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NeuroImage 54 (2011) 2514–2523

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Cognitive state and connectivity effects of the genome-wide significant psychosis

variant in ZNF804A
a, ÿ,1 a a a
Christine Esslinger , Peter Kirsch a,1 , Leila Haddad , Daniel Mier , Carina Sauer d , Susanne Erk b,c ,

c c d It is
Knut Schnell , Claudia Arnold , Stephanie H. Witt , Marcella Rietschel , Sven Cichon ,

a,1
Henrik Walter b,c,1 , Andreas Meyer-Lindenberg
a
Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, University of Heidelberg, J5, 68159 Mannheim, Germany
b
Department of Psychiatry and Psychotherapy, Division of Mind and Brain Research, Charité – Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany
c
Division of Medical Psychology, Department of Psychiatry and Psychotherapy, University of Bonn, 53105 Bonn, Germany
d
Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, University of Heidelberg, J5, 68159 Mannheim, Germany
Department of Genomics, Life & Brain Center and Institute of Human Genetics, University of Bonn, 53105 Bonn, Germany
It is

article info abstract

Article history: Alterations of connectivity are central to the systems-level pathophysiology of schizophrenia. One of the best-
Received 22 February 2010 established genome-wide significant risk variants for this highly heritable disorder, the rs1344706 single
Revised 13 September 2010 nucleotide polymorphism in ZNF804A, was recently shown to modulate connectivity in healthy carriers
Accepted 4 October 2010
Available online 12 October 2010
during working memory (WM) in a pattern mirroring that which was found in overt disease. However, it was
unclear whether this finding is specific to WM or if it is present regardless of cognitive state. Therefore, we
examined genotype effects on connectivity in healthy carriers during rest and an emotion processing task
without WM component.
111 healthy German subjects performed a battery of functional imaging tasks. Functional connectivity with
the right dorsolateral prefrontal cortex during rest and an implicit emotion recognition task was determined
using the seed voxel method and compared to results during WM.
During rest and during the emotional task, a pattern of reduced interhemispheric prefrontal connectivity with
increasing number of rs1344706 risk alleles could be seen that was close to identical to that during WM,
suggesting a state-independent influence of the genetic variant on interhemispheric processing, possibly
through structural effects. By contrast, the abnormal prefronto-hippocampal connectivity was only seen
during the WM task, indicating a degree of task specificity in agreement with prior results in patients with
schizophrenia. Our findings confirm a key role for disturbed functional connectivity in the genetic risk
architecture of schizophrenia and identify cognitive state-dependent and independent components with
regard to WM function.
© 2010 Elsevier Inc. All rights reserved.

Introduction
Since Wernicke, it has been suspected that the presence of
dramatic behavioral impairments despite only subtle regional brain
pathology that characterizes schizophrenia may indicate disturbances
in connectivity, i.e. the interactions of brain regions during distributed
processing of information. This has mainly been studied in the context
of working memory (WM), since cognitive impairment, and especially
impairment of WM function, is a core feature of schizophrenia (Vaz
and Heinrichs, 2006). Abnormal connectivity during WM has indeed
been identified consistently in schizophrenia patients (Crossley et al.,
2009; Ragland et al., 2007; Spoletini et al., 2009; Whitfield-Gabriel
ÿ Corresponding author.
E-mail address: christine.esslinger@zi-mannheim.de (C. Esslinger).
1
These authors contributed equally.
et al., 2009; Wolf et al., 2009). One node specifically highlighted in
these network analyses is lateral prefrontal cortex. Here, a well-replicated
finding is impaired interhemispheric prefrontal connectiv-ity during working
memory in patients (Schlosser et al., 2003a;
Schlosser et al., 2003b) and also in patients and unaffected siblings
during a choice reaction task, pointing towards heritability of the
effect (Woodward et al., 2009). These functional findings are
supported by several studies using structural imaging or diffusion
tensor imaging (Carpenter et al., 2008; Kubicki et al., 2008; Price et al.,
2007). In these studies, reduced volume or tract integrity of the corpus
callosum, especially in the genu, was consistently found.
Another way in which connectivity may influence WM in
schizophrenia is through altered prefronto-temporal interactions.
During verbal encoding (episodic memory), a reduction of functional
connectivity was seen between the dorsolateral prefrontal cortex
(DLPFC) and parahippocampal/superior temporal gyrus (STG) regions

1053-8119/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuroimage.2010.10.012
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C. Esslinger et al. / NeuroImage 54 (2011) 2514–2523
(Wolf et al., 2007), while during WM, an increase in functional coupling between
frontal and temporal regions has been observed (Crossley et al., 2009).
Specifically, schizophrenia patients failed to uncouple right DLPFC from left
hippocampal activation during WM, but not a control condition (Meyer-Lindenberg
et al., 2005b).
Typically, the hippocampus is deactivated during WM tasks, at least in tasks with
non excessive WM load and non familiar stimuli (Esposito et al., 2006; Zarahn et
al., 2005). Therefore, this persistent prefrontal hippocampal coupling in
schizophrenia could be inter-preted as aberrant recruitment of associative
memory capacities during a WM task. These data suggested that schizophrenia,
at least functionally, may not be mainly a “dis”connection syndrome (where
connectivity is uniformly reduced) but rather a “dys”connection syndrome, where
both abnormally increased and decreased coupling may be present during some
cognitive states (Stephan et al., 2006).
Since schizophrenia is highly heritable, with estimates as high as 81%
(Sullivan et al., 2003), it is reasonable to ask whether these findings in manifest
disease extend to genetic risk. Extensive studies in healthy candidate gene variant
carriers provide broad support for an impact of these variants on connectivity
(Bertolino et al., 2006; Buckholtz et al., 2007; Kempf et al., 2008; McIntosh et al.,
2008; Meyer-Lindenberg et al., 2007; Tan et al., 2008). However, candidate gene
studies are open to the objection that they study genetic variants that have not
been unambiguously linked to the schizophrenia disease phenotype. An
opportunity to further establish a role for connectivity in mediating genetic risk was
therefore presented by the recent discovery, through genome-wide association
(GWA) and follow-up, of a genome-wide significant risk single nucleotide
polymorphism for psychosis in the gene ZNF804A (rs1344706) (O'Donovan et al.,
2008).
This variant has been confirmed in subsequent independent GWA study (Purcell
et al., 2009) and large case–control datasets (Riley et al., 2009; Steinberg et al.,
2010). Although not every dataset surveyed has shown positive association, a
recently published meta-analysis including 23 studies (Williams et al., 2010)
showed that the evidence for association between rs1344706 and schizophrenia
surpasses widely accepted benchmarks of significance by several orders of
magnitude (p= 2.5Eÿ11). Therefore, this variant near ZNF804A represents one of
the best-established genetic entry points into studying systems-level mechanisms
in schizophrenia genetics.
In a previous analysis (Esslinger et al., 2009) we found that healthy subjects
carrying the ZNF804A schizophrenia risk allele showed changes in functional
connectivity of right DLPFC during WM that resembled those discussed earlier for
schizophrenia, namely a gene dose dependent reduction in interhemispheric
prefrontal connectivity and an increase in connectivity of DLPFC with contra-lateral
hippocampus. However, the question remained whether this result is specific for
WM (highlighting cognitive state-dependent mechanisms) or broadly present
(therefore arguing for state-independent, for example structural, changes in
connections between brain areas). To answer this question, we studied healthy
subjects from our ongoing study (Esslinger et al., 2009) stratified for ZNF804A
rs1344706 genotype during rest and during a face emotion proces-sing task that
places no demands on WM. Based on the literature discussed earlier, we
hypothesized that diminished interhemispheric connectivity in the ZNF804A risk
allele would be task independent, i.e. found during all three conditions. In contrast,
we expected abnormal prefronto-hippocampal connectivity to be present only
during WM, following our previous findings in patients with schizophrenia (Meyer-
Lindenberg et al., 2005b).
Methods
Subjects
Subjects were taken from an ongoing large scale multicenter imaging genetic
study (Esslinger et al., 2009). All participants were
2515
healthy German volunteers with parents and grandparents of European origin.
Because we applied a strict threshold for movement in the resting condition (less
than 2 mm in translation and/or 2° in rotation), only 111 out of the previously
reported (Esslinger et al., 2009) 115 participants (61 from Mannheim and 50 from
Bonn) could be included in the present analysis. No participant had lifetime or
family history of schizophrenia or affective disorder, 21 were rs1344706 CC
homozygotes, 50 CA heterozygotes and 40 AA homozygotes (C= cytosine, A=
adenine= risk allele, in Hardy– Weinberg Equilibrium: Chi2= .724, df= 1, n.s.).
Scanning site, gender, age, handedness, level of education, and task performance
did not differ significantly between genotype groups (Supplementary Table S1).
Subjects gave written informed consent. The study was approved by the local
ethics committees of the universities of Heidelberg and Bonn.
DNA-extraction and genotyping
Genomic DNA was prepared from whole blood according to standard
procedures. rs1344706 was genotyped using a TaqMan 5ÿ nuclease assay.
Accuracy was assessed by duplicating 15% of the original sample, and
reproducibility was greater than 99.9%.
Functional imaging tasks
Subjects completed a battery of cognitive tasks during MR scanning as well
as resting. The cognitive tasks have been previously described (Esslinger et al.,
2009). Briefly, during the WM task (Callicott et al., 2000) subjects viewed a series
of digits (1–4) presented sequentially for 500 ms (ISI 1500 ms) on a screen via
LCD goggles. In the 0-back control condition, subjects had to press a button
corresponding to the digit presently seen. In the 2-back WM condition they had to
react to the digit seen two instances before the present digit. Four blocks of each
condition were presented alternately. The task lasted 4 min or 124 scans.
The face matching task (FMT) is used to study implicit emotion recognition
(Hariri et al., 2000). Subjects had to match two out of three pictures of angry or
fearful faces (emotional condition) or geometrical shapes (control condition). The
design was four blocks for each condition alternating, each consisting of six trials
and lasting 30 s. The task lasted for 4.2 min or 130 scans.
For resting state examination, subjects were asked to close their eyes, relax
and try not to concentrate on anything. The session lasted 5 min, but to compare
it with the WM task, only the first 124 scans were analyzed. Interviews directly
after the scan revealed that none of the subjects had fallen asleep.
Imaging parameters
BOLD fMRI was performed on two Siemens Trio 3 T scanners at the Central
Institute of Mental Health Mannheim and the University of Bonn. At both sites
identical sequences and scanner protocols were used (parameters: 28 4 mm
slices, 1 mm gap, FOV 192 mm, TR 2 s, TE 30, and flip angle 80°). Quality
assurance (QA) measures were conducted on every measurement day at both
sites according to a multicenter QA protocol (Friedman and Glover, 2006) revealing
stable signals over time. To account for differences in signal-to-noise across sites,
site was used as a covariate for all statistical analyses.
Functional image processing and connectivity analyses
Image processing and statistical analyses were conducted using statistical
parametric mapping methods (SPM5, http://www.fil.ion. ucl.ac.uk/spm/software/
spm5/). Images were realigned to the first image, slice-time corrected, and spatially
normalized into a standard stereotactic space (Montreal Neurological Institute
(MNI) template)
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with volume units (voxels) of 3 × 3 × 3 mm and smoothed with a Statistical inference

9 mm FWHM (full width at half maximum) Gaussian filter.
The procedure for the functional connectivity analyses has been
described previously (Esslinger et al., 2009). First, the maximally
activated voxel during the WM task within a mask of the right DLPFC
(mask descriptions see later) was determined. To create seed
timeseries, first eigenvariates of the timeseries from all voxels (in
case of the WM task of all voxels activated at pb.01) within a sphere of
6 mm around this maximally activated voxel in the WM task were
calculated. We used seed regions that were functionally defined on
the individual level instead of group means or anatomical seed
regions to make sure that the seed region was part of each individual's
working memory network. Identical seed regions were used for all
three tasks in each individual. Using SPM5, seed timeseries were
highpass filtered (128 s) and in case of the WM task, task related
variance was removed using a general linear model approach to avoid
the contribution of task related coactivation to functional connectivity
measures, following our previously published practice in the
estimation of functional and effective connectivity (Meyer-Linden-berg et
al., 2004; Pezawas et al., 2005; Stein et al., 2007). In case of
FMT and rest, no task related activation in DLPFC was expected and no
correction for coactivation was necessary. To account for unspecific
noise, first eigenvariates from masks covering cerebrospinal fluid and
white matter were extracted and entered, together with movement
covariates, into whole brain multiple regression where seed region
activity was the covariate of interest.
To assess the functional connectivity networks within the three
conditions, we included the global signal (the first eigenvariate of all
timeseries within a mask of the whole brain) as covariate of no
interest in the first-level connectivity analyses in order to account for
variations in the global level of whole brain connectivity. In these
within-task analyses with global signal as a first-level covariate, which
are descriptive because the overall level of connectivity has been
covaried for, only positive correlations are reported. Following
standard practice, this covariate was not included in between-task
group analyses, where it would have introduced artificial negative
correlations (Murphy et al., 2009; Weissenbacher et al., 2009).
To compare functional connectivity during the WM task with that
during rest and the FMT, we subjected the maps of partial correlation
with the right DPLFC to a second level mixed effects 3 × 3 analysis of
variance with one within subject factor: condition (WM task vs rest vs
FMT) and one between subject factor: genotype (AA vs AC vs CC).
Scanning site was added to the model as a covariate.
For post-hoc analyses of regional connectivity, connectivity maps
from the relevant t contrast were thresholded at pb.001 and the mean
contrast estimate of the cluster of activated voxels in each anatomical
mask was used to calculate ANOVAs or correlations using SPSS
(version 16.0.2, SPSS Inc., Chicago, Ill.).
For all imaging methods, significance threshold was set to pb.05,
corrected for multiple comparisons within the region of interest
(ROI). We used family-wise error, (FWE), based on Gaussian Random
Fields theory, and false discovery rate (FDR), a widely used frequentist
method, which strongly control for type I error in imaging genetics
(Meyer-Lindenberg et al., 2008). The same anatomical a priori ROIs as
in the previous study were used (Esslinger et al., 2009).
Creation of masks for region of interest (ROI) analyses and seed voxel
extraction
All masks were created using anatomical labels provided by the
Wake Forest University PickAtlas (www.fmri.wfubmc.edu/downloads).
In the case of DLPFC, voxels defined as right or left Brodmann area (BA)
46 and BA 9 were smoothed (9 mm FWHM) and medial areas of BA 9
were subtracted (Supplementary Fig. S1). The masks of right and left
DLPFC contained 578 and 522 voxels, the masks of right and left
hippocampus 262 and 281 voxels.
Results
Behavioral results
There was no significant correlation between number of ZNF804A
risk (adenine) alleles and performance in the two tasks as measured
by reaction times and percentage of correct answers in each condition
(Table 1).
Functional connectivity
Within-task connectivity of the right DLPFC
During all three conditions, WM, rest and emotional task,
functional connectivity maps for the right DLPFC were very similar
and comprised the known areas of the WM network (Table 2):
bilateral DLPFC, premotor areas (BA 6), the pre-supplementary motor
area (BA 6 and BA 9), premotor areas (BA 6 and BA 8), insula (BA 13,
these clusters were reaching into VLPFC in all three conditions), the
inferior and superior parietal lobule, precuneus (BA 40 and BA 7), and
the left cerebellum. There were some differences in connectivity
networks between the three conditions: during the WM task there
was connectivity of the right DLPFC with bilateral postcentral gyrus
(BA 3). Functional connectivity with right striatum was significant
only during the WM task and rest. Only during rest and the FMT there
was significant connectivity with bilateral posterior temporal regions
(BA 37).

Table 1
Performance during the n-back and emotion recognition tasks. Means and standard deviations. C= cytosine, A= adenine= risk allele. R= Pearson's r, two-tailed.

rs1344706 genotype Linear correlation with genotype

CC THAT AA r p

Number of subjects 21 50 40

n-back working memory task

Percent correct answers during 0-back 98.6 (±2.7) 79.6 97.7 (±9.9) 81.0 98.5 (±2.0) 80.6 .012 .898
Percent correct answers during 2-back (±16.4) 562.1 (±20.6) 511.6 (±18.8) 537.1 .016 .870
Reaction times: 0-back (±174.5) 412.6 (±153.2) 415.9 (±149.5) 389.8 .045 .639
Reaction times: 2-back (±261.1) (±260.5) (±264.1) ÿ.037 .700

Emotion recognition task

Percent correct answers during form matching 95.0 (±5.8) 96.4 97.3 (±3.3) 99.3 94.9 (±6.6) 97.7 ÿ.057 .551
Percent correct answers during face matching (±6.4) 1040.7 (±1.5) 1036.7 (±5.1) 1094.1 .050 .607
Reaction times: forms (ms) (±207.7) 1223.0 (±173.4) 1146.9 (±216.8) 1273.3 .115 .232
Reaction times: faces (ms) (±338.9) (±214.3) (±230.8) .115 .231
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Table 2
Connectivity with right DLPFC in the whole group during the three task conditions: n-back working memory task, emotion recognition task and resting state. pb.05, FWE-corrected.
BA= Brodmann area, tmax=maximal activation in the cluster, and MNI=Montreal Neurological Institute.

Hemisphere Anatomical region NOT tmax MNI coordinates

n-back working memory task

Frontal lobe
Left Middle frontal gyrus BA 46 15.56 ÿ42 33 30
Left Middle/inferior frontal gyrus BA 9 14.95 ÿ45 24 36
Left Middle frontal gyrus BA 6 6.75 ÿ33 ÿ3 48
Right Middle/inferior frontal gyrus NOT 9/10 23.43 45 33 33
Right Medial frontal gyrus/SMA NO 6/9 13.68 12 33 39
Right Middle frontal gyrus BA 8 13.36 36 18 54
Right Precentral gyrus BA 4 5.58 60 ÿ12 33
Insular cortex
Left The island BA 13 7.20 ÿ33 21 6
Right The island BA 13 9.55 33 21 0
Parietal lobe
Left Inferior parietal lobule BA 40 9.19 ÿ54 ÿ42 48
Left Superior parietal lobule BA 7 7.10 ÿ30 ÿ57 45
Left Precuenius BA 7 6.45 ÿ9 ÿ66 51
Left Postcentral gyrus BA 3 5.42 ÿ60 ÿ15 30
Right Postcentral gyrus BA 3 6.00 63 ÿ12 27
Right Inferior parietal lobule BA 40 13.65 48 ÿ48 51
Right Superior parietal lobule BA 7 9.96 33 ÿ66 48
Right Precuenius BA 7 6.91 12 ÿ69 51
Cerebellum
Left Posterior lobe, decline 6.72 ÿ9 ÿ78 ÿ30
Subcortical
Right Caudate body 5.94 12 9 18

Emotion recognition task

Frontal lobe
Left Middle frontal gyrus BA 10/46 20.19 ÿ42 33 30
Left Inferior frontal gyrus BA 9 12.47 ÿ51 6 30
Left Middle frontal gyrus BA 6 11.76 ÿ30 ÿ3 48
Left Medial frontal gyrus/SMA BA 6 10.64 ÿ6 15 48
Right Middle frontal gyrus BA 9/10/46 24.79 45 36 30
Right Middle frontal gyrus BA 6 16.35 33 6 57
Insular cortex
Left The island BA 13 11.54 ÿ33 18 9
Right The island BA 13 16.14 33 21 3
Parietal lobe
Left Inferior parietal lobule BA 40 15.06 ÿ42 ÿ45 48
Left Precuenius BA 7 11.60 ÿ24 ÿ63 54
Right Inferior parietal lobule BA 40 19.15 48 ÿ45 48
Right Superior parietal lobule BA 7 10.46 12 ÿ69 54
Right Postcentral gyrus BA 3 5.77 60 ÿ18 33
Cerebellum
Left Posterior lobe, decline 7.16 ÿ12 ÿ78 ÿ30
Left Posterior lobe, inferior semilunar lobule 7.05 ÿ33 ÿ66 ÿ48
Temporal lobe
Left Middle temporal gyrus BA 37 5.55 ÿ48 ÿ63 ÿ6
Right Middle temporal gyrus BA 37 7.48 54 ÿ51 ÿ9

Resting state
Frontal lobe
Left Middle frontal gyrus NOT 9/10 18.19 ÿ42 30 30
Left Middle frontal gyrus BA 6 10.26 ÿ27 6 60
Right Middle/superior frontal gyrus NOT 9/10 24.21 45 33 33
Right Middle frontal gyrus BA 6 15.54 42 9 51
Right Middle frontal gyrus BA 11 6.73 24 45 ÿ12
Right Medial frontal gyrus/SMA BA 6 17.44 6 27 42
Limbic lobe
Right Cingulate gyrus BA 31 6.67 9 ÿ36 42
Right Cingulate gyrus BA 23 5.89 12 ÿ30 33
Insular cortex
Left The island BA 13 8.36 ÿ33 18 9
Right The island BA 13 11.13 33 21 ÿ3
Parietal lobe
Left Inferior parietal lobule BA 40 13.64 ÿ51 ÿ42 48
Left Superior parietal lobule BA 7 10.21 ÿ30 ÿ63 48
Left Precuenius BA 7 5.61 ÿ9 ÿ69 51
Right Inferior parietal lobule BA 40 16.73 51 ÿ45 48
Right Precuenius BA 7 8.37 9 ÿ69 48
Cerebellum
Left Posterior lobe, inferior semilunar lobule 8.24 ÿ36 ÿ63 ÿ48
Left Posterior lobe, decline 7.70 ÿ36 ÿ66 ÿ30
Left Posterior lobe, uvula 7.54 ÿ33 ÿ63 ÿ33

(continued on next page)

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Table 2 (continued)

Hemisphere Anatomical region NOT tmax MNI coordinates

Resting state
Subcortical
Right Putamen 5.24 21 0 15
Right Caudate body 6.03 9 12 15
Temporal lobe
Left Middle temporal gyrus BA 37 6.22 ÿ54 ÿ54 ÿ9
Right Middle temporal gyrus BA 37 6.45 57 ÿ48 ÿ9

Comparisons between connectivity with right DLPFC during the
three tasks revealed increased connectivity in areas of the default mode
network (precuneus, anterior cingulate, inferior parietal lobule (Raichle
et al., 2001)) during rest compared to FMT and increased connectivity
during the FMT compared to rest in bilateral occipital cortex, fusiform
face area, superior parietal lobules and left pre- and postcentral regions.
Comparison between resting state and the WM task showed increased
connectivity in areas related to the default mode network like
precuneus and middle and inferior temporal gyri, and also in the WM
network (Supplementary Fig. S2). The latter was most possibly due to
regressing out the task related variance in theWM data and can thus not
be related to physiological differences in connectivity between the two
states. For the same reason, no regions of increased connectivity during
the WM task compared to rest could be identified.
Association of ZNF804A genotype with interhemispheric prefrontal
connectivity
To compare the impact of ZNF804A genotype on connectivity with
the right DLPFC during the different conditions, we examined
connectivity maps during the three conditions in one second level
ANOVA with the within subject factor task condition and the between
subject factor genotype.
There was a significant main effect of genotype within the a priori
regions of interest, the left DLPFC (MNI coordinates [ÿ39 27 42],
Fmax= 13.22, p= .001) as well as the right DLPFC (MNI coordinates
[42 33 39], Fmax= 11.84, p= .003). P-values are FWE-corrected within
the masks. Voxel-wise post-hoc tests revealed that there was a
consistent effect in terms of lower connectivity with increasing
number of risk (adenine) alleles (Fig. 1A). Both peak voxels in the
DLPFC as well as those in bilateral premotor regions were significantly
less connected in the risk allele carriers in all three conditions
(Table 3). Comparisons of mean contrast estimates in the significant
clusters in right and left DLPFC are depicted in Fig. 1B. During all three
conditions there was a linear decrease in connectivity with increasing
number of risk alleles. Fig. 2 shows the effect of decreasing number of
risk alleles on connectivity with the right DLPFC in the three
conditions separately. There were regions of overlap of all three
conditions within the left DLPFC (26 voxels), right DLPFC (37 voxels),
and right premotor area, BA 6 (11 voxels). No regions showed
significantly decreased connectivity with decreasing number of
rs1344706 risk alleles.

Fig. 1. A: Consistently decreasing connectivity with right DLPFC with number of rs1344706 risk alleles. Main effect over all three tasks, pb.001 uncorrected, cluster sizeÿ10.
B: Means and standard errors of contrast estimates in the cluster in the left (left panel) and the right (right panel) DLPFC between the three groups and genotypes (C= cytosine,
A= adenine= risk allele).
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C. Esslinger et al. / NeuroImage 54 (2011) 2514–2523
Table 3
Decreasing connectivity with right DLPFC with increasing number of rs1344706 risk (adenine) alleles. Main
effect over all three task conditions. FDR corrected p-values (over the whole brain), t-values (tmax) and
coordinates (MNI=Montreal Neurological Institute) given for the peak voxel in each cluster. BA= Brodmann
area.
Anatomical region BA Cluster size p FDR tmax MNI coordinates
Left middle frontal gyrus BA 9 29 .016 5.09 ÿ39 27 42 .021 4.68
Right middle frontal gyrus BA 9 25 42 33 39 .023 4.5 24 ÿ3 72
Right superior frontal gyrus BA 6 21
Left middle frontal gyrus BA 6 1 .048 3.9 ÿ21 15 63
Mean contrast estimates of prefrontal connectivity in the signif-icant
clusters differed between the scanning sites (Bonn and Mannheim): right
DLPFC: F(1,105)= 6.24, p= .014, and left DLPFC: F(1,105)= 4.94, p=
.028, with higher estimates in Bonn. There was also an interaction
between scanning site and genotype: right DLPFC: F(2,105)= 4.80, p=
.01, left DLPFC: F(2,105)= 4.08, p= .020, but this was driven by
heterozygotes. Comparison of homozygotes only revealed no site main
effect in both clusters.
Association of ZNF804A genotype with prefronto-hippocampal
connectivity
In the hippocampus, the region that showed increased connectiv-ity
with the right DLPFC with increasing number of risk (adenine) alleles
during the WM task, no main effect of genotype could be shown. Instead,
there was a significant interaction between task condition and genotype
(Fig. 3A: increasing connectivity during the n-back task, decreasing
connectivity during the other conditions): connectivity of right DLPFC
with left hippocampus: MNI coordinates [ÿ36 ÿ21 ÿ18], tmax= 3.82, p=
.007, connectivity with right hippocampus: MNI coordinates [39 ÿ12 ÿ18],
tmax= 3.66, p= .012.
Voxel-wise post-hoc tests in single conditions revealed a signif-icant
influence of genotype on connectivity with hippocampus only during the
WM task: connectivity with left hippocampus: MNI coordinates [ÿ39 ÿ24
ÿ15] tmax= 4.28, p= .001, with right hippocampus: MNI coordinates [36
ÿ18 ÿ15], tmax= 3.34, p= .032. We found no significant association of
prefronto-hippocam-pal connectivity with increasing number of risk
alleles during the n-back task and FMT. P-values are FWE-corrected
within the masks.
Fig. 3B shows the mean contrasts estimates for the connectivity in
the right and left hippocampal clusters. Increase of connectivity with
increasing number of risk alleles could only be shown during the WM
task. During the emotional task and during rest there were no significant
differences in prefronto-hippocampal connectivity be-tween the genotype
groups.
There were no significant site effects on mean contrast estimates in
right and left hippocampal clusters. This was true for main effects of site
as well as for interactions between site and genotype and three way
interactions between site, genotype and task.
Fig. 2. Red areas: decreased connectivity with right DLPFC with increasing number of rs1344706 risk alleles. A: Working memory task, B: emotion recognition task, and C: resting state. Yellow areas: voxels that showed
this correlation with genotype in all three conditions. All voxels shown at pb.005, uncorrected, cluster sizeÿ10.
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Correlations between connectivity measures across task conditions
To analyze whether subjects with high connectivity in one task
condition also showed high connectivity in the other conditions, we
examined the correlation of connectivity estimates in the DLPFC and the
hippocampus. Interhemispheric DLPFC connectivity and connec-tivity
within the right DLPFC correlated highly and very significantly between
conditions (Table 4).
In contrast, there was only one correlation of prefronto-hippo-campal
connectivity during rest with that during the FMT that did not survive
Bonferroni correction for multiple testing.
Correlations of connectivity measures with behavioral results
Because we did tests for 8 behavioral measures (reaction times and
correct answers for both tasks for both conditions), only p-values less
than .0063 can be regarded as significant. There was a trend for subjects
who had more connectivity within the right DLPFC during rest to be
faster (Pearson's r=ÿ.188, p(two-tailed)= .048), and subjects with more
connectivity between right DLPFC and right hippocampus during the n-
back task (r= .233, p= .014) and right DLPFC and left hippocampus
during the FMT (r= .205, p= .030) tended to be slower in the 0-back
condition. Subjects with more connectivity between right DLPFC and
hippocampus during the FMT tended towards more hits during form
matching (r= .196, p= .039).
Discussion
The aim of this study was to investigate possible interactions of
cognitive state and genotype in the neural systems-level effects of the
genome-wide significant variant for psychosis, ZNF804A rs1344706.
To test this issue we examined functional connectivity with the right
DLPFC during three different conditions: a WM task, an emotion
recognition task with no demand on WM and during rest. We confirmed
strong genotype effects on connectivity and identified both cognitive
state-dependent and state-independent components of this effect, which
agrees with previous results in patients with manifest disease.
Our first main finding was that the decrease of interhemispheric
prefrontal connectivity and of connectivity within the right DLPFC in
subjects with the ZNF804A risk genotype previously demonstrated for
the WM task in this sample (Esslinger et al., 2009) was independent of
the cognitive condition: a very comparable pattern of decreasing
prefrontal functional connectivity could be observed during the emotional
control task and during rest. Furthermore, connectivity measures in all
subjects were very highly correlated between the three conditions.
Correlated activity in function specific large scale cortical networks
(temporally correlated networks, TCNs) has been previously observed
not only during the execution of a specific task, but also during rest
Machine Translated by Google

2520 C. Esslinger et al. / NeuroImage 54 (2011) 2514–2523

Fig. 3. A: Areas showing an interaction between connectivity with right DLPFC and rs1344706 genotype: post-hoc tests show that this reflects increasing connectivity during the
working memory task, decreasing connectivity during the other conditions. All voxels at pb.005, uncorrected, cluster sizeÿ10. B: Means and standard errors of contrast estimates in
the cluster in the left (left panel) and right (right panel) hippocampus between the three groups and genotypes (C= cytosine, A= adenine= risk allele).

( Beckmann et al. , 2005 ; Biswal et al. , 1995 ; Buckner and Vincent , 2005 ).
2007; Calhoun et al., 2008; Cordes et al., 2000; Damoiseau et al.,
2006; Fox and Raichle , 2007 ; Fox et al., 2005; Beans et al., 2009; Lowe
et al., 1998; Seeley et al., 2007). Most of these groups investigating
temporally correlated networks at rest have identified amongst others
a fronto-parietal network that is attributed to executive and working
memory (WM) functions, similar to the one we found present in
resting data and impacted by ZNF804A genotype in the present data.
Table 4
Correlations of the connectivity estimates between the three task conditions.
R= Pearson's r, two-tailed.
Conditions
Interhemispheric DLPFC connectivity
Working memory — rest
Working memory — emotion recognition
Emotion recognition — rest
Connectivity within the right DLPFC
Working memory — rest
Working memory — emotion recognition
Emotion recognition — rest
Connectivity between right DLPFC and right hippocampus
Working memory — rest ÿ.066 Working memory — emotion recognition
ÿ.020 Emotion recognition — rest .232
Connectivity between right DLPFC and left hippocampus
Working memory — rest
Working memory — emotion recognition
Emotion recognition — rest
When examining functional connectivity maps for the whole group
independent of genotype, we could observe very comparable patterns
that only differed in areas that are not consistently part of the WM
network, namely the temporal lobes, postcentral regions and the
striatum. A similar finding of conserved functional networks has
previously been shown in a study using independent component
analysis during the relevant task, rest and irrelevant tasks (Arfanakis
et al., 2000). More recently, Lohmann et al. could show that a network
associated with language was detected during different tasks, but the
posterior superior temporal sulcus was specifically included only
when subjects performed a language task (Lohmann et al., 2010). The
core components of functionally specific TCNs therefore seem to be
consistent within subjects not only during rest, but also during
different tasks. Furthermore, these TCNs are also consistent over time
r p
(Chen et al., 2008). It is still unclear what the neuronal basis for these
very stable networks of temporally coherent fluctuations are, but
.619 4.3 × 10ÿ13
.637 5.4 × 10ÿ14
there is increasing evidence that they are dependent on the integrity
.628 1.6 × 10ÿ13 of white matter tracts. Studies combining functional connectivity MRI
and diffusion tensor imaging (DTI) found good correlation between
these measures (Honey et al., 2009; van den Heuvel et al., 2009) and
.673 5.8 × 10ÿ16
Lowe and colleagues could show that in multiple sclerosis patients,
.739 2.2 × 10ÿ20
.782 4.4 × 10ÿ24
connectivity in the sensorimotor network correlated with the
integrity of the transcallosal motor tract measured with DTI (Lowe
et al., 2008). The repeated findings of decreased size and fiber
.491
integrity in the genu of the corpus callosum in schizophrenia patients
.834
(Gasparotti et al., 2009; Hulshoff Pol et al., 2004; Kubicki et al., 2008)
.014
indicate that our finding of decreased interhemispheric connectivity
in the risk (adenine) allele carriers in all three conditions supports the
.023 .813 assumption of a structural disturbance within the WM network itself
ÿ.010 .916
affecting functional connectivity irrespective of a particular task
ÿ.102 .287
performed.
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C. Esslinger et al. / NeuroImage 54 (2011) 2514–2523
Not only the functional anatomy of the TCN, but most importantly
the genotype effect of decrease in connectivity within the right DLPFC
and across hemispheres with left DLPFC with increasing numbers of
risk (adenine) alleles was stably found during all conditions. Because
we took first eigenvariates of a sphere around the maximum activation
in the right DLPFC as seed timeseries, some correlation within the
right DLPFC was expected and found, but does not explain
differences between the genotype groups. A likely physiological basis
of the reduced within-region functional connectivity might be increased
cortical noise. Increased cortical noise (reduced efficiency) has been
described in a number of fMRI studies during a WM task in
schizophrenia patients (Callicott et al., 2000) and also in subjects at
high genetic risk (Callicott et al., 2003; Rasetti et al., 2009). The
findings of decreased prefrontal signal to noise in schizophrenia and
also of decreased intra-DLPFC coherence fit well into a model of
dopamine regulation of prefrontal signal to noise ratio. This model
suggests that dopamine D1 receptor activity increases input specific
neuronal firing by inducing suppression of surrounding background
activity leading to a high energy barrier that makes it more difficult to
switch between different states of network activity (Durstewitz and
Seamans, 2002, 2008). Some direct evidence of differential regulation
of prefrontal noise and signal through genetic mechanisms impacting
on prefrontal dopamine has been presented from PET imaging
(Meyer-Lindenberg et al., 2005a). We also note that preserved
genotype effects across conditions have been previously observed
for a key regulatory molecule in dopaminergic neurotransmission,
DARPP-32 (Meyer-Lindenberg et al., 2007). Given the evidence
cited earlier for a reduction in WM structural connectivity underlying
the preserved topography of the TCN across tasks, our finding of a
consistent genotype effect for DLPFC connectivity should also prompt
an investigation of possible structural correlates, e.g. through DTI,
especially regarding transcallosal links between prefrontal cortex
across hemispheres.
In addition to a possible effect of variation in rs1344706 on white
matter connectivity between prefrontal cortices, an additional
consideration is whether changes in connectivity might relate to
changes in activation topography across prefrontal cortex. Since we
did not observe significant differences in voxel-wise activation
analyses (Esslinger et al., 2009), and because of the size of the
DLPFC, we chose individual seed voxels at the maximum activation
within the right DLPFC during the WM task. The advantage of this
approach is that it ensures that the selected seed voxel is indeed
part of the individual WM network. Otherwise, the strength of the
coactivation of other WM regions would be dependent on how far
away the seed voxel is located from the individual hotspot of activation
in the DLPFC. Indeed, in a study of the resting state motor network,
Ma et al. (2007) found that the size and location of the TCN depend
on the choice of the seed voxel: their data show that the interindividual
variance in the size of subregions of the TCN was higher when the
seed was selected according to anatomical structure rather than
maximum activation in a motor paradigm. Interestingly, a post-hoc
analysis of the spatial distribution of the seed voxels in the three
genotype groups showed a genotype effect on hotspot z-coordinates
in our study. These were located more ventrally with increasing risk
(adenine) allele dosage (Supplementary Table S2).
This is an indication that the differences in functional connectivity
between the genotype groups might induce (or at least be related to)
a shift of activation in the right DLPFC towards more ventral regions.
This would be consistent with findings in schizophrenia patients, who
contrary to healthy control subjects, engaged more ventral prefrontal
areas with increasing WM load, possibly as a result of an insufficient
specialization of the dorsal prefrontal cortex (Tan et al., 2006). It
should be clearly stated that this subtle effect does not survive
accepted statistical procedures for imaging genetics, as no voxel-
wise activation differences between the genotype groups were found
here or in our previous study (Esslinger et al., 2009). It also does not
2521
contribute significantly to the observed differences in connectivity
between the ZNF804A genotype groups, because the main effect of
reduced interhemispheric connectivity in all three conditions and the
interaction between condition and prefronto-hippocampal connec-
tivity remained significant even when we included the z-coordinate of
the seed voxel as a covariate into the model (data not shown).
Nevertheless, our post-hoc observation indicates that interactions
between regional topography of activation and the connectivity
profiles of the networks these regions participate in are a promising
subject for further studies.
Although scanner models and protocols were the same at both
study sites, we saw a difference in the effect of ZNF804A genotype
in interhemispheric connectivity with stronger effects in Mannheim.
This is unlikely to have influenced our findings since there was no
significant difference in genotype distribution between the sites and
we had included scanning site as a covariate in all analyses.
The second main finding was that the abnormal connectivity
between right DLPFC and left hippocampus in ZNF804A risk
(adenine) allele carriers was cognitive state dependent: it was only
present during the WM task, but not during rest or during the emotion
recognition task. State-dependence was also demonstrated by the
absence of significant correlation of prefronto-hippocampal connec-
tivity (collapsing over all genotypes) measures across the three
conditions. This confirmed our hypothesis, based on the findings in
schizophrenia patients, where abnormally persistent coupling of
prefrontal and hippocampal activity was only seen under WM
conditions but not during the control condition (Meyer-Lindenberg et
al., 2005b).
These findings are also in line with results of a recent study that
showed hippocampal activation during the phonological maintenance
phase of a WM task only in schizophrenia patients but not in controls
(Henseler et al., 2009). Another study with healthy subjects using
EEG and fMRI demonstrated that hippocampus is activated during
WM if higher WM load is imposed and deactivated if only a single
item has to be maintained in WM (Axmacher et al., 2007). Although
these studies looked at activation and not connectivity, the specific
effect of the rs1344706 risk (adenine) allele on prefronto-hippocampal
connec-tivity might be due to an earlier recruitment of the hippocampus
for WM performance in healthy subjects bearing an rs1344706 risk allele.
This would then be a compensatory mechanism by which relatively
impaired WM functions are still preserved. Further evidence that
(para)-hippocampal involvement during WM performance indicates
a change of strategy comes from a study by Glabus et al. (2003).
They could show that there was less coupling between
parahippocampal areas and the WM network in healthy subjects who
used a more verbal strategy to solve the n-back task and who solved
it more successfully. Because we did not ask subjects for their
strategy, we could not investigate this directly, but a difference in
strategy might explain why subjects with the rs1344706 risk allele
failed to decouple the hippocampus during the WM task. Besides
data suggesting that imaging intermediate phenotypes show higher
penetrance (Mier et al., 2009) than behavioral (including
neurocognitive) measures (Flint and Munafo, 2007), this could also
contribute to why we did not see poorer performance during the n-
back WM task in the risk allele carriers despite the disturbance of
interhemispheric connectivity and the reduced intra-DLPFC coherence.
To conclude, in this study of a TCN during different task conditions,
we found that the genome-wide significant risk variant for psychosis,
ZNF804A, impacts systems-level connectivity with both state-inde-
pendent and state-dependent components. A decrease in interhemi-
spheric prefrontal functional connectivity was task independent,
which could be an indication of underlying structural abnormalities.
Conversely, risk allele carriers showed reduced decoupling of
hippocampus specifically during the WM task only, indicating a
dependence on state of information processing and emphasizing a
possible special role for WM impairments in the pathophysiology of
Machine Translated by Google
2522 C. Esslinger et al. / NeuroImage 54 (2011) 2514–2523
schizophrenia. Thus, our study shows that different cognitive states can
differentially modulate the impact of a genetic risk variant for psychiatric
disease on the functional organization of cortical net-works. We hope
that our findings prompt further studies investigating structural changes
associated with ZNF804A as a contribution to the molecular mechanisms
in this component of the neurogenetic risk architecture of schizophrenia.
Acknowledgments
Funding for this study was provided by BMBF (NGFNplus MooDs)
and DFG (SFB 636-B7). We thank Dagmar Gass, Beate Newport, Kyeon
Raab and Carola Opitz von Boberfeld for help with data acquisition.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.1016/
j.neuroimage.2010.10.012.
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