Gray matter abnormalities in subjects at ultra-high risk for schizophrenia and first-episode schizophrenic patients compared to healthy controls

Psychiatry Research: Neuroimaging 173 (2009) 163–169

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Psychiatry Research: Neuroimaging j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p s yc h r e s n s

Gray matter abnormalities in subjects at ultra-high risk for schizophrenia and first-episode schizophrenic patients compared to healthy controls Henning Witthausa, Christian Kaufmannb, Georg Bohner c, Seza Özgürdala, Yehonala Gudlowskid, Jürgen Gallinatd, Stephan Ruhrmann e, Martin Brünea, Andreas Heinz d, Randolf Klingebielc, Georg Juckela,⁎ a Early Recognition and Therapy Centre for Psychoses Bochum (BoFit), Department of Psychiatry, Psychotherapy and Psychosomatic Medicine, Ruhr-University of Bochum, Alexandrinenstrasse 1, 44791, Germany b Department of Psychology, Humboldt-Universität zu Berlin, 12489 Berlin, Germany c Department of Neuroradiology, Charité Campus Mitte, 10117 Berlin, Germany d Department of Psychiatry and Psychotherapy, Charité Campus Mitte, 10117 Berlin, Germany e Department of Psychiatry and Psychotherapy, University of Cologne, 50924 Cologne, Germany

a r t i c l e

i n f o

Article history: Received 26 June 2007 Received in revised form 2 May 2008 Accepted 18 August 2008 Keywords: Prodrome Voxel-based morphometry (VBM) Anterior cingulate cortex (ACC)

a b s t r a c t Neuroimaging studies have revealed gray matter abnormalities in schizophrenia in various regions of the brain. It is, however, still unclear whether such abnormalities are already present in individuals at ultra-high risk (UHR) for transition into psychosis. We investigated this issue using voxel-based morphometry of structural magnetic resonance images (MRI) and compared UHR patients with first-episode patients with schizophrenia and healthy controls. Gray matter volume maps from high-resolution MR T1-weighted whole brain images were analyzed in a cross-sectional study in 30 UHR patients, 23 first-episode schizophrenic patients and 29 controls. UHR patients showed significantly lower gray matter volume in the cingulate gyrus bilaterally, in the right inferior frontal and right superior temporal gyrus, as well as in the left and right hippocampus in comparison to healthy subjects. First-episode patients with schizophrenia showed smaller gray matter volume in the cingulate cortex bilaterally, in the left orbitofrontal gyrus, in the right inferior frontal and superior temporal gyrus, in the right temporal pole, in the left and right hippocampus, in the left parahippocampus, left amygdala, and in the left fusiform gyrus compared to the UHR patients. This study provides further evidence that gray matter brain volume, especially in the anterior cingulate cortex, is already reduced in the prodromal state of schizophrenia. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction A wealth of neuroimaging studies has shown gray matter abnormalities in schizophrenia, which foremost affect the limbic system including the medial temporal lobe, the hippocampus and entorhinal cortex (Bogerts et al., 1990) as well as the prefrontal cortex, particularly the dorsolateral surface (Gur et al., 2000; for a recent review, see Steen et al., 2006; Vita et al., 2006). Much less is known about the onset of morphological changes, and whether or not they indicate vulnerability to schizophrenia preceding manifestation of the disorder. The prodromal phase of schizophrenia has therefore become a target of current research to clarify these questions. A recent study has demonstrated gray matter volume reduction in the fronto-temporal regions (right medial and lateral temporal and

⁎ Corresponding author. LWL Clinic Bochum, Psychiatry – Psychotherapy – Psychosomatic Medicine – Preventive Medicine, University of Bochum, Alexandrinenstr. 1, 44791 Bochum, Germany. Tel.: +49 234 5077 201; fax: +49 234 5077 204. E-mail address: [email protected] (G. Juckel). 0925-4927/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pscychresns.2008.08.002

right inferior frontal cortex) as well as in the anterior and posterior cingulate gyrus bilaterally of ultra-high risk (UHR) subjects who later developed psychosis, compared to UHR subjects who did not. After transition into psychosis, a significant decrease of gray matter volume was noted in the left parahippocampal gyrus, left fusiform gyrus, and left orbitofrontal cortex, and in the cingulate gyrus bilaterally at follow-up (Pantelis et al., 2003). Borgwardt et al. (2007) have compared gray matter volume in UHR subjects, first-episode patients with schizophrenia and healthy subjects, and found gray matter reductions in UHR patients compared to controls in the left insula and superior temporal gyrus, in the midline region which included the posterior cingulate gyrus and precuneus, and in the left medial temporal cortex; those who later developed psychosis showed lower gray matter volume in a region which included the right insula and the adjacent part of the right anterior superior temporal gyrus compared to those who did not develop schizophrenia (Borgwardt et al., 2007). An earlier study of Phillips and colleagues demonstrated — in contradiction to their hypotheses — that UHR subjects who progressed into psychosis had bigger baseline hippocampi, particularly on the left, compared to UHR subjects who did not (Phillips et al., 2002), whereas Borgwardt et al. (2007)

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Table 1 Demographic and clinical data of the 82 study participants.

Age, years Male/female Handedness: right–leftmixed or unknown Antipsychotic medication yes–no PANSS positive PANNS negative Whole-brain volume (cm3)

Statistics

Ultra-high risk (n = 30)

First-episode Healthy schizophrenia controls (n = 29) (n = 23)

25.1 ± 4.3 20/10 25–2–3

26.4 ± 6.1 16/7 13–5–5

25.7 ± 5.2 F(2,81) = 0.402, n.s. 17/12 χ2 = 0.748, n.s. 23–5–1 χ2 = 7.584, n.s.

12–18

6–17

0–29

12.0 ± 3.3 19.3 ± 5.1 13.5 ± 5.1 18.0 ± 5.1 1258 ± 144 1214 ± 107

χ2 = 14.090, P = .001

t = − 6.205, P b 0.001 t = − 3.149, P b 0.005 1262 ± 99 F(2,81) = 1.226, n.s.

failed to confirm this finding. Moreover, the hippocampi of UHR patients who later developed psychosis were larger than those of subjects with first-episode psychosis (Phillips et al., 2002). A main limitation of the study was the decision to have “psychosis" as the outcome variable, but not all of the subjects had developed schizophrenia. In another study patients with first-episode schizophrenia displayed a significant reduction in left hippocampal volume compared with healthy controls. It remained unclear, however, whether this decrease occurred at the prodromal phase or during the period of transition to the first schizophrenic episode, because UHR patients did not show a significant difference in hippocampal volume compared with either healthy controls or first-episode schizophrenic patients (Velakoulis et al., 2006). To test the hypothesis that gray matter volume reductions precede the first clinical manifestation of schizophrenia, we analyzed data from patients who were putatively in the prodromal phase of schizophrenia, the period of time preceding the transition into and manifestation of the first schizophrenic episode. These UHR subjects displayed non-specific, basic (i.e. negative) and attenuated or brief, self-limiting psychotic symptoms, as well as functional impairment and social disabilities (Miller et al., 1999; Klosterkotter et al., 2001; Yung et al., 2004). Specifically we hypothesized that (1) UHR patients would show gray matter volume reduction in temporal and frontal areas including the anterior cingulate cortex (ACC) compared with healthy controls; (2) patients with first-episode schizophrenia would show smaller gray matter volumes than the UHR patients. 2. Methods 2.1. Subjects Eighty-two subjects (30 UHR patients, 23 first-episode schizophrenic patients and 29 healthy controls) aged between 18 and 38 years (mean: 25.7 ± 5.2) took part in the study. Table 1 summarizes the demographic and clinical parameters. Before scanning, less than half of the UHR patients (12/30) had been treated with either risperidone or olanzapine in low dosages for a maximum period of 3 weeks. Six of the 23 patients with first-episode schizophrenia had received typical or atypical antipsychotic medication in clinical dosages, but only for less than 10 days. The others were completely antipsychotic-naïve at the time of investigation just after admission to the hospital. Within 9 months after magnetic resonance imaging (MRI), one of the 30 UHR patients made the transition to schizophrenic psychosis. Fifteen of the UHR patients were continuously treated with antipsychotic medication, in an attempt to slow or prevent the transition to full-blown psychosis. We lost contact with seven patients, who were presumed, based on the clinical information received, to have made the transition to schizophrenia. The UHR subjects were recruited by the Early Recognition and Intervention Center (ERIC), Department of Psychiatry and Psychotherapy of the Charité Berlin, and diagnosed according to the Structured Interview for Prodromal Symptoms (SIPS) (McGlashan et al., 2001). The recruit-

ment criterion was at least one attenuated positive symptom with a severity level of at least “three” (Table 2). First-episode schizophrenic patients were in-patients or had frequent contact with the outpatient clinic. Before being included in the study, all subjects underwent medical examinations and blood and urine tests to exclude physical health problems. The healthy control subjects were screened with the MiniInternational Neuropsychiatric Interview (Sheehan et al., 1998) for mental disorders in themselves or in their family family history. None of them were receiving medication except birth control pills. Subjects with a history of neurological or severe somatic illness, head injury, alcohol dependence, or substance abuse were excluded. We determined handedness using the Edinburgh Handedness Inventory (Oldfield, 1971) and estimated premorbid verbal IQ using the MWT-B (Lehrl, 1978). Psychopathological state was assessed with the Positive and Negative Syndrome Scale (PANSS) (Kay et al.,1987) and the SIPS (McGlashan et al., 2001). The study was approved by the local ethics committee and carried out in accordance with the Declaration of Helsinki (current version: Edinburgh 2000). All subjects gave their informed consent after the study design and procedures had been fully explained to them. A neuroradiologist reviewed all MRI brain scans; no gross abnormalities were observed. 2.2. Structural MRI scanning protocol and data processing MRI was performed on a 1.5-Tesla Siemens MAGNETOM Symphony Scanner. A 3-D structural MRI was acquired on each subject using a T-1 weighted MPRAGE sequence (TR 2280 ms, TE 3.93 ms, TI 1100 ms, flip angle 15°, matrix size 256×256, FOV 256×256 mm2, yielding 160 transversal slices with a thickness of 1 mm, resulting voxel size 1×1×1 mm3). Data were analyzed using MATLAB 6.5 (MathWorks, Natick, MA) and SPM2 (Wellcome Department of Cognitive Neurology, London; http://www.fil.ion.ucl.ac.uk/spm). For preprocessing of structural MRI data, we used an optimized method of MRI to normalize and segment, modulate and smooth the images (Ashburner and Friston, 2000; Good et al., 2001; Silver et al., 2001). SPM2 was applied to determine group differences in regional gray matter volumes and concentrations and to correlate structural changes with psychopathological scales. Before Table 2 Inclusion and exclusion criteria. Ultra-high risk patients Inclusion criteria: 1) Attenuated psychotic symptoms – Presence of at least on of the following symptoms: ideas of reference, odd beliefs or magical thinking, unusual perceptual experiences, odd thinking and speech, suspiciousness or paranoid ideation, odd behavior or appearance (SIPS score of 3,4, or 5) – Frequency of symptoms: at least several times a week – Duration of mental state change for at least 1 week within the last 3 months 2) NO substance abuse – For cannabis there had to be a drug-free period of at least 3 months if a symptom was not definitely present before the use of any drug. The respondent could, however, be included if the symptoms had been present before cannabis use. – If a symptom was definitely present before the use of cannabis, the respondent could be included with present abuse. – For hallucinogenics and amphetamines, the drug-free period had to be 3 months, i.e. new symptoms had to still be present after the drug-free period. Exclusion criteria: acute psychosis – Presence of at least one of the following psychotic symptoms: delusions, formal thought disorders or hallucinations (PANSS score P1–P3, P5–P6 greater/equal 4 within the last 3 months) – Frequency of symptoms: at least several times a week – Duration of mental state change is longer than 1 week First-episode schizophrenic patients Inclusion criteria: acute psychosis (see exclusion criteria for ultra-high risk patients) Exclusion criteria for all patients and healthy controls – Organic mental disorder – Alcohol dependence in history – Cannabis abuse in last 3 months – Verbal IQ below 85 – Gross abnormalities in the brain scan

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preprocessing, all the structural images were checked for artifacts and the center point was placed on the anterior commissure. After conversion into ANALYZE format, MRI scans were spatially normalized; they were transformed into the same stereotactic space by registering each of the images to our T1 template, using the residual sum of squared differences as the matching criterion. The T1 template was created from our 82 subjects in order to reduce any scanner-specific bias. The first step in spatial normalization involved estimating the optimum 12-parameter affine transformation to match images (Ashburner et al., 1997). A Bayesian framework was used, whereby the maximum a posteriori estimate of the spatial transformation was made using prior knowledge of the normal variability in brain size. The second step accounted for global non-linear combination of smooth spatial basis functions (Ashburner and Friston, 1999). A masking procedure was used to weight the normalization to brain rather than non-brain tissue. The spatially normalized images were resliced with a final voxel size of 1×1×1 mm3. Scans were then segmented into gray matter, white matter and cerebrospinal fluid (CSF). SPM2 segmentation employed a mixture model cluster analysis to identify voxel intensities matching particular tissue types (gray matter, white matter, CSF) combined with an a priori knowledge of the spatial distribution of these tissues in normal subjects, derived from probability maps. The segmentation step also incorporated an image intensity nonuniformity correction to address image intensity variations caused by different positions of cranial structures within the MRI head coil (Worsley et al., 1995). Spatial normalization resulted in a growing and shrinking of regional volumes. In order to preserve the volume of a particular tissue within a voxel, a modulation of voxel values with the Jabobian determinant in the segmented images was necessary. Modulated data enabled an analysis of regional differences in volume of gray or white matter or CSF (Good et al., 2001). The normalized, segmented and modulated images were smoothed using a 12-mm FWHM isotropic Gaussian kernel. This step made the subsequent voxel-by-voxel analysis more comparable to a region of interest (ROI) approach, because each voxel in the smoothed images contained the average concentration of the gray matter from the selected voxel and, to a lesser extent, from neighboring voxels (the smoothed volume can be thought of as a weighted ROI). In accordance with the central limit theorem, smoothing also had the effect of making the data more normally distributed, increasing the validity of parametrical statistical tests (Worsley et al., 1995). Moreover, the smoothing step helped to compensate for the inexact nature of spatial normalization.

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the amygdala bilaterally, and for the left parahippocampus and the left fusiform gyrus. For group comparisons we used brain images with all ROI masks named above and defined an overall threshold of significance of P b 0.05 (uncorrected). Afterwards small volume correction was performed using WFU Pickatlas masks and cluster sizes were calculated. The spatial coordinates obtained from the SPM2 results were converted to standard Talairach brain coordinates (with mni2tal function by Matthew Brett; http://imaging.mrc-cbu.cam.ac. uk/imaging/MniTalairach) and then entered into Talairach Daemon Client. To compare the magnitude of mean differences, and to distinguish substantive from statistically significant results, Cohen d standardized effect sizes were calculated from the pairwise comparisons. Mean and standard deviation were taken from the volumes of interest that were defined by a sphere with a diameter of 4 mm. An absolute value of an effect size of 0.20 is typically regarded as small, 0.50 as moderate, and 0.80 as large. 3. Results UHR patients, the first-episode patients with schizophrenia and healthy controls did not differ with regards to age, gender, and handedness. The first-episode patients had significantly higher values of PANSS positive and PANSS negative scores than the UHR group; 18 out of 53 patients received antipsychotic medication before MRI brain scans were performed (see Table 1). Whole-brain volume did not differ significantly between the three groups (F(2, 81) = 1.226, n. s.). Using t-contrasts, we found the regionally specific differences in gray matter volume between the three groups that are reported below. 3.1. Comparison between UHR patients and healthy controls Fig. 1 depicts gray matter volume reduction in UHR subjects compared to healthy controls (significance level set at P b 0.05, uncorrected). The cluster sizes, Z-scores, effect sizes, and Talairach coordinates for these areas are given in Table 3. Significantly smaller volume was found in the right and left ACC and middle portion of the cingulate cortex, in the left posterior cingulate gyrus, in the right inferior frontal and right superior temporal gyrus, as well as in the right and left hippocampus. The largest difference of gray matter

2.3. Data and statistical analysis Group differences in demographic and clinical parameters were assessed by analysis of variance (ANOVA) and chi-square test. Correlations between volumes of interest and psychopathology were calculated using Pearson's correlation coefficient. The normalized, segmented, modulated, and smoothed data were analyzed using statistical parametric mapping (SPM2) employing the framework of the General Linear Model (Friston et al., 1995). To test for regional differences in gray matter between groups, we modeled the data with an analysis of covariance (ANCOVA). Age, sex, and brain volume were entered as covariates; age and sex were included because of their known influence on brain morphology (Good et al., 2001), and brain volume was entered because its inclusion permits correction for different global tissue volume. Brain volumes were calculated by the sum of gray and white matter. For our hypothesisdriven focus on specific areas, results from ANCOVA were masked with ROI masks from the Wake Forest University (WFU) Pickatlas (Maldjian et al., 2003; 2004). The WFU Pickatlas toolbox provided an atlas-based method of generating ROIs. Using the automated anatomical labeling atlas (aal) (Tzourio-Mazoyer et al., 2002), we created ROI masks for the left and right anterior, middle and posterior cingulate gyrus, for the left orbitofrontal and the right inferior frontal gyrus, for the right superior temporal gyrus and temporal pole, for the hippocampus and

Fig. 1. Glass brain image of gray matter reductions in the right and left ACC and middle cingulate cortex, in the left posterior cingulate cortex, in the right inferior frontal and right superior temporal gyrus, and in the hippocampus bilaterally of UHR patients in comparison to healthy controls (SPM indicates statistical parametric map; Puncorr b 0.05). (The gray shades indicate the Z-scores that are presented, in part, in Table 3), darker gray means higher Z-score.

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Table 3 Brain regions in which voxels showed significantly decreased gray matter volume in UHR patients compared with healthy controls (Puncorr b 0.05; df = 1, 76).

Table 4 Brain regions in which voxels showed significantly decreased gray matter volume in first-episode schizophrenia compared with UHR patients (Puncorr b 0.05; df = 1, 76).

Region

Region

ACC Middle cingulate cortex Post. cingulate cortex Inferior frontal gyrus Sup. temporal gyrus Hippocampus

L R L R L R R L R

Cluster Z size (K)

Effect size

Talairach coordinates x

y

z

5049 5391 1350 3733 101 34 2737 12 1524

− 0.33 − 0.35 − 0.33 − 0.40 − 0.13 − 0.21 − 0.26 − 0.19 − 0.25

−8 14 − 11 15 −7 34 49 − 28 30

29 26 8 14 − 38 42 − 17 − 39 − 32

14 25 32 32 23 − 20 6 −1 3

2.93 3.18⁎ 3.06 3.64⁎ 1.87 1.91 2.48 1.82 2.32

BA 24 32 24 24 23 11 22

⁎ Significant at P b 0.05 FWE-corrected.

volume was found in the right ACC and middle portion of the cingulate cortex. These two regions were still significant after correcting for a Family Wise Error (FWE) (P b 0.05; degrees of freedom (df) = 1, 76). There was no brain area where the UHR patients showed significantly more gray matter volume than the controls (with significance of uncorrected Pb 0.05; df =1, 76). 3.2. Comparison between first-episode patients with schizophrenia and UHR patients

Middle cingulate cortex Post. cingulate cortex Orbitofrontal gyrus Inferior frontal gyrus Sup. temporal gyrus Temporal pole Hippocampus Parahippocampus Amygdala Fusiform gyrus

L R L R L R R R L R L L L

Cluster size (K)

Z

9859 10717 1249 748 2950 1892 5271 1575 1408 164 1408 410 1015

3.35⁎ 2.69 2.09 2.32 2.36 2.47 2.94 2.07 2.47 2.26 1.87 1.97 2.34

Effect size

Talairach coordinates x

y

z

− 0.41 − 0.30 − 0.24 − 0.25 − 0.30 − 0.27 − 0.34 − 0.27 − 0.33 − 0.23 − 0.33 − 0.24 − 0.21

− 14 12 0 10 − 20 63 48 51 − 10 42 − 30 − 21 − 30

− 38 − 39 − 40 − 47 49 15 − 19 3 1 − 16 −9 −1 − 62

44 46 32 31 − 13 14 −7 −2 − 21 − 10 − 21 − 22 1

BA 5 7 31 31 11 44 22 22

19

⁎ Significant at P b 0.05 FWE-corrected.

3.3. Comparison between first-episode patients with schizophrenia and controls

Fig. 2 shows brain areas where first-episode schizophrenic patients had less regional gray matter volume than the UHR patients (uncorrected P b 0.05; df = 1, 76). The cluster sizes, Z-scores, effect sizes, and the Talairach coordinates for these areas are given in Table 4. Significant decreases were found in the left and right middle and posterior cingulate cortex, in the left orbitofrontal gyrus, in the right inferior frontal and superior temporal gyrus, in the right temporal pole, in the left and right hippocampus, in the left parahippocampus and left amygdala, and in the left fusiform gyrus. The global maximum of reduced volume was found in the left middle cingulate cortex and it was still significant after FWE correction (P b 0.05; df = 1, 76). We found no significant differences of gray matter volume in the first-episode schizophrenic patients compared to the UHR patients (with significance of uncorrected P b 0.05; df = 1, 76).

Fig. 3 illustrates gray matter volume differences between firstepisode schizophrenic patients and healthy subjects (uncorrected P b 0.05; df = 1, 76). We found smaller gray matter volume in all observed regions except in the amygdala bilaterally. The cluster sizes, Z-scores, effect sizes, and the Talairach coordinates for these areas are given in Table 5. The global maximum of reduced volume was found in the right superior temporal gyrus (PFWE-corr b 0.01). Further significant decreases after FWE correction were found in the whole right and in the left middle and posterior cingulate cortex (PFWE-corr b 0.05) as well as in the right hippocampus (PFWE-corr b 0.01). There was an area of 460 voxels in the right inferior frontal gyrus where the healthy controls had smaller gray matter volume than the schizophrenic patients (with significance of uncorrected P b 0.05 but without significance after FWE correction). There were no significant relationships between gray matter volumes of anterior cingulate regions and positive and negative

Fig. 2. Decreased gray matter volume in the left and right middle and posterior cingulate cortex, in the right inferior frontal and superior temporal gyrus, in the right temporal pole, in the left and right hippocampus, in the left parahippocampus and left amygdala, and in the left fusiform gyrus of patients with first-episode schizophrenia, compared to UHR patients (with significance of uncorrected P b 0.05).

Fig. 3. Decreased gray matter volume in the cingulate cortex bilaterally, in the left orbitofrontal and right inferior gyrus, in the right superior temporal gyrus and temporal pole, in the hippocampus bilaterally, in the left parahippocampus, and in the left fusiform gyrus of first-episode patients with schizophrenia in comparison to healthy controls (with significance of uncorrected P b 0.05).

H. Witthaus et al. / Psychiatry Research: Neuroimaging 173 (2009) 163–169 Table 5 Brain regions in which voxels showed significantly decreased gray matter volume in patients with first-episode schizophrenia compared with control subjects (Puncorr b 0.05). Region ACC

L R Middle cingulated cortex L R Post. cingulated cortex L R Orbitofrontal gyrus L Inferior frontal gyrus R Sup. temporal gyrus R Temporal pole R Hippocampus L R Paraphippocampus L Fusiform gyrus L

Cluster Z size (K)

Effect size

Talairach coordinates x

y

z

8688 6504 14976 16616 2801 1388 6521 5724 19281 2203 1667 3155 1667 608

− 0.39 − 0.43 − 0.47 − 0.45 − 0.41 − 0.39 − 0.33 − 0.40 − 0.52 − 0.32 − 0.29 − 0.42 − 0.29 − 0.21

−1 13 −1 0 0 5 − 25 50 49 55 − 21 41 − 21 − 23

37 25 − 40 −9 − 40 − 34 62 10 − 18 5 31 − 22 − 34 − 34

21 24 40 28 32 30 −5 15 −8 1 −7 −7 −3 − 11

2.98 3.35⁎ 3.48⁎ 3.49⁎ 3.17⁎ 3.11⁎ 2.94 3.15 4.07⁎⁎ 2.32 2.54 3.64⁎⁎ 2.48 2.13

BA 32 32 31 24 31 31 10 44 22 22

36

⁎ Significant at P b 0.05 FWE-corrected. ⁎⁎ Significant at P b 0.01 FWE-corrected.

psychopathology, as measured by the PANSS, as well as the medication state (medicated/unmedicated) in both UHR subjects and first-episode patients. 4. Discussion The findings of our study largely support our hypotheses. UHR patients showed smaller gray matter volumes than controls of the right inferior frontal and superior temporal gyrus (but not the temporal pole) and the cingulate cortex bilaterally (except the right posterior cingulate gyrus). UHR patients also had lower gray matter volume in the hippocampus bilaterally than the healthy volunteers. Our study confirms previous findings that there is no significant difference in the amygdala volume bilaterally between UHR patients and controls. As predicted first-episode schizophrenic patients displayed lower gray matter volume than the UHR patients in the left orbitofrontal and fusiform gyrus, in the left parahippocampus and in the middle and posterior cingulate gyrus bilaterally, but we found no significant gray matter volume difference in the left and right ACC between the two groups. In comparisons of UHR patients to healthy control subjects, only two regions showed significant differences in gray matter volume after FWE correction: the right anterior and right middle cingulate cortex. First-episode schizophrenic patients also had (FWE-corrected) significantly reduced gray matter volume in these two regions compared to controls. No differences in gray matter volume were found between the two patient groups in right ACC (at significance level of uncorrected P b 0.05). Reduction of gray matter volume could not be attributed to age, sex or total brain volume effects. There is considerable evidence that the cingulate gyrus plays an important role in emotional behavior and attention. A study of lesions found that patients after cingulotomy (patients treated with small bilateral lesions in the ACC to alleviate pain) showed deficits of focused and sustained attention as well as mild executive dysfunction (Cohen et al., 1999a,b). Such cognitive deficits are also prominent features of schizophrenia (Dollfus et al., 2002). The cingulate cortex is an integral part of a larger frontal–subcortical brain system (Murray and Lewis, 1987; Morecraft et al., 1993). Functional, anatomical, and histopathological studies have accumulated evidence that the connections between sub-regions of the cingulate cortex and many cortical and subcortical brain regions are altered in schizophrenia (Benes, 1993). Yucel et al. (2003) found morphological abnormalities of the ACC including under-developed left paracingulate sulcus and interrupted left cingulate sulcus in young men at UHR of developing a psychotic illness. These abnormalities did not identify individuals that

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subsequently transit to psychosis because there was no significant difference in morphological abnormalities between those who did and those who did not subsequently develop a schizophrenic illness. On the other hand, there is some evidence that reduction of gray matter volume of the cingulate gyrus predisposes to psychosis. Pantelis et al. (2003) found that UHR patients who had developed psychosis in the following 12 months showed significantly less gray matter in the cingulate cortex bilaterally than those UHR patients who did not develop psychosis. In addition, UHR subjects without a family history of schizophrenia showed smaller left ACC volumes (reduced paracingulate folding and cingulate sulcus interruption), as compared to those with such history (Wood et al., 2005). It remains, however, unclear at what stage of the disorder structural brain abnormalities emerge, i.e. whether or not morphological abnormalities parallel the onset of attenuated psychotic symptoms or predate clinical manifestation. Job et al. (2003) found reductions of gray matter volume bilaterally in the ACC in mostly asymptomatic subjects at genetic high risk of schizophrenia compared with healthy subjects. Furthermore, reduced gray matter volume in prefrontal cortex and low performance of cognitive executive functioning were associated with schizotypical features, i.e. corresponded to the clinical UHR state, in genetically predisposed offspring (Diwadkar et al., 2006). It is therefore plausible to assume that these morphological abnormalities pose a risk factor for psychosis. Pantelis et al. (2003) have argued that the onset of psychosis is a time of active brain changes: An early prenatal and perinatal neurodevelopmental lesion renders the brain vulnerable to anomalous late postpubertal neurodevelopmental processes, as indicated by accelerated loss of gray matter. This is prominent in prefrontal and cingulate regions and appears to be related to premorbid neuropsychological deficits in executive function. The reduction of ACC gray volume in the UHR subjects as well as in the first-episode patients without a tendency toward progression as found in the present study can be interpreted in support of this assumption. According to the hypothesis of Pantelis and coworkers, early neurodevelopmental insults interact with postpubertal brain maturation (including causative stress factors associated with the onset of psychosis) to produce further neurodevelopmental brain structural and functional changes especially within the temporal and frontal lobes. Accordingly, further gray matter loss in temporal and frontal regions was found in first-episode patients with schizophrenia in the present study, as compared to the UHR patients. Our findings that first-episode schizophrenic patients had less gray matter volume in right inferior frontal, left orbitofrontal, and right medial and lateral temporal regions than controls are consistent with previous research. Several studies identified structural brain abnormalities in the temporo-limbic structures and in the frontal lobe of patients with schizophrenia. In a review of MRI findings in schizophrenia, Shenton et al. (2001) found strong evidence for the involvement of the superior temporal gyrus and the medial temporal lobe (which includes the amygdala, hippocampus, and parahippocampal gyrus) and moderate evidence for frontal lobe abnormalities. A meta-analysis of VBM studies that investigated regional deficits in brain volume in schizophrenia concluded that 7 out of 14 studies found significant volume deficits in the right superior temporal gyrus and 5 of 14 in the left inferior frontal gyrus (Honea et al., 2005). Healthy subjects not only have significantly larger gray matter volume of the right superior temporal, the right inferior frontal gyrus and the hippocampus bilaterally than the first-episode schizophrenic patients but also compared with the UHR patients. Our study results support the suggestion of Pantelis and his colleagues that some neuroanatomical abnormalities predate the onset of frank psychotic symptoms (Pantelis et al., 2003). Progressive changes might be happening in these four regions because UHR patients showed even more gray matter volume here than first-episode schizophrenic patients. In contrast to Velakoulis et al. (2006), we we found smaller hippocampal volumes bilaterally in patients at UHR for psychosis

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compared with healthy subjects. Velakoulis et al. reported that UHR patients had normal baseline hippocampal volumes irrespective of subsequent development of psychosis. They concluded that structural changes in the hippocampus (and whole medial temporal gyrus) are not seen until after onset of psychosis. By contrast, our findings concerning hippocampal volume are more consistent with a previous study of Phillips and colleagues, who also reported reduced gray matter in UHR individuals compared to controls (Phillips et al., 2002). Surprisingly, in our study in UHR patients who later developed a psychotic disorder, the volume of the left hippocampus was larger compared with the non-psychotic UHR and the first-episode groups. Contradictory findings have been reported regarding the involvement of the amygdala in the pathogenesis of schizophrenia. In accordance to the study of Velakoulis and colleagues, we found no significant amygdala gray matter volume difference in both the firstepisode and UHR groups compared with healthy volunteers. Consistent with the known role of the amygdala in affect regulation (Gloor, 1997), structural changes of the amygdala rather seem to be involved in nonschizophrenic psychosis: Bilateral amygdala enlargement was present in first-episode nonschizophrenia patients (Velakoulis et al., 2006) and in patients with bipolar disorder (Altshuler et al., 1998; Strakowski et al., 2007). We found, however, that the left amygdalar volume in first-episode schizophrenic patients was smaller than in UHR patients. Manual tracing techniques were used to measure amygdalar volumes of the same individuals a second time. With this different technique first-episode patients displayed significantly smaller left amygdalar volumes than UHR patients and normal controls; no differences between the groups emerged regarding the right amygdalar volume (unpublished data). Our findings suggest disruption of the left amygdala in schizophrenia that might emerge during the prodromal phase. A critical issue is whether the differences between the UHR group and the other groups are being driven by a subgroup of individuals who will convert to schizophrenia. There was one transition to schizophrenia over 9 months of follow-up in the 23 UHR cases (cases with which we did not lose contact). Previous studies found a transition rate of 20 to 40% (Pantelis et al., 2003; Yung et al., 2003; Mason et al., 2004; Borgwardt et al., 2007). The transition rate of our 23 UHR patients was low, because 15 of them were medically treated to prevent conversion. Because we intended to prevent UHR patients from transition into schizophrenic psychosis (that was the aim of ERIC), we cannot present sufficient data to compare whose who did and did not make the transition to schizophrenia. It cannot be ruled out that our study ascertained a largely schizotypal population that is not so much at ultra-high risk for developing schizophrenia but is manifesting a more stable syndrome indicative of a genetic liability to schizophrenia. MRI studies of subjects with schizotypal personality disorder showed gray matter reductions in fronto-temporal regions similar to patients with schizophrenia (Kawasaki et al., 2004; Koo et al., 2006). Several limitations of this study have to be considered. First, the sample size is relatively small, which may restrict the generalization of these findings to a larger group of patients at UHR to transit to schizophrenic psychosis. Secondly, this was not a longitudinal study, so that we do not know exactly if all of our putatively prodromal patients will develop schizophrenic episodes or any kind of frank psychosis in terms of DSM-IV, and, moreover, which kind of psychotic disorder will emerge (Rapoport et al., 1999). These different types of psychosis, i.e. mainly schizophrenic psychoses and the several known clinical subgroups, schizoaffective disorder, psychotic bipolar disorder or psychotic depression, may be associated with different neuroanatomical changes. However, the similarities between the morphological aberrations in our UHR group and in our group with first-episode schizophrenia may indicate that the observed decreases of gray matter volume are more likely associated with an enhanced risk for schizophrenia. The number of participants in our study precluded an

analysis of schizophrenic subgroups. Thirdly, influence of antipsychotics cannot be completely excluded since approximately one third of the patients received treatment. The influence of antipsychotic medication on gray matter volume is still unclear. A few follow-upstudies found a significant correlation between a decrease in gray matter volume and higher cumulative dosage of antipsychotic medication in first-episode schizophrenic patients (Gur et al., 1998; Madsen et al., 1999; Cahn et al., 2002). In contrast to these findings in first-episode patients, there was no relationship between antipsychotic dosage and gray matter volume in patients with chronic schizophrenia (Gur et al., 1998) or in patients with childhood-onset schizophrenia (Rapoport et al., 1999). A meta-analysis of post-mortem studies showed no effects of antipsychotic treatment on brain morphology of patients with schizophrenia (Baldessarini, 1997). It was also shown that typical, but not atypical, antipsychotics have a substantial effect on gray matter volume (Garver et al., 2005; Lieberman et al., 2005). All patients presented here, however, had received atypical antipsychotic medication for only a short period of time prior to scanning, such that the effect of neuroleptics on gray matter volume, if any at all, may be negligible. In addition, no relationship between gray matter volume of ACC and medication state was found in the current study. Finally, in the present study a voxelbased automated image analysis method was used which keeps operator biases to a minimum and permits analysis of the whole brain rather than a few regions of interest. Although there has been some debate about the relative merits of this approach compared with region of interest (ROI) techniques, there is good correlation between results from these methods, and voxel-based analyses have produced relatively consistent results in studies of patients with schizophrenia (Wright et al., 1999; Sigmundsson et al., 2001; Kubicki et al., 2002; Giuliani et al., 2005). Acknowledgement This study was supported in part by an unrestricted grant from the Charities Aid Foundation (Janssen-Cilag Ltd.). The results of the study constitute part of the doctoral thesis of Henning Witthaus. References Altshuler, L., Bartzokis, G., Grieder, T., Curran, J., Mintz, J., 1998. Amygdala enlargement in bipolar disorder and hippocampal reduction in schizophrenia: an MRI study demonstrating neuroanatomic specificity. Archives of General Psychiatry 55, 663–664. Ashburner, J., Friston, K.J., 1999. Nonlinear spatial normalization using basis functions. Human Brain Mapping 7, 254–266. Ashburner, J., Friston, K.J., 2000. Voxel-based morphometry—the methods. Neuroimage 11, 805–821. Ashburner, J., Neelin, P., Collins, D.L., Evans, A., Friston, K., 1997. Incorporating prior knowledge into image registration. Neuroimage 6, 344–352. Baldessarini, R.J., 1997. Meta-analysis of postmortem studies of Alzheimer's disease-like neuropathology in schizophrenia. American Journal of Psychiatry 154, 1180. Benes, F.M., 1993. Neurobiological investigations in cingulate cortex of schizophrenic brain. Schizophrenia Bulletin 19, 537–549. Bogerts, B., Ashtari, M., Degreef, G., Alvir, J.M., Bilder, R.M., Lieberman, J.A., 1990. Reduced temporal limbic structure volumes on magnetic resonance images in first episode schizophrenia. Psychiatry Research 35, 1–13. Borgwardt, S.J., Riecher-Rossler, A., Dazzan, P., Chitnis, X., Aston, J., Drewe, M., Gschwandtner, U., Haller, S., Pfluger, M., Rechsteiner, E., D'Souza, M., Stieglitz, R.D., Radu, E.W., McGuire, P.K., 2007. Regional gray matter volume abnormalities in the at risk mental state. Biological Psychiatry 61, 1148–1156. Cahn, W., Pol, H.E.H., Lems, E.B.T.E., van Haren, N.E.M., Schnack, H.G., van der Linden, J.A., Schothorst, P.F., van Engeland, H., Kahn, R.S., 2002. Brain volume changes in firstepisode schizophrenia — a 1-year follow-up study. Archives of General Psychiatry 59, 1002–1010. Cohen, R.A., Kaplan, R.F., Moser, D.J., Jenkins, M.A., Wilkinson, H., 1999a. Impairments of attention after cingulotomy. Neurology 53, 819–824. Cohen, R.A., Kaplan, R.F., Zuffante, P., Moser, D.J., Jenkins, M.A., Salloway, S., Wilkinson, H., 1999b. Alteration of intention and self-initiated action associated with bilateral anterior cingulotomy. Journal of Neuropsychiatry and Clinical Neurosciences 11, 444–453. Diwadkar, V.A., Montrose, D.M., Dworakowski, D., Sweeney, J.A., Keshavan, M.S., 2006. Genetically predisposed offspring with schizotypal features: an ultra high-risk group for schizophrenia? Progress in Neuro-Psychopharmacology and Biological Psychiatry 30, 230–238.

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