Original Article

Molecular Psychiatry (2007) 12, 1089–1102; doi:10.1038/sj.mp.4002095; published online 25 September 2007

Stress-induced changes in primate prefrontal profiles of gene expression

A M Karssen1,6, S Her1,6,7, J Z Li2, P D Patel3, F Meng3, W E Bunney Jr4, E G Jones5, S J Watson3, H Akil3, R M Myers2, A F Schatzberg1 and D M Lyons1

  1. 1Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA
  2. 2Stanford Human Genome Center and the Department of Genetics, Stanford University, Stanford, CA, USA
  3. 3Molecular and Behavioral Neuroscience Institute and the Department of Psychiatry, University of Michigan, MI, USA
  4. 4Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA
  5. 5Center for Neuroscience, University of California, Davis, CA, USA

Correspondence: Dr DM Lyons, Psychiatry Neuroscience, Stanford University, 1201 Welch Rd, MSLS P104—Mail Code 5485, Stanford, CA 94305-5485, USA. E-mail: dmlyons@stanford.edu

6These authors contributed equally to this work.

7Current address: Korea Basic Science Institute, Chuncheon, South Korea.

Received 22 August 2006; Revised 9 August 2007; Accepted 10 August 2007; Published online 25 September 2007.

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Abstract

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.

Keywords:

mood disorders, cortisol, hypothalamic-pituitary-adrenal axis, oligonucleotide microarray, squirrel monkey

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Introduction

In major depression, dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis is evinced in 40–80% of patients by an increase in circulating levels of the stress hormone cortisol.1, 2, 3, 4, 5 Receptors for cortisol are densely expressed in prefrontal cortex6, 7 where they function as transcription factors that regulate gene expression.8, 9, 10 Hundreds of genes in prefrontal cortex appear to be differentially expressed in humans with a history of major depression based on postmortem analysis of whole-genome microarray data.11, 12, 13, 14 These findings offer potential new insights for the discovery of stress-related genes involved in the pathophysiology of depression,15, 16 but must be considered with caution.

In studies of human postmortem tissue, terminal medical conditions affect gene expression throughout the brain17, 18, 19, 20 and antidepressant medications alter gene expression profiles in prefrontal cortical tissue.21 Animal models that utilize rodents are informative22 but not sufficient because prefrontal cortex and white matter differ in rodents compared to humans and other primates.23, 24, 25 Relative to rodents, receptors for cortisol occur at higher levels in primate neocortex6, 26 and remarkably low levels of glucocorticoid receptor are found in rhesus monkey hippocampus.27 Neuroimaging evidence likewise implicates ventromedial prefrontal cortex as a key region for understanding the biology of stress and depression.28, 29, 30, 31, 32 Here, we investigate prefrontal profiles of gene expression in squirrel monkeys exposed to intermittent social stress-induced episodes of hypercortisolism using microarray technology designed for the human transcriptome.

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Materials and methods

Experimental design

A total of 22 male squirrel monkeys (Saimiri sciureus) that were born and raised at the Stanford University Animal Research Facility were randomized to the following two treatment conditions in adulthood at approx9 years of age (range 7.2–10.6 years). In one condition, monkeys were exposed to six intermittent social separations that each lasted 3 weeks in duration. During each social separation session, monkeys were individually housed and could see, hear, smell, but not touch other unfamiliar monkeys. After each intermittent separation, new male pairs were subsequently formed and maintained for 9 weeks. New pair formations33 and social separations34 are known to increase cortisol levels in adult male squirrel monkeys. In the no-stress control condition, adult monkeys were housed with the same male companion in stable same-sex pairs.

As part of other studies, half of the monkeys in each adult treatment condition were previously exposed to postnatal stress or postnatal no-stress conditions described elsewhere in detail.35 Randomization of monkeys to the adult social stress versus no-stress conditions was stratified by prior postnatal conditions to provide similar size samples in the 2 times 2 factorial design (Figure 1). Based on evidence that postnatal experiences alter gene expression in the forebrain of rodents,36, 37 postnatal and adult stress effects were examined in the analysis of data from monkeys. All procedures were conducted in accordance with the Animal Welfare Act, and were approved by Stanford University's Administrative Panel on Laboratory Animal Care.

Figure 1.
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Schematic representation of the 2 times 2 factorial design.

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Longitudinal measures of cortisol

Blood samples for cortisol determinations were collected from 6 of 12 monkeys exposed to adult social stress and 4 of 10 monkeys from the adult no-stress condition. Half of the monkeys in each adult condition were previously exposed to postnatal stress or the postnatal no-stress control. Monkeys sampled 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 serial blood samples were collected from each monkey according to the following schedule: 1 day prior to the first and fifth social separations; 1, 3 and 17 days during the first and fifth separation sessions and 1, 3 and 17 days after completion of the first and fifth separations during new pair formations. Matched samples were collected at simultaneous time points in the adult no-stress condition. All samples were collected as described elsewhere38 between 1530 and 1630 hours to control for diurnal variation.39 Cortisol levels were subsequently measured in duplicate using a radioimmunoassay established for squirrel monkey research.40

Brain tissue collection

Brain tissues were collected from six monkeys in each of the two adult treatment conditions 12 weeks after the end of the final social separation while all of the monkeys were housed in same-sex pairs. In each of the two adult treatment conditions, 2–4 monkeys were previously exposed to either postnatal stress or the no-stress postnatal control. Adult monkeys were anesthetized with an intramuscular injection of 10 mg kg-1 ketamine, followed by euthanasia with an intravenous overdose of 120 mg kg-1 pentobarbital. Craniotomies were performed, brains were removed, and the left and right cerebral hemispheres were separated by a mid-sagittal incision. Left cerebral hemispheres were placed in a custom-designed acrylic brain matrix and cut coronally into blocks that were frozen in isopentane on dry ice at -40 °C. One block contained all prefrontal tissue from the anterior commissure to the frontal pole. All brain tissue collections occurred between 0800 and 0930 hours to control for diurnal variation in gene expression profiles.

Serial tissue sections cut 20 micrometers thick were thaw-mounted onto Superfrost Plus glass slides and stored at -80 °C. From each animal, a 1-in-10 series of sections that contained white matter tissue extending from the anterior striatum to the frontal pole was randomly selected for dissection at 0 °C 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 -80 °C. 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 correlation coefficients greater than 0.90.41

Figure 2.
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Prefrontal regions on serial sections from the left cerebral hemisphere of a hemisected squirrel monkey brain.

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Microarray hybridization

A total of 36 Affymetrix (Mountain View, CA, USA) Human Genome U133A 2.0. GeneChips were used to analyze gene expression profiles in ventromedial prefrontal cortex, dorsolateral prefrontal cortex and prefrontal white matter samples from each of the 12 adult monkeys. No pooling of samples across different animals was necessary. To accommodate the workload, tissue samples were processed in two separate batches that each contained an equal number of randomly selected animals from each treatment condition. Tissue samples were homogenized with a motorized pellet pestle, and total RNA from each sample was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Quantification was carried out by spectrophotometric analysis, and gel electrophoresis was used to verify the integrity of each RNA sample. All samples were amplified with the two-cycle method, reverse transcribed into cDNA, labeled with biotinylated nucleotides and hybridized to HG U133A 2.0 microarrays at the Stanford University Genomic Facility according to the manufacturer's instructions. All 36 RNA samples were of high and comparable quality as determined by (i) the ratio of 28S:18S ribosomal RNAs, (ii) signal intensity ratios from probes for the 3' and 5' ends of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin transcripts used as quality controls on Affymetrix microarrays and (iii) the absence of outlier microarrays determined using a color-coded correlation matrix described elsewhere.42

Microarray data processing

The original design of the HG U133A 2.0 microarray was based on information from UniGene build 133 (April 2001 release). To account for recent advances in genomics, signal intensity data were interpreted by use of a custom chip description file (filename U133Av2_HS_UG_5) based on UniGene build 183 (April 2005 release) and freely available at http://brainarray.mhri.med.umich.edu/Brainarray/Database/CustomCDF/
genomic_curated_CDF.asp
. Design of the custom chip description file is described elsewhere in detail.43 Briefly, every probe within each probe set on the U133A 2.0 microarray was individually analyzed and reassigned to new probe sets to ensure that all probes within a given set detect sequences within a single UniGene cluster from build 183. The corresponding UniGene cluster then served as the probe set identification name. In contrast to the default Affymetrix chip description file, which interrogates 12 757 unique Unigene clusters, the custom chip description file contained 11 839 unique Unigene probe sets.

Scanned chip data were initially processed by MicroArray Suite 5 (MAS5, Affymetrix) to convert raw image files (.DAT) into probe signal value files (.CEL). Probe signal value files were subsequently processed using two different computational protocols: the robust multiarray average (RMA) protocol44 available at http://www.bioconductor.org, and the dChip perfect match-only protocol45 available at http://biosun1.harvard.edu/complab/dchip. Log2 transformed expression values determined separately by RMA and by dChip were imported into R software (http://www.r-project.org), normalized using quantile normalization,46 and median-centered to correct for technical batch-related variation.42

Statistical analysis

To identify similarities and differences between the prefrontal regions of interest, unsupervised hierarchical cluster analysis and paired t-tests were performed with data collected from monkeys randomized to the adult no-stress condition. Experience-dependent differences in gene expression within each region were then assessed using two-way analysis of variance (ANOVAs) with adult social stress versus no-stress conditions stratified by prior postnatal stress versus postnatal no-stress conditions in a 2 times 2 factorial design. Genes with significant postnatal or adult social stress main effects (P<0.05) in both RMA and dChip data sets were considered to be differentially expressed. Postnatal-by-adult social stress interactions were also examined but our power to detect the interactions was less than that for either of the two main effects.

In addition to analyzing individual genes, Gene Set Enrichment Analysis (GSEA) was used to evaluate RMA and dChip data at the level of functional gene sets.47 Established gene sets were downloaded in December 2005 from the gene ontology (GO) database (ftp://ftp.geneontology.org/pub/go/godatabase/archive/latest-termdb/). GO terms are nested categories that summarize known biological processes and molecular functions associated with a given gene, as well as its cellular location. GO terms for gene sets with fewer than 8 and more than 250 genes in our data were excluded from further consideration, leaving 900 gene sets for biological processes, 449 gene sets for molecular functions and 207 gene sets for cellular location. For each of these gene sets, GSEA calculates a weighted score that reflects the extent to which members of the gene set occur toward the extremes of the gene list rank-ordered by differential expression between the conditions of interest. The rank-ordered gene list consisted of all probe sets detected as present on one or more of the microarrays that were used to interrogate each region of interest, and not just probe sets above an arbitrary cutoff in terms of differential expression. Statistical significance of the enrichment score for each gene set on the list rank-ordered by differential expression was determined by condition-based permutation tests with 1000 random permutations used to build the null distribution. This statistical approach preserves gene–gene correlations and provides a more accurate null model compared to other approaches developed to analyze GO information.47 To minimize the frequency of false positive results, only gene sets identified as significant (P<0.05) in both RMA and dChip data were considered enriched. All test statistics from GSEA and the ANOVAs described above for the microarray data analysis were evaluated with two-tail probabilities.

Quantitative real-time polymerase chain reaction (qPCR) confirmation of microarray expression data

qPCR was used to confirm differential expression using the same RNA samples that were assayed by microarray. Samples were prepared using RNAase-free DNAase (Promega, Madison, WI, USA), and control reactions with not reverse transcribed RNA confirmed the absence of genomic DNA. Primers were selected within or near the Affymetrix target sequence, and designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA). Primers that did not reliably produce single products as indicated by the dissociation curve were discarded. Random hexamer primed cDNA was made from 500 ng total RNA using SuperScript II reverse transcriptase (Invitrogen) following the manufacturer's instructions. The reaction was diluted 10-fold with water and stored at -20 °C. At the second step, 2 mul diluted cDNA (from approximately 5 ng of starting RNA) was added to a 10 mul PCR reaction containing 10 pmol each of forward and reverse primers for each gene tested (Supplementary Table 1), and 5 mul of 2 times SYBER GREEN PCR Master Mix (Applied Biosystems, Warrington, UK), in a 384-well plate. Samples were amplified on an ABI 7900 (Applied Biosystems, UK) for 40 cycles at 94 °C for 15 s followed by fluorescence capture at 60 °C for 1 min. The critical threshold or point at which signal fluorescence exceeds background for each gene sample was compared against a 10-fold serial dilution standard curve of cDNA control sample amplified in parallel. The expression level for each gene was subsequently normalized to the mean expression level of GAPDH and beta-actin. To validate the microarray results, predicted directional differences in gene expression (up or downregulation) were assessed with the qPCR data using directional one-tail t-tests. The relationship between qPCR and microarray data was also evaluated with a Pearson's product moment correlation.

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Results

A total of 36 microarrays were used to compile gene expression profiles for three prefrontal regions in 12 adult squirrel monkeys. Approximately half of the 11 839 screened genes were called present by MAS5 software on one or more of the microarrays used to interrogate each region, that is, 5539 genes were called present in ventromedial prefrontal cortex, 5501 genes in dorsolateral prefrontal cortex and 5625 genes in prefrontal white matter. Many of these genes were expressed in all three regions of interest (Figure 3). A total of 5389 genes were not called present on any array, and these were excluded from further consideration. Both RMA and dChip data sets for all genes called present in each region are available for public use at http://www.pritzkerneuropsych.org/data/data.htm.

Figure 3.
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Venn diagram depicts the unique and overlapping subsets of genes called present in ventromedial prefrontal cortex (vmPFC), dorsolateral prefrontal cortex (dlPFC) and prefrontal white matter (WM). Approximately half of the 11 839 screened genes were called present on at least one of the microarrays used to interrogate each region.

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Genes enriched in white matter compared to prefrontal cortex

Of the genes called present in prefrontal white matter, 792 were expressed at higher levels (P<0.05) in white matter compared to cortex in both RMA and dChip data sets for monkeys from the adult no-stress condition. The percentage of genes differentially expressed in RMA and confirmed by dChip was 80%. Expression levels were, on average, 73% higher in white matter compared to cortex for the 792 genes differentially expressed in both RMA and dChip data sets (Supplementary Table 2). Many of the genes enriched in white matter are associated with myelination as determined by GSEA (Supplementary Table 3).

Genes enriched in cortex compared to prefrontal white matter

Of the genes called present in ventromedial and/or dorsolateral prefrontal cortex, 1481 were expressed at higher levels (P<0.05) in one or both regions of cortex compared to prefrontal white matter in both RMA and dChip data sets for monkeys from the adult no-stress condition. The percentage of genes differentially expressed in RMA and confirmed by dChip was 83%. Expression levels were, on average, 42% higher in cortex compared to white matter for the 1481 genes differentially expressed in both RMA and dChip data sets (Supplementary Table 4). Biological process and molecular GO terms identified by GSEA as overrepresented in cortex compared to prefrontal white matter included gene sets involved in energy-dependent proton transport, glycolysis, homeostasis, synaptic transmission and voltage-gated ion channel activity (Supplementary Table 5). Overrepresented cellular component GO terms included gene sets localized to the synapse and voltage-gated channels.

Differences between regions of prefrontal cortex

Genes enriched in cortex compared to white matter were further examined with hierarchical clustering of the data for monkeys from the adult no-stress condition. Samples of prefrontal cortex from the same anatomical region in different monkeys tended to cluster into regional groupings (Figure 4). Paired t-tests identified 477 genes that were consistently expressed in both RMA and dChip data at higher levels (P<0.05) in ventromedial versus dorsolateral prefrontal cortex (Supplementary Table 6). Conversely, 268 genes were expressed at higher levels (P<0.05) in dorsolateral versus ventromedial prefrontal cortex according to both RMA and dChip data (Supplementary Table 7). The percentage of genes differentially expressed in RMA and confirmed by dChip data was 59%. Expression levels differed, on average, by a modest 16% between ventromedial and dorsolateral prefrontal cortex for the combined list of 745 genes differentially expressed in both RMA and dChip data sets. Biological process and molecular GO terms identified by GSEA as overrepresented in ventromedial compared to dorsolateral prefrontal cortex included gene sets involved in neural plasticity, chromatin remodeling and transcriptional repression (Table 1). Of the 19 overrepresented GO terms for dorsolateral prefrontal cortex, 10 included gene sets related to phosphatase activity (Supplementary Table 8).

Figure 4.
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Hierarchical cluster analysis of dorsolateral (dlPFC) and ventromedial (vmPFC) prefrontal cortical samples from six monkeys in the adult no-stress condition. The dendrogram topography is highly robust, and does not differ appreciably with either dChip (shown) or RMA data. All 1481 genes enriched in prefrontal cortex compared to prefrontal white matter were included in the cluster analysis.

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Adult social stress-induced changes in gene expression

In ventromedial prefrontal cortex, a significant adult social stress main effect (P<0.05) was discerned for 440 genes in the data set from RMA, and 985 genes were identified in the data set from dChip (Figure 5). The number of genes in each data set expected to show a significant main effect by chance is 277, that is, 0.05 times 5539 genes called present in ventromedial prefrontal cortex. The percentage of genes with a significant main effect for adult social stress in RMA and confirmed by dChip was 49%, that is, 216 genes were differentially expressed in both data sets (Table 2). Far fewer genes in prefrontal white matter (22 genes) and dorsolateral prefrontal cortex (23 genes) showed a significant adult social stress main effect in both RMA and dChip data sets (Figure 5).

Figure 5.
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Venn diagrams depict the unique and overlapping subsets of genes discerned in robust multiarray average (RMA) and dChip data as differentially expressed (P<0.05) after exposure to adult social stress compared to the adult no-stress condition for three prefrontal regions. The numbers of genes differentially expressed after exposure to adult social stress exceeds chance levels for ventromedial prefrontal cortex (vmPFC), but not dorsolateral prefrontal cortex (dlPFC) or prefrontal white matter (WM).

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Most of the affected genes in ventromedial prefrontal cortex (Table 2) were downregulated to a modest extent by exposure to adult social stress. For the 171 genes diminished in both RMA and dChip data sets, expression levels were, on average, 16% lower in monkeys exposed to adult social stress compared to adult no-stress control. Downregulation of gene expression in ventromedial but not dorsolateral prefrontal cortex corresponds with our finding of regional differences in the capacity for transcriptional repression. As indicated above, genes involved in transcriptional repression and chromatin remodeling are overrepresented in ventromedial compared to dorsolateral prefrontal cortex for monkeys from the adult no-stress control condition (Table 1).

Fourteen of 21 biological process and molecular GO terms identified by GSEA in ventromedial prefrontal cortex as overrepresented in monkeys exposed to adult social stress compared to the no-stress condition are involved in catabolism and ligase activity (Table 3). Overrepresented cellular component GO terms included gene sets localized to mitochondria. Of the core genes involved in catabolism and ligase activity discerned by GSEA, several ubiquitin conjugation enzymes (that is, UBE2A, UBE2D2, UBE2E1) and ubiquitin ligases (that is, SMURF2, UBR2, UBE3A) were also identified by the above-mentioned two-way ANOVAs as having a significant adult social stress main effect in both RMA and dChip data sets.


Postnatal effects on gene expression

In ventromedial prefrontal cortex, a significant postnatal stress main effect (P<0.05) was discerned for 237 genes in the data set from RMA, and 387 genes were identified in the data set from dChip. As indicated above, the number of genes in each data set expected to show a significant main effect by chance alone is 277. The percentage of genes in ventromedial prefrontal cortex with a significant postnatal stress main effect in RMA and confirmed by dChip was 27%, that is, 65 genes were differentially expressed in both data sets. Only 15 genes in prefrontal white matter and 59 genes in dorsolateral prefrontal cortex had a significant postnatal stress main effect (P<0.05) in both RMA and dChip data sets. Postnatal stress did not robustly modify the subsequent effects of adult social stress. Less than 5% of the genes with a consistent adult stress main effect showed a postnatal-by-adult stress interaction in both RMA and dChip data sets.

qPCR validation of microarray data

To test the accuracy of the microarray data for genes identified by RMA and confirmed by dChip, 19 regional pairwise comparisons (Table 4) and 13 adult social stress main effects (Table 5) were re-assessed with qPCR using the same RNA samples that were assayed by the microarray. Twenty three of the 32 qPCR comparisons were consistent with the microarray-predicted direction for a validation rate of 72%. A total of 16 additional pairwise comparisons that were not significantly different according to the microarray were also examined by qPCR, and 5 were found to differ significantly according to qPCR for a microarray false negative rate of 31%. Microarray data often underestimated fold-difference scores (Tables 4 and 5), but gene expression differences measured by the microarray were highly correlated with those that were measured by qPCR (r=0.92, n=48 comparisons, P<0.001).



Adult social stress-induced episodes of hypercortisolism

As expected from previous research, cortisol levels were consistently increased during the first and fifth separations compared to when the same adult monkeys were sampled in previous pairs (Figure 6). Intermittent social separations also appeared to sensitize the HPA-axis response, as cortisol levels were 70% greater 1 day after the fifth compared to the first separation (paired-t=3.06, P=0.028). During each adult social separation, cortisol levels returned to baseline then increased for a short time thereafter during the formation of new pairs. Cortisol levels in the adult no-stress condition did not differ significantly over time, and postnatal effects were not statistically significant.

Figure 6.
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Longitudinal measures of cortisol in adult monkeys exposed to intermittent social stress. Plasma cortisol levels are presented for six monkeys sampled during the first (left panel) and fifth (right panel) recurrent sessions of social separation and new pair formation (meanplusminuss.e.m.). Asterisks signify cortisol levels that are greater than the levels in previous pairs (*P<0.05; **P<0.01).

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Discussion

In monkeys exposed to adult social stress-induced episodes of hypercortisolism, 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. Adult social stress did not affect gene expression beyond that expected by chance in dorsolateral prefrontal cortex or prefrontal white matter. Prefrontal profiles of gene expression in monkeys from the adult no-stress condition were consistent, however, with microarray data from recent studies of healthy humans as discussed below. These results demonstrate that the application of human arrays to nonhuman primates recovers information from thousands of genes, and may represent a useful new strategy for understanding the biology of stress in humans with major depression.

Human arrays used in monkey research do of course suffer from a loss of sensitivity and coverage of the transcriptome because hybridization is impaired by inter-species gene sequence differences. For example, 46% of all probe sets were called present in squirrel monkey dorsolateral prefrontal cortex (Figure 3) compared to 59% in a similar size sample of humans (unpublished data). Comparable differences have been reported in humans and rhesus monkeys for different tissue types.48, 49 Observed species differences may reflect genuine differences in gene expression, or methodological artifacts related to gene sequence differences. However, when groups of samples are compared within a single species, gene sequence differences consistently carry the same effect across all samples and no longer represent a confounding factor.42

In regional comparisons of squirrel monkeys randomized to the adult no-stress condition, 792 genes were expressed in both RMA and dChip data sets at higher levels in prefrontal white matter compared to prefrontal cortex (Supplementary Table 2). More than 550 of these genes were similarly classified as white matter genes in an independent microarray study of human prefrontal tissue.50 Conversely, we identified 1481 genes expressed at higher levels in one or both regions of prefrontal cortex compared to prefrontal white matter (Supplementary Table 4). Of these genes, 1251 were previously examined in healthy humans,50 and 80% were classified as enriched in human and monkey prefrontal cortex.

Additional evidence that human arrays recover valid information from monkeys was obtained with GSEA, which instead of analyzing individual genes, evaluates transcriptional profiles at the level of functional gene sets.47 As expected, 7 of 8 biological process gene sets identified by GSEA as overrepresented in prefrontal white matter compared to prefrontal cortex are involved in myelination (Supplementary Table 3). Gene sets involved in phosphatase activity are overrepresented in dorsolateral compared to ventromedial prefrontal cortex (Supplementary Table 8). Phosphatase activity has been linked to a core cognitive function of dorsolateral prefrontal cortex, that is, working memory.51 Ventromedial prefrontal cortex mediates emotional and autonomic responses to psychological stress.52, 53, 54 Gene sets involved in transcriptional repression are overrepresented in ventromedial compared to dorsolateral prefrontal cortex (Table 1). This regional difference in the capacity for transcriptional repression corresponds with our finding that adult social stress-induced hypercortisolism downregulates the expression of hundreds of genes in ventromedial, but not dorsolateral, prefrontal cortex in monkeys (Figure 5). Exogenous glucocorticoids likewise downregulate the majority of glucocorticoid responsive genes in rodent hippocampus.8, 55, 56

In monkey ventromedial prefrontal cortex, a subset of related genes differentially expressed after exposure to adult social stress was reliably identified in dChip and RMA data. These genes are ubiquitin conjugation enzymes (UBE2A, UBE2D2, UBE2E1) and ligases (SMURF2, UBR2, UBE3A) that target ubiquitin to protein substrates for selective degradation by the proteasome. Ubiquitin proteasome pathways regulate brain proteins involved in synaptic plasticity,57 cell cycle progression58 and diverse aspects of nuclear receptor signaling.59, 60 In addition to proteolysis of nuclear receptors, ubiquitin conjugation enzymes and ligases also function as co-regulatory factors in nuclear receptor signaling and chromatin remodeling.59, 61 These findings are of interest because cortisol effects are mediated by two nuclear receptors and dysregulation of this dual receptor system is thought to play a role in the pathophysiology of stress and depression.62, 63

Despite indications that ventromedial prefrontal cortex is involved in depression,28, 29, 30 microarray studies of humans have not examined this prefrontal region. In humans with a history of major depression, whole-genome expression analysis has focused on dorsolateral prefrontal cortex (Brodmann's area 8/9),11, 12, 14, 64 ventrolateral prefrontal cortex (BA47),64 orbital frontal cortex (BA11),14 anterior cingulate (BA24)11, 12 and the frontal pole (BA10).13 The ventromedial prefrontal region examined in monkeys is primarily comprised of agranular cortex lacking a well-developed granular layer IV,65, 66 and roughly corresponds to BA25, BA12 and medial portions of BA11 in human prefrontal cortex based on neuropsychological functions and anatomical connections.23, 67, 68, 69, 70, 71

Microarray studies of prefrontal cortex in humans with a history of major depression have identified abnormalities in gamma-aminobutyric acid (GABA) signaling11 and catabolism of polyamines.14 In monkeys exposed to adult social stress, three GABA-related genes are diminished in ventromedial prefrontal cortex compared to monkeys from the no-stress condition (Table 6). The spermidine/spermine N1-acetyltransferase (SSAT) candidate gene for suicidal depression14 is likewise diminished in monkeys exposed to adult social stress, based on directional one-tail tests of RMA and dChip data. SSAT is the rate-limiting enzyme in the catabolism of polyamines, and is involved in the polyamine stress response evoked by emotional stimuli or exogenous glucocorticoids.72 Also in keeping with our findings in monkeys (Table 6), diminished expression of ubiquitin-related genes has been observed in orbital frontal and not dorsolateral prefrontal cortex in humans with alternating episodes of mania and depression in bipolar disorder.73 Differential expression of prefrontal cholecystokinin (CCK), neuropeptide Y, and related receptors in bipolar disorder, depression and suicide74, 75, 76 corresponds with our finding that stress affects these same neuropeptide systems in primate prefrontal cortex (Table 6). Stress-induced regulation of transcription factors in monkey ventromedial prefrontal cortex (Table 6) likewise concurs with findings from human postmortem brain research,13, 77, 78 and with new advances in understanding the molecular mechanisms of antidepressant drugs.79


These results should be interpreted in the context of several potential limitations. The sample of monkeys is entirely male, and the reported gene expression profiles may or may not hold true for females. Moreover, the regional differences discerned between monkey dorsolateral and ventromedial prefrontal profiles of gene expression (Supplementary Tables 6–8) are at odds with microarray surveys that failed to find broad differences between neocortical regions in humans and chimpanzees.50, 80, 81, 82, 83 In rodents, broad regional differences in gene expression have been reported in carefully controlled and adequately powered studies of the hippocampus.84, 85, 86, 87, 88 Our study examined prefrontal tissue of the highest possible quality, collected at the same time of day, within a small age range from adult monkeys born and raised in the same research facility. Cluster analysis clearly identified differences between monkey dorsolateral and ventromedial prefrontal profiles of gene expression in both RMA and dChip data sets (Figure 4).

Cluster analysis also identified adult social stress effects in ventromedial prefrontal cortex for dChip but not RMA data (dendrograms not presented). This discrepancy reflects our finding that fewer genes are differentially expressed in both RMA and dChip for adult social stress effects compared to the regional comparisons. That regional comparisons are more robust than those for adult stress effects is consistent with the fold-difference scores we determined for stress responsive genes (Table 2). Many of the genes expressed in brain tissue occur at low levels55 or are restricted to subpopulations of cells.88 Fold-difference scores are therefore 'diluted' in microarray studies of bulk brain tissue collected by supra-cellular dissections.42 Furthermore, microarrays tend to underestimate fold-difference scores relative to qPCR based on the 48 pairwise comparisons that we examined in monkeys. Small fold-difference scores commonly occur in microarray studies of human brain tissue and peripheral blood cells in which psychiatric case-control comparisons rarely exceed 2-fold thresholds.11, 12, 13, 14, 15, 64, 73, 89

Microarrays screen thousands of genes and many false positive type I errors are expected by chance. Procedures for controlling False Discovery Rates (FDR) are increasingly used in microarray studies designed to test one hypothesis per gene, but FDR procedures have not been well characterized for factorial microarray experiments that test multiple effects. An alternative approach was therefore applied based on evidence that different methods of calculating gene expression may provide different results.42 To minimize false positives, only genes or functional gene sets identified as nominally significant (P<0.05) in both RMA and dChip data were considered as having potential biological relevance. To test the accuracy of this approach, 48 pairwise comparisons were re-assessed by qPCR using the same RNA samples assayed by microarray. Thirty four of the 48 qPCR comparisons were consistent with the microarray-predicted result, including CCK, NR3C2 and the recently discovered90 ubiquitin ligase UBE3C (Tables 4 and 5). Microarray and qPCR data were also highly correlated. Permutation analysis of our microarray data using procedures described elsewhere91 suggests that more than 85% of the 216 stress responsive genes identified by dChip and confirmed by RMA in ventromedial prefrontal cortex (Table 2) are not false positive type I errors due to chance alone. Nevertheless, microarrays primarily provide exploratory data and many specific candidate genes must be independently confirmed.

In summary, we emphasize the value of this study as a model of stress rather than hypercortisolism or depression per se. Certain key features of depression are probably unique to humans, for example, low self-esteem, feelings of worthlessness, and suicidal ideation. Furthermore, despite indications that hypercortisolism may be a marker of severity or depressive subtypes,1, 2, 92 dysregulation of the HPA-axis is not a formal feature of depression in DSM-IV. Although the effects of adult social stress in monkeys are consistent with evidence that cortisol regulates hundreds of genes in certain brain tissue types,8, 9, 10 our study does not establish that gene expression changes are directly caused by hypercortisolism. Other physiological adaptations induced by adult stress may have contributed to the changes discerned in ventromedial prefrontal gene expression. Comparisons between these data from monkeys and those for ventromedial prefrontal cortex in humans with a history of major depression and related psychiatric disorders may help to distinguish the molecular signature of stress from other confounding factors in human postmortem brain research.

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Acknowledgements

This work was funded by Public Health Service Grant MH47573 and the Pritzker Neuropsychiatric Disorders Research Consortium, which is supported by the Pritzker Neuropsychiatric Disorders Research Fund LLC. A shared intellectual property agreement exists between the Pritzker Neuropsychiatric Disorders Research Fund LLC and the University of Michigan, the University of California and Stanford University to encourage the development of appropriate findings for research and clinical applications.

Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)