Ethical approval

The study was approved by the joint South London and Maudsley National Health Service Foundation Trust Ethics Committee and all participants gave written consent to participate after full details of the study were explained.

Participants

Twenty-six individuals meeting criteria for an at-risk mental state (ARMS) were recruited from OASIS (Outreach and Support in South London),19 a clinical service for people at risk of developing psychosis within the South London and Maudsley National Health Service Foundation Trust. The diagnosis was based on Personal Assessment Crisis Evaluation criteria,20 as assessed by two expert clinicians using the comprehensive assessment of at-risk mental states (CAARMS)21 and confirmed at a consensus clinical meeting. All participants were antipsychotic naive at the time they took part in the study while five were taking antidepressant medication. Seventeen control subjects were recruited over the same period from the same sociodemographic area. Participants were aged 18 to 30 years and were excluded if their intelligence quotient was below 70, if they had a history of a neurological disorder or severe head injury or if they met DSM-IV criteria for an alcohol or substance dependence disorder other than nicotine. An additional exclusion criterion for control subjects was a family history of psychosis.

All the UHR participants were followed up by OASIS for at least 2 years after first contact and monitored for signs of transition to psychosis.

Clinical measures

CAARMS,21 Positive and Negative Syndrome Scale (PANSS),22 Hamilton Anxiety Rating Scale (HAM-A)23 and Hamilton Depression Rating Scale (HAM-D)24 were used on the day of scanning to assess and rate symptom severity.

Salivary cortisol

Salivary cortisol was collected in a naturalistic, non-clinical environment. Participants received verbal and written step-by-step instructions to use Salivettes (Sarstedt, Leicester, UK) and return them in a pre-paid envelope. Participants were instructed to wake up before 1000 h to collect saliva samples immediately at awakening (0 minutes) and then after 30 and 60 minutes. They were asked to abstain from consuming alcohol the night preceding collection and asked not to eat, drink, brush their teeth or engage in physical activity during the 60-minute collection period. Samples were stored at a temperature of −20 °C until they were centrifuged at 3500 rpm for 10 minutes at 6 °C to separate saliva from the pad. Saliva was then transferred from the Salivettes to microtubes and stored at −80 °C until a continuous, automated, competitive chemiluminescence immunoassay was performed using the Immulite immunoassay analyzer system (DPC; www.diagnostics.siemens.com)25 to determine free cortisol concentration. The percentage cross-reactivity of the antiserum with cortisone and prednisolone was 0.35% and 27.5%, respectively. The area under the curve for the cortisol awakening response (CAR) was calculated using cortisol levels at 0, 30 and 60 minutes after awakening with formulae described by Pruessner et al.26 The validity of the sampling is dependent upon timing, with delayed collection leading to an underestimation of peak response.27 A negative difference between the samples at time 0 and 30 minutes (Δ30) is considered indicative of delayed collection of the first sample with recommended exclusion from the analysis.28 Any participant with a missing sample or one characterized by very low salivary volume (<200 μl), one reportedly collected 15 minutes before or after the indicated time point or providing a negative Δ30 was therefore excluded from the study.

Demographic and cortisol measures were compared between the two groups using independent sample two-tailed t-tests, as variables were normally distributed. Chi-square was used for categorical variables.

Image acquisition and analyses

Volumetric magnetic resonance images were acquired using a General Electric (Milwaukee, WI, USA) 3 T magnetic resonance system. A whole-brain three-dimensional coronal inversion recovery prepared spoiled gradient echo scan was acquired with echo time 2.82 ms, repetition time 6.96 ms, inversion time 450 ms and flip angle 20°.

Group-related differences in grey matter volume (GMV) were analysed using voxel-based morphometry, implemented in SPM8 software (http://www.fil.ion.ucl.ac.uk/spm) running under Matlab 7.4 (MatWorks, Natick, MA, USA). T1-weighted volumetric images were preprocessed using the Diffeomorphic Anatomical Registration Through Exponentiated Lie algebra (DARTEL)29 SPM8 toolbox, iteratively registering grey matter by nonlinear warping to a template generated using DARTEL to obtain a high-dimensional normalization.29 A homogeneity check across the sample was followed by smoothing with an 8-mm full-width at half maximum (FWHM) Gaussian kernel. The normalization protocol included a ‘modulatory step’ to preserve information about the absolute grey matter values.30 We then looked for grey matter voxels in the normalized modulated smoothed data that correlated with CAR in all subjects. Age, gender and antidepressant medication were modelled in the analysis to reduce the potential impact of these variables on the findings. To identify specific changes not confounded by global volumetric differences, the proportional scaling option was used. We also looked for any existing differences in the relationship between cortisol response and cortical grey matter between UHR participants and controls. We thus used the general linear model to look for brain voxels in which this correlation differed according to the clinical status of the participants (UHR/control).

Use of a priori biological information to guide statistical inferences

Neuroimaging studies usually involve the analysis of multiple univariate comparisons, posing a multiple-comparison problem. We here corrected our results based on the expression of corticoid receptors in the brain, using this to threshold our results. Our rationale was that regions that had high levels of these receptors were more likely to be influenced by cortisol, reducing the likelihood that a correlation with local grey matter volume would be a false positive. We used data from the Allen Brain Atlas,18 a multimodal atlas integrating neuroanatomical and gene expression information in humans. Briefly, the Atlas is based on tissue samples collected postmortem from anatomically diverse regions of six healthy adult human brains. Microarray analyses of the samples gave information on the RNA expression levels of a large number of genes for each of the regions sampled. The information on gene expression distribution across different regions of the brain was then used to build a whole-brain atlas. We retrieved the normalized transcription rates of the GRs and MRs on all the available sampled regions, then divided the brain into anatomically defined regions following a widely used template.31 Where one of the template regions included more than one sampling site, microarray information from the multiple samples was averaged. In addition, the Brain Allen project designed their microarray analysis such that more than one probe would target the expression of a specific gene. In this case, the expression levels of the GR and MR genes were inferred to be the average expression of the different probes targeting them. We took the mean across subjects, and on the basis that one-third of regulated genes are responsive to both receptor types,32 we averaged measures for both GR and MR expression in one a priori mask (Figure 1). Average transcription of the receptors across class and subjects were used to determine a priori probabilities of a false positive as described below.

Figure 1 Method used to include a priori biological information. We used data from the Allen Brain Atlas (Allen Brain Atlas methodology summarized in (a–c) and described in detail in ref. 18) to create a new brain mask (d) used to flexibly threshold our results according to the expression of cortisol-binding receptors. (a) Information about expression levels of glucocorticoid (GR) and mineralocorticoid (MR) receptors was obtained from several parts of the brain of six healthy adults from the Allen Brain Atlas. (b) Samples obtained from the same region of interest of the template used were averaged. (c) Expression rates of probes targeting the same gene (MR or GR) were averaged. (d) Brain mask ranking regions according to their average expression of glucocorticoid and mineralocorticoid receptors in the healthy brain. The brain is shown in radiological convention (where the left side of the figure is the right side of the brain). Full size image

We assumed that a biological relationship with cortisol levels would be most likely in brain areas where the expression of cortisol receptor genes was highest. In these areas, the statistical threshold was set at P<0.01, uncorrected for multiple comparisons. At the other extreme, in areas with the lowest expression of these genes, the statistical threshold was set at P<0.05, Bonferroni-corrected for all voxels within the mask. All areas were ranked according to their expression level and assigned a statistical threshold between P<0.01 uncorrected and P<0.05 Bonferroni-corrected. This range of probabilities was divided into equally sized steps, and a threshold was assigned to each region according to its rank.