Piccolo genotype modulates neural correlates of emotion processing but not executive functioning

Abstract

Major depressive disorder (MDD) is characterized by affective symptoms and cognitive impairments, which have been associated with changes in limbic and prefrontal activity as well as with monoaminergic neurotransmission. A genome-wide association study implicated the polymorphism rs2522833 in the piccolo (PCLO) gene--involved in monoaminergic neurotransmission--as a risk factor for MDD. However, the role of the PCLO risk allele in emotion processing and executive function or its effect on their neural substrate has never been studied. We used functional magnetic resonance imaging (fMRI) to investigate PCLO risk allele carriers vs noncarriers during an emotional face processing task and a visuospatial planning task in 159 current MDD patients and healthy controls. In PCLO risk allele carriers, we found increased activity in the left amygdala during processing of angry and sad faces compared with noncarriers, independent of psychopathological status. During processing of fearful faces, the PCLO risk allele was associated with increased amygdala activation in MDD patients only. During the visuospatial planning task, we found no genotype effect on performance or on BOLD signal in our predefined areas as a function of increasing task load. The PCLO risk allele was found to be specifically associated with altered emotion processing, but not with executive dysfunction. Moreover, the PCLO risk allele appears to modulate amygdala function during fearful facial processing in MDD and may constitute a possible link between genotype and susceptibility for depression via altered processing of fearful stimuli. The current results may therefore aid in better understanding underlying neurobiological mechanisms in MDD.

Citation: Transl Psychiatry (2012) 2, e99, doi:10.1038/tp.2012.29

& 2012 Macmillan Publishers Limited All rights reserved 2158-3188/12 http://www.nature.com/tp

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Piccolo genotype modulates neural correlates of emotion processing but not executive functioning

S Woudstra1,2,3,4,5, Z Bochdanovits2,5, M-J van Tol6,7, DJ Veltman1,5, FG Zitman3, MA van Buchem4,8, NJ van der Wee3,4, EM Opmeer6, LR Demenescu6,9, A Aleman6,10, BW Penninx1,3,5,6 and WJ Hoogendijk1,5,11

Major depressive disorder (MDD) is characterized by affective symptoms and cognitive impairments, which have been associated with changes in limbic and prefrontal activity as well as with monoaminergic neurotransmission. A genome-wide association study implicated the polymorphism rs2522833 in the piccolo (PCLO) geneinvolved in monoaminergic neurotransmissionas a risk factor for MDD. However, the role of the PCLO risk allele in emotion processing and executive function or its effect on their neural substrate has never been studied. We used functional magnetic resonance imaging (fMRI) to investigate PCLO risk allele carriers vs noncarriers during an emotional face processing task and a visuospatial planning task in 159 current MDD patients and healthy controls. In PCLO risk allele carriers, we found increased activity in the left amygdala during processing of angry and sad faces compared with noncarriers, independent of psychopathological status. During processing of fearful faces, the PCLO risk allele was associated with increased amygdala activation in MDD patients only. During the visuospatial planning task, we found no genotype effect on performance or on BOLD signal in our predened areas as a function of increasing task load. The PCLO risk allele was found to be specically associated with altered emotion processing, but not with executive dysfunction. Moreover, the PCLO risk allele appears to modulate amygdala function during fearful facial processing in MDD and may constitute a possible link between genotype and susceptibility for depression via altered processing of fearful stimuli. The current results may therefore aid in better understanding underlying neurobiological mechanisms in MDD. Translational Psychiatry (2012) 2, e99; doi:http://dx.doi.org/10.1038/tp.2012.29

Web End =10.1038/tp.2012.29 ; published online 3 April 2012

Introduction

Major depressive disorder (MDD) is a common, multifactorial disorder and is one of the leading causes of years lost due to disability.1,2 Its core symptoms are affective, manifesting themselves in depressed mood accompanied by low self-esteem and loss of interest in normally enjoyable activities. Other MDD symptoms include cognitive impairments, such as concentration and planning problems.3 The presence and severity of both affective and cognitive symptoms can be evaluated by neuropsychological assessments,4 and may be reected in altered regional brain activity as measured using fMRI.57

Twin studies have shown that up to 40% of MDD is genetically determined.8 Much of the candidate gene literature has focused on genes from the monoaminergic neuro-transmitter system, such as the serotonin transporter, monoamine oxidase A and tryptophan hydroxylase 2.914

A recent study by our consortium demonstrated that candidate genetic association studies are not well replicated,15 which suggests that a hypothesis-free approach is more useful to identify possible genetic variants that

contribute to MDD. Previously, we conducted a genome-wide association study (GWAS) in MDD using B500 000 single-nucleotide polymorphisms (SNPs), showing one SNP (rs2522833) located at position 82453708 (hapmap genome build 37.1) in the piccolo gene (PCLO) in particular to be of interest in the genetic model for MDD.16 This association was replicated in some studies,1618 but not in others.19,20 The

rs2522833 SNP changes the hydrophilic, uncharged amino acid serine to the charged amino acid alanine in the calcium-binding C2A domain of PCLO and may affect protein stability.21 The PCLO protein is localized at the cytomatrix of the presynaptic active zone and is important in monoaminergic neurotransmission in the brain.16,22 Recently, Schuhmacher et al.23 showed that the PCLO SNP rs2522833 is associated with antidepressant treatment response, supporting the involvement of PCLO in MDD. The mechanism through which this PCLO risk allele contributes to MDD is not known, but its primary role in monoaminergic neurotrans-mission suggests that it may affect activity of brain circuitry involved in emotional information processing and/or executive functioning in MDD.24

1Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Medical Genomics, VU University Medical Center, Amsterdam, The Netherlands; 3Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands; 4Leiden Institute for Brain and Cognition, Leiden University, Leiden, The Netherlands; 5Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands; 6BCN Neuroimaging Center and Department of Neuroscience, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 7Leibniz Institute for Neurobiology, Otto von Guericke University, Magdeburg, Germany; 8Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands; 9Department of Psychiatry and Psychotherapy, RWTH Aachen University, Aachen, Germany; 10Department of Psychology, University of Groningen, Groningen, The Netherlands and 11Department of Psychiatry, Erasmus MC, Rotterdam, The NetherlandsCorrespondence: S Woudstra, Department of Psychiatry, MF-A422, VU University Medical Center, PO Box 7057, Amsterdam 1007 MB, The Netherlands.

E-mail: mailto:[email protected]

Web End [email protected] Keywords: emotion processing; executive function; genome-wide association; major depressive disorder; neuroimaging genetics; PCLO

Received 20 December 2011; revised 15 February 2012; accepted 3 March 2012

Piccolo genotype and emotion processing S Woudstra et al

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Abnormal emotion processing of, for example, facial stimuli is one of the intermediate phenotypes that can be considered closer to the neurobiological substrate of MDD than the clinical diagnosis itself.6 Identication of emotional facial expressions is reportedly impaired in MDD,25 and functional imaging studies have shown aberrant activity of emotional face processing related areas, including the amygdala, striatum, insula, parahippocampal area and dorsolateral prefrontal cortex (PFC), using an emotional faces task in healthy controls (HC).26 It is thought that serotonin has an important role in emotional face processing and dysregulation of the serotonergic system is associated with altered amygdala activity as well as the occurrence of MDD.9,27,28

Another intermediate phenotype for MDD may be executive dysfunction, including impaired working memory and planning/problem solving. Previous studies in MDD have shown abnormal task performance and increased activity in task-related brain areas,7,29,30 using the Tower of London visuo-

spatial planning task (ToL) and the n-back working memory

task.3134 Apart from the PFC, other areas involved in

planning include the striatum, and a visuospatial processing system, including the precuneus and inferior parietal cortex.29,34 The association of executive function with monoaminergic neurotransmission is thought to be bidirectional: PFC function is shaped by monoaminergic neurotransmitters, but ascending modulatory monoaminergic systems may also be affected by the PFC.24 Studies suggests that the PFC is able to recruit monoamine systems in specic circumstances, for example, to optimize the coping strategy for stress.35,36

Over the last few years, imaging genetics has shown to be a powerful tool in unraveling neurobiological pathways in various psychiatric disorders. However, to date, studies in MDD have been rare,10,37,38 and the role of the PCLO risk allele

on emotional information processing and executive function has never been studied. Given that PCLO is associated with MDD, we hypothesized that the PCLO risk allelerelative to the nonrisk alleleis associated with similar changes in activity in emotional processing and in executive function related areas as described for MDD. In addition, we aimed to investigate the relationship of PCLO with current psycho-pathology during emotional face processing and executive function.

Materials and methods

Subjects. A sample of 159 subjects participating in the Netherlands Study for Depression and Anxiety (NESDA) was selected to take part in this genetic neuroimaging study. Details of the NESDA study are extensively described elsewhere.39 Genotype data were collected through the genetic association information network (GAIN) study for MDD, in which 1862 cases and 1857 controls were genotyped.40

Magnetic Resonance Imaging (MRI) data were available from the MRI NESDA study, in which 301 subjects aged between 18 and 57 years participated.41 During a 1-h protocol, subjects underwent MR imaging while performing different tasks, including the ToL paradigm, the emotional faces paradigm and a verbal memory task, structural imaging and resting state imaging.

Exclusion criteria were: presence of MRI contraindications, DSM-IV axis I disorder other than MDD, Panic Disorder or social anxiety disorder (except generalized anxiety disorder) lifetime, or any DSM-IV disorder (for HC), dependence or recent abuse of alcohol and/or drugs, hypertension, major internal and/or neurological disorders and use of psychotropic medication (other than stable use of a selective serotonin reuptake inhibitor (SSRI) or incidental use of benzodiaze-pines). To assess depressive symptom characteristics and severity scores, the Inventory of Depressive Symptomatology42 and the Montgomerysberg Depression Rating Scale,43 were used. For the present report, we included only subjects that met the 6-month recent DSM-IV criteria for current MDD, or no lifetime DSM-IV diagnosis. Owing to missing genotype data, technical problems during scanning and/or insufcient task performance, our nal sample for this study consisted of 126 subjects for the emotional face processing study and 159 subjects for the executive function study (see Supplementary Table 1 for an overview). All participants provided written informed consent and the study was approved by the Ethics Committees at the VU University Medical Center, Amsterdam, the Leiden University Medical Center and the University Medical Center Groningen.

Demographics. The 126 subjects in the emotional faces study included 91 PCLO risk allele carriers (PCLO ; 25

homozygous and 66 heterozygous for the C risk allele) and 35 homozygous A nonrisk allele carriers (PCLO ). The ratio

of PCLO and PCLO in the executive function study did

not signicantly differ (110 PCLO ; 33 homozygous and 77

heterozygous for the C risk allele, and 49 homozygous A nonrisk allele carriers; PCLO ) from the ratio in the

emotional faces study (w2 [df 1] 0.31; P40.05). For the

analyses, we formed two groups: one group consisting of subjects carrying the risk allele, and one group consisting of subjects not carrying the risk allele. From this point forward, we will refer to these groups as PCLO and PCLO ,

respectively. PCLO groups did not differ with regard to age, gender, education, MDD/HC ratio, depression severity or SSRI use/duration (see Table 1). Thirty-four individuals were using SSRIs, and 15 individuals were using nonpsychotropic medication other than SSRIs. All subjects reported Western European ancestry.

Genotyping. Genotyping for the genetic association information network study was performed by Perlegen and is described in detail elsewhere.16 Observed genotypes in our sample did not deviate from HardyWeinberg equilibrium (emotional face processing paradigm): w2 [df 2] 0.37;

P40.05; CC: AC: AA 35:66:25; P40.05; executive fun

ctioning paradigm: w2 [df 2] 0.07; CC: AC: AA 33:77:49;

P40.05; http://www.oege.org/software/hardy-weinberg.html

Web End =http://www.oege.org/software/hardy-weinberg.html ).

Emotional information processing paradigm. An emotional faces task, based on the event-related fMRI paradigm used by Wolfensberger et al.44 was used in the present study and has been extensively described elsewhere.5 Briey, 24 stimuliselected from the Karolinska Directed Emotional

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Table 1 Demographic characteristics of total sample

Characteristics Tower of London planning task Emotional face paradigm

Genotype GenotypeTotal PCLO+ PCLO v2 P-value Total PCLO+ PCLO v2 P-value

Number of subjects 159 110 49 126 90 36Gender (%female) 64.1 64.5 63.3 0.024 40.05 61.9 61.1 63.8 0.08 40.05

Age (mean years s.d.) 38.17 (10.7) 37.9 (10.2) 38.8 (11.9) 40.05 38.1 (10.3) 38.0 (10.2) 38.4 (10.7) 40.05 Education (mean years s.d.) 12.5 (3.1) 12.8 (3.3) 11.9 (2.8) 40.05 12.6 (3.1) 12.8 (3.2) 12.1 (2.8) 40.05 Scan center (A/L/G) 42/62/55 28/46/36 14/16/19 1.21 40.05 24/54/48 19/40/31 5/14/17 1.99 40.05

Diagnosis (MDD/HC) 118/41 84/26 35/15 0.86 40.05 96/30 69/21 27/9 0.04 40.05 IDS (mean score s.d.) 18.90 (13.5) 20.06 (13.2) 16.27 (14.0) 40.05 19.46 (13.9) 20.05 (13.6) 17.91 (14.8) 40.05

MADRS (mean score s.d.) 11.89 (10.4) 13.01 (10.3) 9.39 (10.1) 0.042 12.78 (10.4) 13.53 (10.3) 10.80 (10.4) 40.05

Duration of SSRI use (months s.d.)

22.91 (37.6) 27.77 (38.8) 18.80 (36.5) 40.05 23 (36.9) 24.75 (37.7) 18.8 (36.5) 40.05

SSRI use (no/yes) 119/40 82/28 37/12 40.05 92/34 67/24 25/10 40.05

Abbreviations: HC, healthy controls; IDS, inventory of depressive symptomatology; MADRS, Montgomerysberg Depression Rating Scale; MDD, major depressive disorder; PCLO, piccolo; SSRI, selective serotonin reuptake inhibitor.

Faces System45were chosen for each of ve facial expressions (angry, fearful, sad, happy and neutral), comprising 12 female and 12 male faces.

Each particular face was not presented more than four times. The control condition (scrambled faces) was presented 80 times. While functional MRI data were collected, subjects were asked to indicate each faces gender using a button box. During the presentation of scrambled faces, participants had to press left or right buttons in conformity with the instruction presented on the screen, that is, an arrow pointing to the left or to the right. The reaction time (RT) was recorded and used as performance measure.

Executive function paradigm. An event-related parametric version of the ToL task was used to investigate the neural correlates of planning, an aspect of executive functioning.7,34

Briey, while functional MRI data were collected, subjects were asked to work out the minimum number of steps neededranging from one to ve movesto get from the start position to the goal position, which were both presented on a screen simultaneously. In the baseline condition, subjects were asked to count the number of yellow and blue beads. RT and proportion of correct answers were used as performance measures.

MRI data acquisition. The functional neuroimaging methods have been comprehensively reported elsewhere.5,7

In summary, T2*-weighted echo-planar images sensitive to the blood oxygenation leveldependent (BOLD) effect were acquired using similar Philips 3T MR systems (repetition time (TR) 2300 ms, echo time (TE) 30.0 ms (UMCG: 28.0 ms),

situated at different locations (Amsterdam, Leiden, and Groningen, the Netherlands). The echo-planar images volumes were acquired at 35 slices (UMCG: 39 slices), interleaved acquisition, 3 mm thickness. The matrix sizes were 96 96 voxels (UMCG: 64 64 voxels). The in-plane

resolution was 2.29 2.29 mm2 (UMCG: 3 3 mm2). The

images were acquired parallel to the anterior-posterior commissure plane. A T1-weighted anatomical MRI was also acquired for each subject (TR 9 ms, TE 3.5 ms, matrix

size 256 256, voxel size 1 1 1 mm3).

AnalysisPerformance. Faces paradigm: RT were analyzed with SPSS (SPSS, Chicago, IL, USA) by means of a repeated-measures analysis of covariance (ANCOVA), using RT per valence as dependent factor, group (PCLO , PCLO ) as

between subject factor and age, scan location, and years of education as covariates. Signicance for behavioral analyses was set at 5%, and post hoc paired tests were Bonferroni-corrected for multiple comparisons. Post hoc analyses were performed to test for interaction using a univariate general linear model.

ToL paradigm: performance data (accuracy and RT) were analyzed with SPSS (SPSS). Performance was analyzed by means of a repeated-measures ANCOVA, using the proportion correct scores and mean response times per trial type as dependent factors, group (PCLO , PCLO ) as between

subject factor, and age as a covariate. Signicance for behavioral analyses was set at 5%, and post hoc paired tests were Bonferroni-corrected for multiple comparisons.

Imaging data analysis. Image processing was performed using Statistical Parametric Mapping (SPM; SPM5http://www.fil.ion.ucl.ac.uk/spm/

Web End =http:// http://www.fil.ion.ucl.ac.uk/spm/

Web End =www.l.ion.ucl.ac.uk/spm/ ) software implemented in Matlab7.5.0 (The Matlab, Natick, MA, USA). Details of preprocessing and rst level single-subject analyses have been described elsewhere.5,7 Briey, following temporal and spatial preprocessing stimulus responses were modeled using delta functions convolved with a synthetic hemodynamic response function. For both paradigms, trial duration was also modeled.46 In the faces task, four contrast images were generated for each subject: angry4neutral, fearful4neutral, sad4neutral and happy4neutral. These contrasts ensured that visual input only differed in its emotional content and were entered in a second-level mixed effect analysis. For the ToL task, activation maps associated with increased task load were calculated per subject, and contrast images were likewise included in a second-level mixed-effect analysis.

Statistical association analysis. The amygdala and ventral striatum were determined a priori as regions of interest (ROI) for the emotional faces task. Positive effects of task across

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whole brain are reported at a threshold of Po0.05 familywise error corrected for multiple comparisons (PFWEo0.05). We

conducted an ANCOVA for the faces task with genotype as an independent factor, to test whether the PCLO risk allele is associated with altered activity in the amygdala during processing of negative faces. Age and scan location were used as covariates. A threshold of Po0.05 FWE corrected using the small volume correction (PFWE_SVCo0.05) procedure

implemented in SPM5 for the amygdala was considered signicant. For this small volume correction, we used the automated anatomical labeling atlas,47 implemented in the Wake Forest University Pick Atlas toolbox. To test whether the PCLO risk allele is associated with altered increased activity in the amygdala and ventral striatum during processing of positive faces, we conducted an ANCOVA with genotype as an independent factor. Age and scan location were used as covariates. A threshold of PFWE_SVCo0.05 (using the

automated anatomical labeling atlas, implemented in the Wake Forest University Pick Atlas toolbox) for amygdala and ventral striatum was considered signicant.

For the executive function task, regions activated with increasing task load were determined a priori as ROIs according to the areas described in van den Heuvel et al.34

(including dorsolateral PFC). Main effects of increasing task load are reported at a threshold of Po0.05 whole-brain FWE corrected for multiple comparisons. Next, an ANCOVA using genotype as independent factor was conducted, to test whether the PCLO risk allele is associated with altered brain activity associated with increasing task load. Age, gender, education and scan location were used as covariates. Owing to dorsolateral PFC size, a threshold of PFWE_SVCo0.05

(10 mm radius around the peak voxel that was found in the main effect of condition) was considered signicant.

We conducted post hoc analyses for both tasks to test whether interaction of genotype with current psychopathology occurred. A threshold of PFWE_SVCo0.05 (6 mm radius

sphere for amygdala and striatum and 10 mm radius sphere for cortical areas around the peak voxel) in the ROIs was considered signicant.

Results

Processing emotional facesPCLO genotype effect on task performance. We did not nd signicant genotype or genotype x valence effects on task performance (RT) during emotional face processing.

Additional genotype x diagnosis x valence effects on RT revealed interaction effects between PCLO genotype and MDD diagnosis for angry, fear and sad faces (RT_angry: PCLO*MDD; P 0.0595; RT_fear: PCLO*MDD; P 0.0493; RT_sad:

PCLO*MDD; P 0.0395; see Supplementary Table 3).

BOLD fMRI resultsMain effect of task during processing of faces. Processing angry faces compared with neutral faces was associated with robustly increased activity of bilateral amygdala as well as bilateral putamen, ventrolateral PFC, superior and middle temporal lobe and inferior parietal cortex. Processing fearful faces compared with neutral faces was similarly associated with increased activity of bilateral amygdala as

well as of bilateral parietal cortex, right temporal cortex, bilateral occipital cortex, bilateral amygdala, decreased activity of ventral striatum and right parahippocampal gyrus. Processing sad faces compared with neutral faces activated bilateral amygdala, striatum, insula, parahippocampal area and dorsolateral PFC.

For happy faces, we observed activation in bilateral amygdala and ventral striatum as well as in bilateral occipital cortex, inferior parietal cortex and posterior cingulate cortex.

PCLO genotype effect during processing of faces. We found signicantly increased activity in the left amygdala in the PCLO group, relative to the PCLO group, during process

ing of negative faces (angry vs neutral: (MNI coordinates x 21 y 0 z 15, Z 3.6, P

FWE_SVC

0.004, (Figure 1); fear vs neutral: MNI coordinates x 18 y 0 z 18,

Z 3.24, P

FWE_SVC

0.014, (Figure 2); and trendwise for sad vs neutral: MNI coordinates x 15 y 3 z 18, Z 2.6,

PFWE_SVC 0.067).

We did not observe signicant effects of PCLO within our

ROIs during processing of happy faces. However, we found subtreshold increased activity in the left amygdala during processing of happy faces compared with neutral faces (MNI coordinates x 24 y 3 z 15; Z 2.47; P

FWE_SVC

o0.09, Puncorrected 0.007). In addition, during processing

of happy faces we found a near-signicantly decreased activity for the PCLO group in the right ventral striatum

(MNI coordinates x 9 y 6 z 9; Z 2.67; P

FWE_SVCE

0.004).

We found similar results as reported for negative vs neutral faces, and plotting our data suggested that these effects were indeed driven by negative faces.

Interaction of PCLO genotype with current psychopathology during processing of faces. Post hoc analyses showed a

0.05 (P 0.058) P

uncorrected

Figure 1 Effect of piccolo (PCLO) genotype on emotion processing of angry vs neutral faces (MNI coordinates (x 21 y 0 z 15), Z 3.6, PFWE 0.004).

Activation shown at Po0.005, uncorrected.

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Figure 2 Effect of piccolo (PCLO) genotype on emotion processing of fearful vs neutral faces (MNI coordinates (x 18 y 0 z 18), Z 3.246,

PFWE 0.014). Activation shown at Po0.005, uncorrected.

Figure 3 Parameter estimates and 95% condence intervals showing direction of interaction effect of genotype and current psychopathology during processing of fearful faces.

Figure 4 Positive effect of increasing task load on activation during planning. Areas involved in the effect of increasing task load include the dorsolateral prefrontal cortex (PFC), middle PFC, precuneus and inferior parietal lobe. Familywise error corrected at Po0.05. Colors indicate Z-scores.

signicant genotype by depression interaction (F(1,119)

9.50; Z 2.8) during processing of fearful faces compared

with neutral faces: PCLO demonstrated increased amyg

dala activity in the MDD group (Z 3.02; pFWE o0.05), but

not in HC (Z 1.1; Figure 3). There was no interaction during

processing of sad or angry faces or during processing of happy faces. Notably, we found no main effect of diagnosis on amygdala activation, which is similar to our previous study.5

Executive functioningPCLO genotype effect on task performance. No effect of genotype was observed on task performance (mean RT: PCLO : 8.30 s2.34; PCLO : 8.19 s2.11; P 0.76;

repeated measures ANOVA for RT per trial type: P 0.094; proportion of correct answers: PCLO : 0.92

0.08; PCLO : 0.900.11; P 0.22; repeated measures

ANOVA for proportion of correct answer per trial type: P 0.488).

BOLD fMRI resultsMain effect of task during executive functioning. In accordance with previous studies,7,34 increasing task load during the ToL was associated with robust activity in the right dorsolateral PFC, right middle PFC, bilateral precuneus and bilateral inferior parietal lobe (Figure 4; Supplementary Table S2).

PCLO genotype effect during executive functioning. We found no signicant effect of genotype in the predened ROIs.

Interaction of PCLO genotype with current psychopathology during executive functioning. Explorative post hoc analyses showed a genotype by diagnosis interaction effect in the left medial dorsolateral PFC (Brodmanns area 9, MNI coordinates x 27 y 30 z 24, Z 3.77, PFWEo0.05).

Increased activity in PCLO group compared with PCLO

was found in the MDD group but not in HC.

Confounding factors. As is known from other studies, SSRI use may alter regional brain function in psychiatric diseases.48 We conducted post hoc analyses for both the emotional face paradigm and the planning paradigm to test for possible effects of SSRI use. During processing of the various emotional faces (angry vs neutral, fear vs neutral, sad vs neutral and happy vs neutral), we found similar results after excluding subjects using SSRIs from the main analyses. Notably, we replicated the effect of PCLO for the left

amygdala.

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Also, post hoc analyses of imaging data of our executive functioning paradigm revealed similar results after excluding subjects using SSRIs as in the main analyses (data not shown).

Discussion

We report, for the rst time and in a large group of subjects, an effect of the PCLO risk allele for MDD on amygdala activity during processing of angry, fearful and (trend wise for) sad facial expression, but not during happy facial processing. During angry and sad facial processing, PCLO risk allele carriers demonstrated increased amygdala activation, independent of psychopathological status. During the processing of fearful faces, only MDD patients with the PCLO risk allele showed increased amygdala activation, whereas no effect of genotype was observed in HC. At a behavioral level, MDD patients with the PCLO risk allele performed near-signicantly faster than MDD patients with the nonrisk allele, whereas in HC, no signicant differences between PCLO genotype were observed during perception of negative emotions. We found no main effect of genotype on executive function, or on its neural substrate, although explorative analyses indicated MDD-specic effects of PCLO risk allele carriers in the medial prefrontal area.

Dysregulation of the amygdala during emotional information processing has previously been associated with MDD.4952 However, imaging ndings have been inconsistent, as several studies failed to demonstrate altered amygdala activity during emotional information processing of faces.5,5357 We suggest that genetic heterogeneity may

explain, at least in part, the inconsistencies seen in the literature. Increased amygdala responsiveness during negative face processing and decreased ventral striatum activity in response to positive stimuli have also been reported in MDD studies. Our ndings provide further evidence for PCLO being involved in the pathogenetic mechanisms underlying MDD. We found a signicant interaction between genotype and current psychopathology, demonstrating increased activity in the amygdala in MDD patients relative to HC during processing of fearful faces. Involvement of the amygdala in the neural response to fearful faces, but not to angry or sad faces has been suggested by some studies,5860 but not by

others.61,62

Our study showed lateralization effects in the amygdala in the PCLO risk allele group, which may reect a bias towards a more specic, sustained emotional reaction in the left amygdala, rather than the general emotional reaction in which the right amygdala is reportedly involved.63 It has also

been proposed that lateralization may be associated with the temporal dynamics of the amygdala in response to emotional stimuli. Whereas the left amygdala is presumably involved in stimulus evaluation, the right amygdala has been implicated in the rapid detection of emotional stimuli.64 Following this line of argument, we suggest that the PCLO variant may modulate evaluation of emotional faces, rather than their rapid detection.

In MDD, treatment with SSRIs may reduce negative bias in perception and memory.6567 In addition, it is thought that changes in serotonergic neurotransmission can modulate

identication of emotional faces, particularly fearful faces.65,66

Recently, the PCLO risk allele was found to enhance the biological response to SSRIs among MDD patients.23 How

ever, the mechanism of PCLO modulation on serotonin transmission is unclear and may not involve the serotonin transporter, also in view of the inconsistent ndings associated with other serotonergic polymorphisms.68,69

We found no effect of the PCLO risk allele on performance or BOLD signal during an executive paradigm, the ToL planning task. However, post hoc, we found an interaction between genotype and current psychopathology during performance of the ToL in the left middle dorsolateral PFC, which was also found to be associated with depression severity.7 However, these results should be interpreted with care, as these ndings were based on explorative analyses. Another potential limitation of our study is that our results cannot be readily generalized outside the eld of planning and problem solving, and emotional faces processing eld.

Taken together, our ndings during emotional face processing and planning indicate that PCLO is associated with emotion processing rather than executive function. Although our ndings suggested selectivity of PCLO for serotonergic neurotransmission relative to other monoamines, such a hypothesis is in need of empirical testing. Therefore, future fMRI studies should seek to employ combined cognitive-emotional paradigms coupled with pharmacological manipulation to further investigate the role of PCLO in emotional and cognitive processing.

In conclusion, the PCLO risk allele is associated with increased activity in the left amygdala during emotional information processing of negative faces, but not with executive function or its neural substrate. We suggest that our data support the role of the PCLO risk allele as a risk factor for MDD through altered amygdala activity and, possibly, altered serotonergic neurotransmission in brain areas related to emotional information processing.

Conict of interest

S Woudstra, Z Bochdanovits, MJ van Tol, DJ Veltman, FG Zitman, MA van Buchem, EM Opmeer, LR Demenescu, BWJ Penninx declare no conict of interest. NJA van der Wee received speaking fees from Eli Lilly and Wyeth; and served on advisory panels of Eli LIlly, Pzer, Wyeth and Servier. A Aleman received an investigator-initiated unrestricted research grant from Brystol-Myers Squibb and speakers bureau honoraria from AstraZeneca, Brystol-Myers Squibb, Glaxo-SmithKline. WJ Hoogendijk lectured and advised for, and received unrestricted research grants from Ely Lilli, Lundbeck, Servier, Bristol-Myers Squibb and Organon for which payments were made to the independent research foundation of GGZ ingeest. WJ Hoogendijk holds a patent for gene-expression proling in peripheral blood as a biomarker for depression. All of these activities are not directly related to the present study and, therefore, do not form a conict of interest.

Acknowledgements. The infrastructure for the NESDA study (http://www.nesda.nl

Web End =www.nesda.nl) is funded through the Geestkracht program of the Netherlands Organization for Health Research and Development (Zon-Mw, Grant number 10-

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000-1002) and is supported by participating universities and mental health care organizations (VU University Medical Center, GGZ inGeest, Arkin, Leiden University Medical Center, GGZ Rivierduinen, University Medical Center Groningen, Lentis, GGZ Friesland, GGZ Drenthe, Scientic Institute for Quality of Healthcare (IQ healthcare), Netherlands Institute for Health Services Research (NIVEL) and Netherlands Institute of Mental Health and Addiction (Trimbos Institute).

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