ben et al. (2005), who found increasedactivation of right lateral OFC (BA 47) and dorsolateral PFC(BA 9), whereas attenuation was seen in OFC (BA 11), an areawhich showed increased activation in our study. Neverthe-less, Del Ben et al. saw medial OFC activation related toresponse inhibition though it was not enhanced by citalo-pram, which might be due to task- and design-relateddifferences. Del Ben et al. used a go/no-go task, whereaswe used a stop signal paradigm. The go/no-go task does notconsist of a stop signal delay and consequently measuresprepotent responses, i.e., action restraint. The stop changetask is more precise in measuring response inhibition ofongoing processes, i.e., action cancellation. Because of thedifferent paradigms used in both studies, different contrastswere designed. Consequently, one can expect differingresults. Del Ben et al. only reported a more complexno-go minus go contrast, whereas the contrastStopInhibitescitalopram4StopInhibitplacebo that we generatedwas not investigated in their study. We only saw increasedactivation in right medial OFC in this condition. However, inour similar contrast StopInhibit minus Goescitalopram4StopInhi-bit minus Goplacebo, we found activation patterns particularlyin subcortical regions, which is consistent with previousfindings relating subcortical regions to response inhibition(Aron and Poldrack, 2006; Li et al., 2008; Del Ben et al.,2005; Kelly et al., 2004). The OFC is strongly connected tosubcortical regions (Alexander and Crutcher, 1990; Cools et al.,2008). The study of Del Ben et al. (2005) and our study alsodiffer in the substance used for pharmacological modulationand its administration route: intravenous administration of theSSRI citalopram used by Del Ben et al. versus the oraladministration of escitalopram we used. Although escitalo-pram is the pure therapeutically active enantiomer of theracemate citalopram, escitalopram has a greater selectivityfor the human 5-HT transporter. So it might be possible thatcitalopram produces more unspecific effects of activation.Another reason for differing results might be the differentways of drug administration (oral versus intravenous). We usedan acute administration of escitalopram, which producesextended 5-HT availability in the synaptic cleft by blockingthe 5-HT transporter. However, Rubia et al. (2005) found asimilar activation pattern to that of Del Ben et al. (2005).Rubia et al. likewise used a go/no-go task but used acutetryptophan depletion for 5-HT modulation. This method inter-feres in the serotonergic system by producing a decrease ofcentral 5-HT via decreased tryptophan availability in the brain.This reduction is mild and only transient (Cools et al., 2008).Evers et al. (2006) saw no effect of acute tryptophan depletionon brain activation in a go/no-go task and ascribed this todifferences in paradigm and design. However, our findings arepartly consistent with a study by Anderson et al. (2002) usingm-chlorophenylpiperazine (mCPP) for 5-HT modulation. Theydemonstrated increased brain activation associated with mCPPin a no-go minus go contrast in right ventral lateral PFC, butalso in the caudate, a region showing activation enhancementin our study in the corresponding condition StopInhibit minusGoescitalopram4StopInhibit minus Goplacebo.

The caudate is reached by ascending serotonergic projec-tions from the raphe nuclei, which could be the reason forincreased activation in this region with escitalopram (Feldmanet al., 1997). However, recent findings suggest that thecaudate plays a role in the controlled execution of movement.Li et al. (2008) found greater activity in the caudate inassociation with short SSRT versus long SSRT, and this was alsocorrelated with medial prefrontal activity. We would alsosuggest that more research is needed to specify the definiterole of the caudate. Consistent with findings in previousliterature we found activation in insula and thalamus (Aronand Poldrack, 2006; Del-Ben et al., 2005; Kelly et al., 2004).The thalamus as a component of the fronto-striato-thalamic-pathway is supposed to gate and facilitate cortically initiatedmovements (Alexander and Crutcher, 1990). However, Li et al.(2008) suggested that thalamus and basal ganglia might delaySSRT. The insula has been implicated in response selectionprocesses under conditions of reduced preparation (Kelly et al.,2004). But Anderson et al. (2002) found attenuations in rightinsula and thalamus, whereas we located increased activationsin right thalamus and left insula. This might be likewise due totask- and substance-related differences as discussed before.

8395-HT in stopping and changing behaviourmCPP is a non-selective 5-HT receptor ligand binding todiverse 5-HT receptors and even 2-adrenoceptors, withgreatest affinity for 5-HT2C and possibly 5-HT1B. It is there-fore an unspecific substance for modulation of 5-HT, mostnotably in comparison to escitalopram. For comparison ofboth substances one also has to keep in mind that mCPP isan agonist. Whereas agonists bind to a certain receptor andalter the receptors' activity, antagonists simply bind thereceptor without producing a response itself though block-ing agonist-mediated responses. Nevertheless, Vllm et al.(2006) also found enhanced activation in right lateral OFCand left insula, though the latter region did not remainstatistically significant after small volume correction.

Vllm et al. used mirtazapine, an antidepressant sub-stance blocking presynaptic 2-adrenic autoreceptor, whichleads to increased noradrenaline (NA) release and enhancesserotonergic transmission. Since mirtazapine blocks 5-HT2Creceptors as well, its main effects on 5-HT are mediated viapostsynaptic agonistic action on 5-HT1A receptors. Althoughthe authors related alterations of brain activations to theserotonergic effects of mirtazapine, most likely the adre-nergic receptor effects also influenced the results. Anotherinteresting aspect is that we found enhanced activations inBA 24 and BA 8, areas that have been related to errordetection and conflict monitoring and therefore have beensuggested to reflect response conflict in a go/no-go task(Kelly et al., 2004; Rubia et al., 2003). This might implicatea serotonergic influence not only on pure inhibitory controlbut also on other components belonging to response inhibi-tion. Taken together, all results are based on slight differ-ences but indicate a serotonergic involvement in responseinhibition as all contrasts were able to be corrected formultiple comparisons via Monte Carlo simulation.5.2. Response re-engagement

For response re-engagement, we found higher activity inmedial temporal and inferior frontal areas as well asin precuneus and right insula. The stronger activation intemporal gyrus during changing with escitalopram might bedue to an increase in selective attention processes, anassumption which is supported by the results from Druekeet al. (2009) and Rubia et al. (2005). An activation of rightinferior PFC indicates also a serial order of response inhibi-tion and re-engagement as it was proposed by Verbruggenet al. (2008). Boehler et al. (2010) associated insula activitywith correct information processing during task performance,which might be a possible explanation for insula activityduring response re-engagement with escitalopram. Takenthese points together, a 5-HT modulation of response re-engagement seems to emphasize inhibitory and attentionalcomponents as well as structures which are responsible forcorrect information processing. During failed response reen-gagement with escitalopram, we found strongest activationin insula which might also be an indicator for the involvedinhibition processes during failed changing. Additionally,insula activation is typically found during error processing(Wrase et al., 2007; Li et al., 2008). Li hypothesized theemotional part of error processing to be responsible for insulaactivation which might be in line with the fact that seroto-nergic modulations are used for the treatment of affectivedisorders (Burke et al., 2002). All these assumptions onneural level remain speculative as the results of our studyfor response re-engagement could only be understood asexplanatory changes with escitalopram which is due to thefact that these contrasts could not be corrected for multiplecomparisons with Monte Carlo simulation.

Finally, some limitations should be discussed. Methodolo-gical concerns come from the oral dosage of 10 mg escita-lopram which is at least the very lowest dosage for thetreatment of major depression. It might be possible thathigher doses would lead to greater effects on behavioural aswell as on neural level. We chose the dosage of 10 mg as thisdosage was well tolerated in other studies from our group(Drueke et al., 2009, 2010). Another consideration concernsthe timing of testing that was conducted after a 200-minperiod according to the pharmacokinetic profile of escita-lopram (Aronson and Delgado, 2004), but it might bepossible that either the maximum plasma concentrationvaries across the participants or that a correlation betweenbehavioural/neural effects and plasma concentration is notexisting. Another possible explanation for the slight differ-ences might be the possibility that the StopChange para-digm is relatively robust against serotonergic modulationsand that other tasks are more sensitive for changes in thisneurotransmitter system.

To sum up, several studies provide evidence for 5-HT-mediated alteration of brain activation associated withresponse inhibition. Enhancement of activation in rightlateral OFC was primarily found across substances, incontrast to our own results. These studies used a go/no-gotask investigating action restraint. However, all studiesincluding our findings support a 5-HT related enhancementin right PFC, particularly in right OFC in general. It remainsunclear why our results implicate an involvement of medialOFC. The medial PFC is strongly innervated by serotonergicprojections from the rostral raphe nuclei (for review,Hornung, 2003; Feldman et al., 1997). As escitalopram ismore selective for the 5-HT transporter than other sub-stances used so far, its effects might be responsible fordislocation from lateral to medial regions in the OFC. Ourfindings of increased activation of subcortical regions con-firm suggestions of their involvement in response inhibition.The basal ganglia have been implicated in response inhibi-tion in previous research (Rieger et al., 2003; Gauggelet al., 2004; Eagle et al., 2008; Aron and Poldrack, 2006).Our results are consistent with an orbitofrontal-striatal-circuitry that has been previously implicated in stopping(Eagle et al., 2008; Chamberlain et al., 2006).

Furthermore, we found a 5-HT related modulation, mostlikely because of the numerous serotonergic projectionsascending from the raphe nuclei to the basal ganglia(Feldman et al., 1997). Altogether, our results clearly supportan influence of 5-HT on response inhibition. We found 5-HTmodulated activation of brain regions that have been con-sistently associated with response inhibition, providing thefirst evidence for an influence of 5-HT on action cancellation.Role of the funding source

This study was supported in part by a research grant fromH. Lundbeck A/S (Copenhagen, Denmark). Lundbeck was

B. Drueke et al.840not responsible for creation of the study, the choice ofinvestigators, the control of allocation schedule, the con-duct of the trial, the collection and monitoring of data, theanalysis and interpretation, nor for writing the manuscript.

Contributors

Authors B. Drueke, S. Gauggel and G. Grnder designed the studyand wrote the protocol. Authors S. Schlaegel, A. Seifert andO. Moeller managed the literature searches and analyses. AuthorsB. Drueke, M. Boecker, S. Schlaegel and A. Seifert undertook thestatistical and fMRI analysis, and author B. Drueke wrote the firstdraft of the manuscript. All authors contributed to and haveapproved the final manuscript.

Conflict of interest

Dr. Grnder has served as a consultant for Bristol-Myers Squibb(New York, NY), Cheplapharm (Greifswald, Germany), Eli Lilly(Indianapolis, Ind), Forest Laboratories (New York, NY, USA),Lundbeck (Copenhagen, Denmark), Otsuka (Rockville, Md.), andServier (Paris, France). He has served on the speakers' bureau ofBristol-Myers Squibb, Eli Lilly, Otsuka, Roche (Basel, Switzerland),and Servier (Paris, France). He has received grant support fromAlkermes, Bristol-Myers Squibb, Eli Lilly, and Johnson & Johnson. Heis the co-founder of Pharma-ImageMolecular Imaging TechnologiesGmbH, Dsseldorf.

Dr. B. Drueke, Dr. S. Schlaegel, A. Seifert, Dr. O. Moeller,Dr. S. Gauggel and Dr. M. Boecker declare no conflicts of interest.

Acknowledgements

We thank Dr. Michaela Siebert and Dr. Mario Staedtgen for proof-reading of the manuscript.

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The role of 5-HT in response inhibition and re-engagementIntroductionExperimental proceduresParticipantsDesignDrugsExperimental procedureMRI scanning procedureStopchange paradigmBehavioural data analysisImaging data analysis

ResultsBehavioural results

fMRI resultsResponse inhibition (StopInhibit)Failed response inhibition (StopRespond)Response re-engagement (ChangeRT)Failed response re-engagement (ChangeRespond)Go-trialsResponse inhibition minus Go

DiscussionResponse inhibitionResponse re-engagement

Role of the funding sourceContributorsConflict of interestAcknowledgementsReferences