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SUPPLEMENTARY MATERIALAmygdala abnormalities in first-degree relatives of individuals with schizophrenia unmasked by benzodiazepine challengeDaniel H. Wolf, MD,PhD1, Theodore D. Satterthwaite, MD,MA1, James Loughead, PhD1, Amy Pinkham, PhD5, Eve Overton, BA1, Mark A. Elliott, PhD2, Gersham W. Dent, PhD4, Mark A. Smith, MD4, Ruben C. Gur, PhD,1,2,3 Raquel E. Gur, MD,PhD1,21 Department of Psychiatry, University of Pennsylvania, Philadelphia PA 19104, USA2 Department of Radiology, University of Pennsylvania, Philadelphia PA 19104, USA3 Philadelphia Veterans Administration Medical Center, Philadelphia PA 19104, USA4 AstraZeneca Pharmaceuticals LP, Wilmington DE 19850, USA5 Department of Psychology, Southern Methodist University, Dallas TX 75275, USA Contents of Supplementary Materials:Supplementary MethodsDetails on participant assessment, image acquisition, image processing and image analysis.Supplementary Table S1Behavioral performance: accuracy and reaction time.Supplementary Table S2Significant clusters from voxelwise analysis of emotion identification and memory tasks. Supplementary Fig. S1Illustration of acquired brain volume and regions of interest.Supplementary Fig. S2Illustration of task activation (vs. fixation baseline) for emotion identification and memory tasks.Supplementary Fig. S3Illustration of memory task performance relative to expected chance performance levels.Supplementary Fig. S4Illustration of inverse correlation between age and emotion identification performance.Supplementary Fig. S5Illustration of correlation between drug-induced reduction in hippocampal activation and drug-induced reductions in memory performance.Supplementary Fig. S6Whole brain global perfusion results.Supplementary ReferencesSupplementary methodsSubject assessment The clinical evaluation included the Diagnostic Interview for Genetic Studies (DIGS)(Nurnberger et al. 1994) and Family Interview for Genetics Studies (FIGS)(Maxwell 1992). Schizotypy was assessed with the Structured Interview for Schizotypy (SIS)(Kendler et al. 1989) included in the DIGS; total SIS scores were calculated as the sum of global items (SIS scores were unavailable for 1 family member). Anxiety was assessed using the State Trait Anxiety Inventory (STAI)(Spielberger et al. 1983); state anxiety was measured three times each study session: prior to drug administration (baseline), just before scanning, and immediately after the scanning session. Neurocognitive function was assessed with the Computerized Neurocognitive Battery (Gur et al. 2001a; Gur et al. 2001b; Gur et al. 2010). Interviews were administered by trained assessors with demonstrated reliability on symptom scales (reliability criterion 0.90 intraclass correlation). Participants had no current Axis I psychiatric disorders, or any history of a psychotic disorder. Axis II disorders were also exclusionary, with the exception of Cluster A personality disorders for family member participants, as Cluster A personality traits might reflect genetic risk for schizophrenia. Family members had a first-degree relative with DSM-IV Schizophrenia as determined through the FIGS interview, supporting medical records, and when possible a DIGS assessment of the ill family member. Control participants had no family history of a psychotic disorder or bipolar disorder in first- or second-degree relatives. Participants had no history of alcohol or other substance use disorders within the past 6 months, no history of benzodiazepine abuse or dependence at any time, and no recent substance use by history and confirmed by urine drug screen on each study day. Use of alcohol in the 24 hours preceding the study was exclusionary, as was use of psychoactive substances or medications within the prior 2 weeks, except for nicotine and caffeine where subjects maintained regular use to avoid withdrawal effects. Participants had no history of neurological or medical disorders or injuries affecting brain function, or which would contraindicate MRI scanning or benzodiazepine administration. Imaging acquisition procedures and parametersSubjects were placed in the scanner supine, earplugs were used to muffle scanner noise, head fixation was ensured by a foam-rubber device mounted on the head coil, and pulse and respiration monitors were attached. Stimuli were rear-projected to the center of the visual field using a PowerLite 7300 video projector (Epson America, Inc.; Long Beach, CA) and viewed through a head coil mounted mirror. Stimulus presentation was synchronized with image acquisition using the Presentation software package (Neurobehavioral Systems, Inc., Albany, CA). Subjects provided responses with a non-ferromagnetic response device (fORP, Current Designs, Inc., Philadelphia, PA) using their dominant hand. Button press responses and response latencies were recorded. Whole-brain structural data were obtained with a 5-minute magnetization-prepared, rapid acquisition gradient-echo T1-weighted image (MPRAGE, TR 1620ms, TE 3.87 ms, FOV 180x240 mm, matrix 192x256, effective voxel resolution of 1 x 1 x 1mm). Pulsed Arterial Spin-Label (PASL) perfusion MRI was used to measure absolute CBF at rest with eyes open viewing a black screen (Wang et al. 2003; Wong et al. 1998). Forty resting label/control PASL image pairs were acquired with the following parameters: FOV=220 mm, matrix=64X64, TR=4s, TE=17ms, flip angle=90, QUIPSS II (TI1 = 700 ms, TI2 = 1900 ms), 20 slices (6mm thick with 1.2mm gap). BOLD fMRI data was obtained as a slab single-shot gradient-echo (GE) echoplanar sequence using the following parameters: TR/TE=3000/32 ms, FOV=240 mm, matrix= 128 X 128, slice thickness/gap=2/0mm, 30 slices, effective voxel resolution of 1.875 x 1.875 x 2mm. Slices were obtained obliquely (axial/coronal) in order to reduce signal distortion in ventral brain regions. Online geometric distortion correction of the echo-planar images was performed using the DiCo sequence of Maxim Zaitsev (Zaitsev et al. 2004). This sequence implements a point-spread-function mapping method (Zeng and Constable 2002) acquired with a one minute reference scan.ImScribe automated field-of-view (FOV) prescriptionAn automated FOV-determining algorithm (ImScribe) was developed by M. Elliott in MATLAB (available at /mediawiki/index.php/ImScribe), utilizing the coregistration modules from SPM2 (http:/www.fil.ion.ucl.ac.uk/spm/). The program performs rigid-body (intra-subject) or affine (inter-subject) coregistration of a scout localizer to a study-specific template, and returns the optimally matched FOV for the functional images. Template structural and functional scans were acquired with a well-chosen FOV for the functional slab. During each subjects first fMRI session, the subjects structural scan (MPRAGE) was imported into the ImScribe program for coregistration to the structural template. The computed affine spatial transformation was then applied to the template functional FOV to determine the optimal FOV prescription in subject space. On the second scan day, the same procedure was used except that the subjects structural image and functional FOV from the first day was used as the template for rigid-body coregistration. The entire process required 2SD across all subjects) on both motion and tSNR measures were excluded; this corresponded to tSNR 0.25 mm. In all these cases, visual inspection confirmed severe motion-related artifact. As a result, two controls were excluded from all analyses; one additional control was excluded only from the identification task fMRI analysis, and one family member was excluded only from the recognition task fMRI analysis. A different family member lacked complete perfusion data due to technical problems, and was excluded from perfusion analysis.Region of interest (ROI) definitionAll structural ROIs were defined using the probabilistic Harvard-Oxford Structural Atlas in FSL. The bilateral amygdala ROI was thresholded at 75 (% probability); bilateral hippocampus was thresholded at 50. The right fusiform cortex ROI was obtained from the temporal occipital fusiform cortex atlas region thresholded at 25; this fusiform subdivision includes the coordinates reported in the literature for the fusiform face area (Fox et al. 2009) and corresponds closely to the fusiform activation area observed in this study. The strong activation clusters observed in both functional tasks in posterior orbitofrontal cortex extending into anterior insula were not well captured by available anatomical atlas divisions; therefore the bilateral orbitofrontal ROI was defined functionally, by thresholding the group map of activation across all conditions and all subjects at Z4.26 (p PlaceboCerebellar vermisB4243.67-2, -72, -16ThalamusB3943.976, -24, 2Cerebellar crus R2413.8938, -78, -32Placebo Drug-Control Family-Family Control-Interaction-EMOTION MEMORYHEM# VOXZMAXCOORDDrug Placebo-Placebo DrugHippocampusR3943.5622, -6, -24Lateral occipital cortexL3043.43-34, -88, 8Inferior frontal gyrusL2083.11-52, 24 18Control Family-Family Control-Interaction-Abbreviations: HEM hemisphere, R right, L left, VOX number of voxels in each significant cluster, ZMAX peak Z statistic value in each cluster, COORD peak voxel x, y, z coordinates in MNI spaceSupplementary Fig. S1 Functional imaging data were acquired in an oblique slab that provided high-resolution coverage of ventral brain regions involved in affective processing. Regions of interest included the amygdala (red), hippocampus (blue), orbitofrontal cortex (green), and fusiform cortex (yellow)Supplementary Fig. S2 Compared to fixation, both identification and memory tasks activated a distributed network including bilateral inferior frontal gyrus, orbitofrontal cortex, anterior insula, thalamus, and visual cortex; bilateral amygdala was robustly task-activated as wellSupplementary Fig. S3 Emotion memory task accuracy is shown by individual subject, separated by group and by drug condition. Lower horizontal dashed line indicates chance accuracy (0.33, or 33% correct responses, 20/60). Upper horizontal dashed line indicates threshold for statistically better-than-chance performance (0.45, or 45% correct responses, 27/60), as determined by one-sample binomial test implemented in STATA, one-tailed p0.6 for all main and interaction effects). This indicates that regional effects of drug in the BOLD data are not driven by nonspecific cerebral perfusion effects. Error bars indicate standard error of the meanSupplementary referencesFox CJ, Iaria G, Barton JJ (2009) Defining the face processing network: optimization of the functional localizer in fMRI. Hum Brain Mapp 30:1637-1651Gur RC, Ragland JD, Moberg PJ, Bilker WB, Kohler C, Siegel SJ, Gur RE (2001a) Computerized neurocognitive scanning: II. The profile of schizophrenia. Neuropsychopharmacology 25:777-788Gur RC, Ragland JD, Moberg PJ, Turner TH, Bilker WB, Kohler C, Siegel SJ, Gur RE (2001b) Computerized neurocognitive scanning: I. Methodology and validation in healthy people. Neuropsychopharmacology 25:766-776Gur RC, Richard J, Hughett P, Calkins ME, Macy L, Bilker WB, Brensinger C, Gur RE (2010) A cognitive neuroscience-based computerized battery for efficient measurement of individual differences: standardization and initial construct validation. J Neurosci Methods 187:254-262Jenkinson M, Bannister P, Brady M, Smith S (2002) Improved optimization for the robust and accurate linear registration and motion correction of brain images. Neuroimage 17:825-841Jenkinson M, Smith S (2001) A global optimisation method for robust affine registration of brain images. Med Image Anal 5:143-156Kendler KS, Lieberman JA, Walsh D (1989) The Structured Interview for Schizotypy (SIS): a preliminary report. Schizophr Bull 15:559-571Maxwell ME (1992) Manual for the FIGS. Clinical Neurogenetics Branch, Intramural Research Program, National Institute of Mental Health, Clinical Neurogenetics Branc