Molecular Psychiatry (2007) 12, 1089–1102 & 2007 Nature Publishing Group All rights reserved 1359-4184/07 $30.00 www.nature.com/mp ORIGINAL ARTICLE Stress-induced changes in primate prefrontal profiles of gene expression AM Karssen1,6, S Her1,6,7,JZLi2, PD Patel3, F Meng3, WE Bunney Jr4, EG Jones5, SJ Watson3, H Akil3, RM Myers2, AF Schatzberg1 and DM Lyons1 1Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA; 2Stanford Human Genome Center and the Department of Genetics, Stanford University, Stanford, CA, USA; 3Molecular and Behavioral Neuroscience Institute and the Department of Psychiatry, University of Michigan, MI, USA; 4Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA and 5Center for Neuroscience, University of California, Davis, CA, USA Stressful experiences that consistently increase cortisol levels appear to alter the expression of hundreds of genes in prefrontal limbic brain regions. Here, we investigate this hypothesis in monkeys exposed to intermittent social stress-induced episodes of hypercortisolism or a no-stress control condition. Prefrontal profiles of gene expression compiled from Affymetrix microarray data for monkeys randomized to the no-stress condition were consistent with microarray results published for healthy humans. In monkeys exposed to intermittent social stress, more genes than expected by chance appeared to be differentially expressed in ventromedial prefrontal cortex compared to monkeys not exposed to adult social stress. Most of these stress responsive candidate genes were modestly downregulated, including ubiquitin conjugation enzymes and ligases involved in synaptic plasticity, cell cycle progression and nuclear receptor signaling. Social stress did not affect gene expression beyond that expected by chance in dorsolateral prefrontal cortex or prefrontal white matter. Thirty four of 48 comparisons chosen for verification by quantitative real-time polymerase chain reaction (qPCR) were consistent with the microarray-predicted result. Furthermore, qPCR and microarray data were highly correlated. These results provide new insights on the regulation of gene expression in a prefrontal corticolimbic region involved in the pathophysiology of stress and major depression. Comparisons between these data from monkeys and those for ventromedial prefrontal cortex in humans with a history of major depression may help to distinguish the molecular signature of stress from other confounding factors in human postmortem brain research. Molecular Psychiatry (2007) 12, 1089–1102; doi:10.1038/sj.mp.4002095; published online 25 September 2007 Keywords: mood disorders; cortisol; hypothalamic-pituitary-adrenal axis; oligonucleotide micro- array; squirrel monkey Introduction postmortem analysis of whole-genome microarray data.11–14 These findings offer potential new insights In major depression, dysregulation of the hypothala- for the discovery of stress-related genes involved in mic–pituitary–adrenal (HPA) axis is evinced in 40– the pathophysiology of depression,15,16 but must be 80% of patients by an increase in circulating levels of considered with caution. the stress hormone cortisol.1–5 Receptors for cortisol In studies of human postmortem tissue, terminal are densely expressed in prefrontal cortex6,7 where medical conditions affect gene expression throughout they function as transcription factors that regulate the brain17–20 and antidepressant medications alter gene expression.8–10 Hundreds of genes in prefrontal gene expression profiles in prefrontal cortical tissue.21 cortex appear to be differentially expressed in hu- Animal models that utilize rodents are informative22 mans with a history of major depression based on but not sufficient because prefrontal cortex and white matter differ in rodents compared to humans and 23–25 Correspondence: Dr DM Lyons, Psychiatry Neuroscience, Stan- other primates. Relative to rodents, receptors for ford University, 1201 Welch Rd, MSLS P104—Mail Code 5485, cortisol occur at higher levels in primate neocortex6,26 Stanford, CA 94305-5485, USA. and remarkably low levels of glucocorticoid receptor E-mail: [email protected] 27 6 are found in rhesus monkey hippocampus. Neuroi- These authors contributed equally to this work. maging evidence likewise implicates ventromedial 7Current address: Korea Basic Science Institute, Chuncheon, prefrontal cortex as a key region for understanding the South Korea. 28–32 Received 22 August 2006; revised 9 August 2007; accepted 10 biology of stress and depression. Here, we August 2007; published online 25 September 2007 investigate prefrontal profiles of gene expression in Social stress affects gene expression AM Karssen et al 1090 squirrel monkeys exposed to intermittent social no-stress condition. Half of the monkeys in each adult stress-induced episodes of hypercortisolism using condition were previously exposed to postnatal stress microarray technology designed for the human tran- or the postnatal no-stress control. Monkeys sampled scriptome. for cortisol determinations were not used to study gene expression to avoid confounding blood sample collection and adult social stress effects. A total of 14 Materials and methods serial blood samples were collected from each Experimental design monkey according to the following schedule: 1 day A total of 22 male squirrel monkeys (Saimiri sciureus) prior to the first and fifth social separations; 1, 3 and that were born and raised at the Stanford University 17 days during the first and fifth separation sessions Animal Research Facility were randomized to the and 1, 3 and 17 days after completion of the first and following two treatment conditions in adulthood at fifth separations during new pair formations. B9 years of age (range 7.2–10.6 years). In one Matched samples were collected at simultaneous condition, monkeys were exposed to six intermittent time points in the adult no-stress condition. All 38 social separations that each lasted 3 weeks in samples were collected as described elsewhere duration. During each social separation session, between 1530 and 1630 hours to control for diurnal 39 monkeys were individually housed and could see, variation. Cortisol levels were subsequently mea- hear, smell, but not touch other unfamiliar monkeys. sured in duplicate using a radioimmunoassay estab- 40 After each intermittent separation, new male pairs lished for squirrel monkey research. were subsequently formed and maintained for 9 weeks. New pair formations33 and social separations34 Brain tissue collection are known to increase cortisol levels in adult male Brain tissues were collected from six monkeys in each squirrel monkeys. In the no-stress control condition, of the two adult treatment conditions 12 weeks after adult monkeys were housed with the same male the end of the final social separation while all of the companion in stable same-sex pairs. monkeys were housed in same-sex pairs. In each of As part of other studies, half of the monkeys in each the two adult treatment conditions, 2–4 monkeys adult treatment condition were previously exposed to were previously exposed to either postnatal stress or postnatal stress or postnatal no-stress conditions the no-stress postnatal control. Adult monkeys were described elsewhere in detail.35 Randomization of anesthetized with an intramuscular injection of À1 monkeys to the adult social stress versus no-stress 10 mg kg ketamine, followed by euthanasia with an À1 conditions was stratified by prior postnatal condi- intravenous overdose of 120 mg kg pentobarbital. tions to provide similar size samples in the 2 Â 2 Craniotomies were performed, brains were removed, factorial design (Figure 1). Based on evidence that and the left and right cerebral hemispheres were postnatal experiences alter gene expression in the separated by a mid-sagittal incision. Left cerebral forebrain of rodents,36,37 postnatal and adult stress hemispheres were placed in a custom-designed effects were examined in the analysis of data from acrylic brain matrix and cut coronally into blocks monkeys. All procedures were conducted in accor- that were frozen in isopentane on dry ice at À40 1C. dance with the Animal Welfare Act, and were One block contained all prefrontal tissue from the approved by Stanford University’s Administrative anterior commissure to the frontal pole. All brain Panel on Laboratory Animal Care. tissue collections occurred between 0800 and 0930 hours to control for diurnal variation in gene expres- Longitudinal measures of cortisol sion profiles. Serial tissue sections cut 20 micrometers thick were Blood samples for cortisol determinations were thaw-mounted onto Superfrost Plus glass slides and collected from 6 of 12 monkeys exposed to adult stored at À80 1C. From each animal, a 1-in-10 series of social stress and 4 of 10 monkeys from the adult sections that contained white matter tissue extending from the anterior striatum to the frontal pole was randomly selected for dissection at 0 1C under a stereo-zoom microscope. Prefrontal tissue from three defined regions on each section was scraped into separate RNAase-free tubes for subsequent storage at À80 1C. The boundaries used to identify each region (Figure 2) were identical to those previously shown in squirrel monkey neuroimaging research to have high interobserver reliabilities, that is, intraclass correla- tion coefficients greater than 0.90.41 Microarray hybridization A total of 36 Affymetrix (Mountain View, CA, USA) Figure 1 Schematic representation