ORIGINAL RESEARCH published: 17 January 2019 doi: 10.3389/fpsyt.2018.00778 Dynamic Patterns of Threat-Associated Gene Expression in the Amygdala and Blood Adriana Lori 1† , Stephanie A. Maddox 2,3† , Sumeet Sharma 1,2 , Raül Andero 4,5,6 , Kerry J. Ressler 1,2,3 and Alicia K. Smith 1,7* 1 Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, United States, 2 Neurobiology of Fear Laboratory, Division of Depression and Anxiety Disorders, McLean Hospital, Belmont, MA, United States, 3 Department of Psychiatry, Harvard Medical School, Boston, MA, United States, 4 Institut de Neurociènces, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 CIBERSAM, Corporació Sanitaria Parc Taulí, Sabadell, Spain, 6 Department of Psychobiology and Methodology in Health Sciences, Universitat Autònoma de Barcelona, Bellaterra, Spain, 7 Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA, United States Stress and trauma profoundly influence psychiatric biobehavioral outcomes. The identification of treatment and biomarker targets would be accelerated by a broad Edited by: understanding of the biological responses to these events. The goal of this study was Richard Eugene Frye, Phoenix Children’s Hospital, to determine genes responsive to auditory fear conditioning (FC), a well-characterized United States amygdala-dependent rodent model of threat-exposure, in the presence or absence of Reviewed by: prior stress history, providing insight into the physiological processes underlying response Polymnia Georgiou, University of Maryland, Baltimore, to trauma. RNA-sequencing was performed in blood and amygdala from mice that United States underwent fear conditioning with (Immo+FC) and without (FC) prior immobilization stress, Eva E. Redei, a paradigm that induces HPA axis, and behavioral stress sensitization. In the amygdala, Northwestern University, United States Stefania Schiavone, 607 genes were regulated by FC vs. home-cage (HC) controls, and 516 genes differed in University of Foggia, Italy stress-sensitized mice (Immo+FC vs. FC). In the former, we observed an enhancement *Correspondence: of specific biological processes involved in learning and synaptic transmission, and in Alicia K. Smith alicia.smith@emory.edu the latter processes associated with cell proliferation and the cellular response to drugs. In the blood of stress-sensitized animals, 468 genes were dynamically regulated when † These authors have contributed equally to this work compared to FC, and were enriched for the biological pathways of inflammation and cytokine signaling. This study identified genes and pathways that respond to threat in Specialty section: the amygdala and blood of mice with and without a prior stress history and reveals the This article was submitted to impact of stress history on subsequent inflammation. Future studies will be needed to Molecular Psychiatry, a section of the journal examine the role of these dynamically regulated genes may play in human clinical stress Frontiers in Psychiatry and trauma-related disorders. Received: 18 September 2018 Keywords: threat, fear, PTSD (post-traumatic stress disorder), amygadala, stress Accepted: 24 December 2018 Published: 17 January 2019 Citation: INTRODUCTION Lori A, Maddox SA, Sharma S, Andero R, Ressler KJ and Smith AK Post-traumatic stress disorder (PTSD) is a pervasive and debilitating psychiatric disorder that (2019) Dynamic Patterns of Threat-Associated Gene Expression in develops in vulnerable individuals after exposure to variable levels of trauma. A prior stress event, the Amygdala and Blood. particularly early life trauma or abuse, has also been shown to cause poor psychiatric outcomes Front. Psychiatry 9:778. in adults, increasing risk for and the severity of PTSD (1, 2). The characteristic features of PTSD, doi: 10.3389/fpsyt.2018.00778 such as hypervigilance and heightened startle reactions (DMS-V), associate with a patient’s inability Frontiers in Psychiatry | www.frontiersin.org 1 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression to regulate their fear response in the presence of a non- alterations that are observed with human clinical trauma- threatening situation (3, 4). Human neuroimaging studies and relevant disorders (20, 21), we utilized a robust mouse model animal models have well established that the amgydala plays of stress exposure and auditory fear conditioning to identify a central role in the processing of fearful and threatening changes in gene expression in the amygdala response to fear stimuli and in mediating the constellation of responses that conditioning with and without prior stress. are associated with fear and threat-related behaviors (5, 6). Ultimately, our goal was to identify correlated patterns of Accumulating and compelling evidence now suggests that PTSD gene expression between blood and brain under these conditions is associated with dysregulation of the amygdala, generally to facilitate interpretation of blood-based studies of PTSD and hyperactivity, in response to trauma-relevant or emotionally to provide new insight into the pathophysiology stress-related salient cues (7, 8). disorders. Recent studies have identified differential gene expression patterns in blood between PTSD cases and trauma-exposed METHODS controls, reporting possible genes, and pathways associated with PTSD, several of which show dysregulation of the immune Animals system and glucocorticoid pathways (9). However, there is All experiments were performed on adult male wild-type limited knowledge of the degree of correlation between gene C57BL/6J mice aged 2–3 months obtained from The Jackson expression changes, accompanying trauma, and psychiatric Laboratory. Male mice were group-housed in a temperature- conditions (e.g., PTSD) in the blood and those in the controlled vivarium with set-point maintained at 72◦ F (±1◦ ) brain. Additionally, because of the obvious limitations of and relative humidity controlled at 40–50%, with ad libitum availability of human brain tissues, there had been paucity access to food and water. Each experimental group consisted of brain-based transcriptomic studies. While transcriptomic of 12 mice maintained on a 12-h light/dark cycle, with all studies from human post-mortem issues can aide in examining behavioral procedures being performed during the light cycle. persistent, long-lasting changes in gene expression relevant to All procedures used were approved by the Emory University specific disease states, they do not permit examination of the Institutional Animal Care and Use Committee (IACUC) and in transcriptional changes which occur in brain regions relevant compliance with National Institutes of Health Guide for the Care for stress and trauma-related disorders at times proximal to and Use of Laboratory Animals. trauma. In this regard, traumatic memory formation in animal Mouse Immobilization Stress (Immo) and models can facilitate identification of genes whose expression is Fear Conditioning (FC) comparable between the amygdala and blood. As such, studies Immobilization stress (Immo) and fear conditioning (FC) were employing rodent models of stress and threat exposure may conducted following the protocol from Andero et al. (4). Briefly, present a powerful approach toward bridging this gap. Moreover, immobilization procedures were conducted in a room separate studies examining the molecular mechanisms associated with from housing and behavioral paradigms. Each animal was the formation and persistence of threat-relevant memories have immobilized by restraining their four limbs with tape in a prone largely utilized Pavlovian fear-conditioning (FC), employing as position to metal arms attached to a wooden board for 2 h. All conditioned stimulus either novel auditory cues to result in a cage-mate animals received the same treatment—either Immo largely amygdala dependent memory, or spatial, and contextual or handling. Handling lasted ∼1 min per mouse and consisted cues that integrate hippocampal and amygdala regions to regulate of letting the animal walk on top of their home cage and in the learning (5, 10). As much work has shown in human clinical hands of the experimenter. After Immo, animals were returned studies has noted the importance of the amygdala in the to their home cage (HC) where they remained undisturbed for pathophysiology of trauma-relevant disorders (11–13) and the a week prior to fear conditioning (FC), which was performed in impact of prior stress history on later risk for the development Immo animals and a subset of naïve animals. For auditory fear of PTSD accompanying trauma (14), we were most interested in conditioning mice were habituated to white-light illuminated, examining the molecular changes occurring within the amygdala standard rodent modular test chambers (ENV-008-VP; Med in the time period proximal to trauma. To meet this objective Associates Inc., St. Albans, VT) with an inside area of 30.5 cm we used paradigm previously utilized by our group which has (L) × 24.1 cm (W) × 21.0 cm (H) for 10 min on 2 consecutive confirmed that mice immobilized for one 2 h session, 1 week days prior to fear conditioning. Fear conditioning consisted of prior to auditory FC have impaired fear extinction and retention, five trials of a novel tone conditioned stimulus (CS; 30 s tone, phenotypes that are seen in human clinical PTSD (15, 16). In 6 kHz, 70 dB), which co-terminated with a foot-shock (500 ms, addition to the observed behavioral phenotype, mice exposed 0.6 mA) unconditioned stimulus (US). The tone conditioned to the immobilization paradigm had hypothalamic-pituitary- stimulus was generated by a Tektronix function generator audio adrenal (HPA) axis hypersensitivity, and transient changes in oscillator delivered through a high-frequency speaker (Motorola, plasma corticosterone levels (4, 17). HPA axis abnormalities, such Model 948) attached to the side of each chamber. The Pre-CS as low levels of cortisol in urine and plasma or higher suppression period lasted 180 s and a variable inter-trial interval (ITI) was of cortisol in response to dexamethasone have been also reported used between each CS-US pairing to result in a total conditioning in PTSD patients (18, 19). As the prior (immobilization) stress session which lasted 840 s. The apparatus was cleaned with history model replicates many of the behavioral and hormonal Quatricide R after each mouse. Frontiers in Psychiatry | www.frontiersin.org 2 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression Mice were sacrificed under basal conditions (HC group) or RNA Extraction and Sequencing 2 h following auditory fear conditioning (Immo+FC and FC RNA extraction, QC, library preparation, and sequencing were alone groups)—a time point that our group has consistently conducted by the Yerkes Non-Human Primate Genomics Core utilized for looking at changes in transcriptional processes in (Atlanta, GA). Amygdala punches were homogenized with the amygdala following auditory fear conditioning—with a brief a bead milling homogenizer, and total RNA was isolated exposure to isoflurane anesthesia, <30 s, followed by decapitation and purified from each sample with the RNeasy Mini Kit and trunk blood collection. Trunk blood from two mice of the (Qiagen, CA) following the manufacturer’s instructions. RNA same behavioral group was collected into a single 3 ml EDTA quality and quantity were verified with the 2100 BioAnalyzer BD-Vacutainer tubes. A 250 µL aliquot of each blood sample PicoChip (Agilent Technologies, Santa Clara, CA) before was allocated for complete blood count, and the remaining sequencing, and all samples had an RNA Integrity Number sample was stored at −80◦ C. Brains were immediately frozen (RIN) score of nine or higher. For blood samples, globin on dry ice and stored at −80◦ C, and 1 week later brains were mRNA transcripts were depleted using the GLOBINclearTM - mounted on a sliding, freezing microtome using Tissue-Tek Mouse kit (Ambion, Austin, TX) according to the manufacturer’s OTC, and sectioned slowly to approximately Bregma −1.34 mm instructions. Briefly, 1 µg of total RNA was treated with (22) to reveal the amygdala. One millimeter of bilateral amygdala biotinylated oligonucleotides to selectively deplete the α- and punches, centered on the basolateral nucleus were taken and β-globin sequences. Subsequently, streptavidin paramagnetic immediately frozen in microcentrifuge tubes on dry ice and beads were added, to capture the hybridized globin mRNA stored at −80 degrees for later RNA extraction. The bilateral biotinylated probes, resulting in an average 25% loss of total amygdala punches (and the blood) from 2 mice of the same RNA. Further purification of the globin depleted RNA was behavioral group were pooled together, thus resulting in a total performed with SPRI magnetic beads as per manufacturer’s of 6 pooled samples for each behavioral condition that were recommendation. sequenced. As the murine basolateral amygdala is slightly larger Libraries were prepared using the Illumina (Illumina Inc. San than 1 mm, we cannot exclude the possibility that other amygdala Diego, CA) TruSeqTM RNA kit as per manufacturer’s instructions. subregions were including in these tissue samples. Briefly, 250 ng of total RNA was used for library preparation. FIGURE 1 | Fear conditioning induces gene expression differences in amygdala and blood. Volcano plots and heatmaps show changes in gene expression in the amygdala (A) and blood (B) after fear conditioning compared to Home Cage (HC vs. FC). The horizontal axis of the Volcano plot is log2 fold change for differently expressed genes, and the vertical axis is the negative-log10 of the p-values are plotted. Each dot represents a gene, with red dots showing genes reaching an FDR corrected p-value of 0.05, and green dots representing genes with FDR < 0.05 and absolute fold change >1; orange dots have an absolute fold change>1 but do not reach experiment-wide significance; black dots are genes whose expression is similar between the two groups. Frontiers in Psychiatry | www.frontiersin.org 3 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression TABLE 1 | Enrichment biological process analyses for differential express genes in fear conditioning mice compared to control (FC vs. HC). GO number Description FEa p-valueb AMYGDALA GO:0007611 Learning or memory 8.20 4.18 × 10−4 GO:0008306 Associative learning 8.47 0.010 GO:0007268 Chemical synaptic transmission 3.65 0.008 GO:0003007 Heart morphogenesis 5.99 0.008 GO:0035556 Intracellular signal transduction 2.44 0.012 GO:0055085 Transmembrane transport 2.49 0.023 GO:0007507 Heart development 2.80 0.025 GO:0048511 Rhythmic process 3.81 0.025 GO:0007612 Learning 5.28 0.030 GO:0007157 Heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules 6.03 0.027 GO:0045944 Positive regulation of transcription from RNA polymerase II promoter 1.75 0.036 GO:0006811 Ion transport 2.03 0.036 GO:0006351 Transcription, DNA-templated 1.50 0.041 GO:0045053 Protein retention in Golgi apparatus 27.87 0.040 GO:0007626 Locomotory behavior 3.87 0.045 BLOOD GO:0007156 Homophilic cell adhesion via plasma membrane adhesion molecules 12.4 1.63 × 10−26 GO:0002376 Immune system process 3.07 0.009 a FE, Fold Enrichment. b p-value following Benjamini-Hochberg correction for multiple testing. The TruSeq method (low-throughput protocol) employs two 5% to account for multiple testing in all analyses (q < 0.05). rounds of poly-A based mRNA enrichment using oligo-dT Volcano plots were generated in R. Differentially expressed magnetic beads followed by mRNA fragmentation using cations genes were further evaluated for the enrichment of biological at high temperature. First and second strand cDNA synthesis processes using DAVID 6.8 (25). Differences in cell counts was performed followed by end repair of the blunt cDNA between groups were evaluated using an independent t- ends. One single “A” base was added at the 3′ end of the test. cDNA followed by ligation of barcoded adapter unique to each sample. The adapter-ligated libraries were then enriched using PCR amplification. The amplified library was validated using RESULTS a High Sensitivity DNA chip on the Agilent Bioanalyzer. The In this study, two groups of mice (with and without a history libraries were further quantified on Qubit R 2.0 Fluorometer of immobilization stress; Immo), were trained in an auditory (Life Technologies, Grand Island, NY) using the High Sensitivity fear conditioning paradigm (Immo+FC and FC, respectively); dsDNA assay. Each library contained the same amount of RNA, a third group of naïve, home-cage control animals was handled and eight sample pools were multiplexed in each lane of the and removed from the vivarium but not exposed to any flowcell. PhiX was used as an internal control on each lane behavioral intervention (HC). All mice were sacrificed together to monitor the error statistics, and sequencing was performed 2 h after last fear conditioning (or an equivalent time of on the Illumina HiSeq1000 system employing a paired-end 101 day after handling), and gene expression patterns from blood cycles run. and amygdala were compared. Examination of the freezing behaviors of animals in the Immo-FC and FC groups did Statistical Analysis not reveal any significant differences in baseline, pre-tone CS Alignment to the 10 mm UCSC Mouse Assembly was performed freezing (t = −0.39, p > 0.05) or tone CS freezing across the using STAR version 2.3 (23); parameters were set using the auditory conditioning session (t = 0.43, p > 0.05). Table S1 annotation as a splice junction reference. Sample reads were shows the total number of expressed genes in both tissues assembled into transcript models using cufflinks (v2.1.1), which across the three different groups (HC, FC or Immo+FC). were then merged and processed with cuffdiff v2.1.1 (24) We observed tissue-specific gene expression, with an overall to produce per sample FPKM expression levels and estimate higher number of genes expressed in the amygdala relative to differential expression between the sample groups. Each tissue the blood in all three groups (HC, FC, Immo+FC). Overall (amygdala and blood) was processed separately to allow for 11,353 genes were expressed in both tissues, with 580 uniquely identification of tissue-specific differences for each behavioral expressed in blood and 4,271 uniquely expressed in the condition. The false discovery rate (FDR) was controlled at amygdala. Frontiers in Psychiatry | www.frontiersin.org 4 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression TABLE 2 | Genes differentially expressed in FC compared to HC in amygdala and blood. Gene Amygdala Blood Fold change p-value q-value Fold change p-value q-value Nxpe4 −0.39 0.0003 0.010 −1.27 5.00E−05 0.002 Plxnd1 −0.22 0.0017 0.047 −0.82 5.00E−05 0.002 C1ql3 0.24 0.0012 0.037 −2.41 0.0001 0.004 Zhx2 0.29 0.0012 0.037 0.68 0.0001 0.006 Pbrm1 −0.26 0.0001 0.005 −0.68 0.0005 0.015 Adam8 0.47 0.0006 0.020 0.62 0.0008 0.022 Zbtb40 0.37 0.0002 0.007 0.67 0.0009 0.026 Myo5a −0.30 0.0001 0.003 −0.53 0.001 0.030 Dmxl2 −0.42 0.0001 0.003 −0.66 0.002 0.045 Kif1b −0.25 0.0012 0.036 −0.53 0.003 0.054 Sorl1 −0.37 0.0001 0.003 0.51 0.004 0.078 Sdc4 0.26 0.0008 0.025 0.46 0.005 0.088 Thada −0.33 0.0010 0.032 0.49 0.006 0.098 Tra2a 0.32 0.0001 0.003 −0.45 0.006 0.106 Insr −0.22 0.0015 0.044 −0.49 0.008 0.120 Galnt9 0.38 0.0001 0.003 −0.58 0.009 0.137 Myadm 0.22 0.0013 0.039 −0.48 0.011 0.152 Abhd2 −0.25 0.0007 0.023 0.50 0.013 0.172 Klf11 −0.41 0.0009 0.028 −0.48 0.016 0.195 Zfp280c −0.31 0.0001 0.005 −0.50 0.020 0.225 Bptf −0.25 0.0001 0.003 0.38 0.020 0.229 Numb 0.26 0.0005 0.017 0.39 0.022 0.241 Pds5a −0.29 0.0001 0.005 0.39 0.023 0.248 Sdf2l1 0.58 0.0001 0.003 0.44 0.023 0.248 Egr3 0.33 0.0001 0.003 0.89 0.024 0.251 Arhgef12 −0.24 0.0004 0.015 −0.40 0.025 0.260 Kat6a −0.28 0.0001 0.003 0.36 0.030 0.288 Guf1 −0.27 0.0006 0.021 −0.45 0.036 0.320 Phka2 −0.47 0.0001 0.003 −0.42 0.040 0.344 Zfp445 −0.21 0.0016 0.046 −0.33 0.044 0.362 Dusp1 −0.54 0.0001 0.003 0.37 0.045 0.364 Fkbp5 0.24 0.0007 0.024 0.35 0.048 0.377 Bold Gene Names: Adam8, a disintegrin and metallopeptidase domain 8; C1ql3, C1q-like 3; Dmxl2, Dmx-like 2; Myo5a, myosin VA; Nxpe4, Neurexophilin and PC-Esterase Domain Family, Member 4; Pbrm1, Polybromo 1; Plxnd1, Plexin D1; Zbtb40, Zinc finger and BTB domain containing 40; Zhx2, Zinc fingers and homeoboxes 2. b C1ql3 (C1q-like 3) changes in different direction in brain and blood. Fear Conditioning Induces Robust Gene learning or memory (p = 4.18 × 10−4 ), and associative learning Expression Differences in Amygdala and (p = 0.01). In blood, FC results in expression differences of 352 genes Blood We first investigated differences in response to fear conditioning in blood relative to HC (Table S4; FDR < 0.05; Figure 1B), (FC vs. HC). Figure 1A shows that FC induced gene expression with the majority of genes identified (84.3%) having lower changes in the amygdala, with 607 genes differentially expressed expression levels in FC when compared to HC. Only two when compared to HC (Table S2; FDR < 0.05). FC resulted biological processes were enriched among these differentially in a down-regulation of gene expression in the majority of expressed genes (Table 1; Table S3), homophilic cell adhesion these genes (76.6%). We then evaluated differentially expressed of plasma membranes of adjacent cells (p = 1.6 × 10−26 ), and genes for enrichment of biological process and identified 15 immune system processes (p = 0.009). Comparison of genes processes that were enriched after multiple test correction regulated in amygdala (FDR < 0.05) with those regulated in (Table 1, Table S3). Among these processes, there was an blood (p < 0.05) revealed 32 genes differentially expressed in enrichment of specific biological processes including memory FC vs. HC, 9 of which reached FDR significance in both tissues formation and consolidation, and neurotransmission, with (Table 2). Frontiers in Psychiatry | www.frontiersin.org 5 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression FIGURE 2 | Fear conditioning and prior immunization induce gene expression differences in amygdala and blood. Volcano plots and heatmaps show changes in gene expression in the amygdala (A) and blood (B) of animals that experienced immobilization (Immo+FC) prior to fear conditioning (FC). The horizontal axis of the Volcano plot is log2 fold change for differently expressed genes, and the vertical axis is the negative-log10 of the p-values are plotted. Each dot represents a gene, with red dots showing genes reaching an FDR corrected p-value of 0.05, and green dots representing genes with FDR < 0.05 and absolute fold change >1; orange dots have an absolute fold change>1 but do not reach experiment-wide significance; black dots are genes whose expression is similar between the two groups. Prior Stress Sensitization Induces Gene (monocytes, neutrophils, and lymphocytes) between each group. Expression Changes in Response to Fear Although there were no differences in blood cell composition between the HC and FC groups, Immo+FC had a higher Conditioning proportion of neutrophils and a lower proportion of lymphocytes To evaluate how prior stress exposure may alter the relative to both FC (p = 0.013) and HC (p = 0.007; Figure 4). transcriptional processes that accompany fear conditioning, We then compared the genes whose expression differed in we compared amygdala expression in the Immo+FC to the FC group (Figure 2A). We identified 516 genes that had differences the amygdala (FDR < 0.05) with those that differed in blood. in expression levels (FDR < 0.05; Table S5, Figure 2A), 84.3% of We identified 27 genes (Table 4) that change in Immo+FC which were higher in FC. Among those, we observed genes that vs. FC in both tissues, 20 (74%) of which occurred in the have been associated with PTSD in previous human studies (e.g., same direction in both tissues. Among the 10 genes that DRD2 and HTR2a) (26–29) or linked to anxiety or PTSD-like remained significant after multiple test correction in both behaviors in humans or animal models (Igf2, Grm2, Clock, tissues, Dmxl2, Trps1, Fgd4, and Thbd have similar expression Trhr) (30–32) (Table S5; Figure 3). Enrichment analyses of patterns in Immo+FC and HC. Interestingly, the remaining the 516 differentially expressed genes revealed four biological genes in which immobilization (Immo+FC) had induced pathways, including cell proliferation and cellular response to changes in expression seem to be involved in immune response drugs (Table 3; Table S6). (Lbp and Lnc2), anxiety behavior and schizophrenia (Pde7b), In blood, prior stress history (Immo+FC) associates with corticosterone homeostasis, and steroid transportation (Lcn2 expression differences in 468 genes relative to FC (Figure 2B), and Soat1; Figure 5). While we cannot state whether or not the majority of which (97%) increased in expression compared these genes are also regulated by immobilization alone, the to FC (FDR < 0.05; Table S7). Enrichment analysis revealed observation of regulation in both stress exposed and non 39 pathways including immune response, inflammation, and exposed animals following fear conditioning suggests that these cytokine signaling pathways (Table 3). To contextualize these genes are similarly transcribed in the amygdala following fear differences, we compared the proportion of blood cell types conditioning. Frontiers in Psychiatry | www.frontiersin.org 6 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression FIGURE 3 | Genes in the Amygdala traditionally associated with PTSD or psychiatric stress phenotypes. Box and Whiskers plots of gene expression in FC and Immo+FC animals for (a) 2 genes traditionally associated with PTSD (Htr2a and Drd2), and (b) 4 genes associated with stress phenotypes (Igf2, Grm2, Trhr, and Clock). On the y-axis, gene expression values from the different animal groups. The box represents their quartiles; whiskers the variability outside the upper and lower quartiles; the horizontal line represents the median. DISCUSSION results provide confidence in our approach, as they are consistent with established pathways relevant to amygdala-mediated fear This study utilized a translational animal model to examine learning. However, our observation of fear conditioning induced molecular alterations associated with threat exposure in both the down-regulation of gene transcription in the amygdala is blood and amygdala of mice with and without a prior stress noteworthy as one might expect that associative learning would history. As much work has noted that, while trauma-related increase transcription to support plasticity necessary for memory disorders can occur in individuals following exposure to a single formation. As fear conditioning has been found to result in trauma, individuals with a history of early stress and trauma multiple waves of transcriptional processes occurring across the are more likely to develop PTSD following subsequent trauma minutes to hours that follow (36), these data should be viewed exposure (2, 33). We examined RNA expression changes in two as a static snapshot of the dynamic transcriptional processes that different tissues: (1) the amygdala, often considered the “hub” of accompany fear conditioning, i.e., 2 h for the current study. Other the fear and threat response in humans and animals (34, 35) and recent studies have utilized time-points more proximal (30 min (2) the blood, the most common tissue used in human PTSD or 1 h) to fear conditioning and more distant (6 h and 24 h) to studies. Identification of a common gene expression response examine transcriptional changes, and have also demonstrated pattern presents a valuable step in translational biology, toward conditioning related down-regulation and up-regulation of gene bridging the disconnect between how peripheral gene expression targets (10, 37–39). Therefore, while 2 h has traditionally been changes are relevant to PTSD-related behavioral alterations in used by our group for examining transcriptional processes humans. following fear conditioning in the amygdala, these data must In this study, fear conditioning resulted in differences in indeed be considered as only a subset of the dynamic and highly enrichment of genes implicated in learning and memory as well interwoven molecular processes, including translational and as general cellular processes in the amygdala (Table 1). These epigenetic processes, which also accompany fear conditioning. Frontiers in Psychiatry | www.frontiersin.org 7 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression TABLE 3 | Enrichment of biological process among genes associated with prior stress immobilization. GO number Description FEa p-valueb AMYGDALA GO:0008285 Negative regulation of cell proliferation 3.43 0.004 GO:0006457 Protein folding 5.61 0.010 GO:0006986 Response to unfolded protein 9.39 0.014 GO:0042493 Response to drug 3.18 0.029 BLOOD GO:0002376 Immune system process 6.51 2.29 × 10−25 GO:0045087 Innate immune response 6.01 6.40 × 10−23 GO:0006954 Inflammatory response 5.83 3.38 × 10−18 GO:0052697 Xenobiotic glucuronidation 39.59 5.18 × 10−8 GO:0032496 Response to lipopolysaccharide 4.97 1.61 × 10−6 GO:0030593 Neutrophil chemotaxis 8.39 1.40 × 10−5 GO:0006955 Immune response 3.93 1.72 × 10−5 GO:0006935 Chemotaxis 6.04 1.70 × 10−5 GO:0042742 Defense response to bacterium 4.66 1.54 × 10−5 GO:0032760 Positive regulation of tumor necrosis factor production 9.06 1.47 × 10−5 GO:0009813 Flavonoid biosynthetic process 16.97 4.82 × 10−5 GO:0052696 Flavonoid glucuronidation 16.97 4.82 × 10−5 GO:0071223 Cellular response to lipoteichoic acid 29.69 1.19 × 10−4 GO:0009615 Response to virus 6.36 4.56 × 10−4 GO:0050729 Positive regulation of inflammatory response 7.07 0.0017 GO:0032755 Positive regulation of interleukin-6 production 7.71 0.0028 GO:0002755 MyD88-dependent toll-like receptor signaling pathway 16.70 0.0028 GO:0019221 Cytokine-mediated signaling pathway 4.27 0.0033 GO:0055072 Iron ion homeostasis 8.48 0.0045 GO:0050830 Defense response to Gram-positive bacterium 5.27 0.0051 GO:0031663 Lipopolysaccharide-mediated signaling pathway 10.06 0.0062 GO:0042127 Regulation of cell proliferation 3.34 0.006 GO:0045766 Positive regulation of angiogenesis 4.42 0.009 GO:0050873 Brown fat cell differentiation 9.17 0.009 GO:0010628 Positive regulation of gene expression 2.57 0.009 GO:0050728 Negative regulation of inflammatory response 5.12 0.013 GO:0042535 Positive regulation of tumor necrosis factor biosynthetic process 17.13 0.013 GO:0002548 Monocyte chemotaxis 7.79 0.020 GO:0034341 Response to interferon-gamma 10.28 0.019 GO:0071222 Cellular response to lipopolysaccharide 3.20 0.021 GO:0008360 Regulation of cell shape 3.84 0.022 GO:0007165 Signal transduction 1.70 0.026 GO:0006898 Receptor-mediated endocytosis 5.94 0.026 GO:0051607 Defense response to virus 3.47 0.026 GO:0043123 Positive regulation of I-kappaB kinase/NF-kappaB signaling 3.59 0.035 GO:0032494 Response to peptidoglycan 22.27 0.036 GO:0050766 Positive regulation of phagocytosis 6.49 0.041 GO:0035456 Response to interferon-beta 19.79 0.050 GO:0045410 Positive regulation of interleukin-6 biosynthetic process 19.79 0.050 a FE, Fold Enrichment. b p-value following Benjamini-Hochberg correction for multiple testing. In response to fear conditioning, prior acute stress pathways in blood. Several studies have shown a strict causal immobilization conducted the previous week, induces distinct association between immune response, inflammation, and PTSD gene expression differences involved in immune activation (40). Compared to mice that underwent FC alone, mice exposed Frontiers in Psychiatry | www.frontiersin.org 8 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression reported by PTSD patients (47). Similarly, Grm2 (metatropic glutamate 2 receptor) has been associated with anxiety-like behaviors in several rodent models, and activation of these receptors in the amygdala has been found to be necessary for fear related behaviors. Highly selective mGluR2/3 agonists depress excitatory neurotransmission in the amygdala (48), suggesting that such agonists may be potentially therapeutic for PTSD patients by reducing amygdala hyperactivity (32, 49). Finally, we observed regulation of the Thyrotropin Releasing Hormone Receptor1 (Trhr1) in Immo+FC animals; a novel finding that is of interest given its role in the hypothalamic-pituitary-thyroid axis and the observation that clinical dysfunction of the thyroid hormone system can manifest with anxiety behaviors (50). These data support the utility of this approach in examining blood and brain-based alterations FIGURE 4 | Prior stress sensitization causes immune dysregulation. Percent in threat exposure occurring with and without a prior stress of monocytes, neutrophils, and lymphocytes in the three different groups (HC, history. FC, and Immo+FC) are shown. Bars indicate standard errors. As one of our main objectives was to identify common biological responses to fear and stress in both brain and blood, we evaluated shared genes in amygdala and blood. Although to prior immobilization showed gene expression patterns there was a limited overlap in individual genes that responded to consistent with immune dysregulation. This was supported by each condition (Table 4), we found six genes in which changes the observation that blood from Immo+FC mice had a higher of expression were specifically associated with immobilization. proportion of neutrophils, an essential part of the innate immune Among these genes, Pde7 encodes phosphodiesterase-7, known system, and an indicator of inflammation. The neutrophil- for its regulation of T-cell function and association with lymphocyte ratio has recently been used as an indicator of regulation of immune response, and its inhibitors may help chronic low-grade inflammation, and known to associate with patients with immunological and neuro-inflammatory disorders clinical outcomes in neuropsychiatric disorders (41, 42). Our (51). In rodents, inhibition of Pde7 regulates anxiety behaviors, data suggested that immune response genes and pathways are mediated by increasing levels of hypothalamic thyrotropin- responsive to prior immobilization stress both in the blood and releasing hormone (52). Notably, a dual Pde7 and GSK-3β amygdala. inhibitor significantly improves episodic and spatial memory In examining transcriptional differences in the amygdala of and enhances fear memory, as well as facilitating paired-pulse mice with and without prior stress exposure, we observed a inhibition and latent inhibition, both behaviors that have been number of genes that have previously been associated with PTSD. found to be impaired in psychosis, suggesting that inhibition of In particular, DRD2, and HTR2a are of interest as both the Pde7 and GSK-3β enhances cognition (53). Lipopolysaccharide dopaminergic and serotonergic systems have been traditionally Binding Protein (Lbp) and Lipocalin 2 (Lnc2) are involved implicated in the pathophysiology of PTSD (28, 43, 44). Indeed, in an acute phase of immunological response and metabolic dopamine dysregulation has been implicated in various PTSD inflammation (54). Sterol O-Acyltransferase 1 (Soat1) plays symptoms (e.g., attention, vigilance, arousal, sleep), and DRD2 an important role in cholesterol homeostasis regulation and has been associated with PTSD diagnosis (26, 29). Similarly, metabolism, and it has been extensively studied as a target as serotonin and norepinephrine reuptake inhibitors (SSRI for hypercholesterolemia and Alzheimer’s disease (55). The and SNRI) remain the first line pharmacotherapy for PTSD, observation of immune response and inflammation-related genes and numerous candidate gene studies have identified a link is of particular interest given the recent emerging appreciation between variants in serotonin related genes, including HTR2a of the role of immune-response and inflammatory pathways (27, 28), our observation of differential expression of HTR2a in psychiatric disorders, including depression, autism, and is consistent with the previous literature and support for trauma-related disorders (56, 57). Recent work examining the this neurotransmitter system in the consequences of threat consequences of early-life inflammation via lipopolysaccharide and trauma exposure. We also identified genes (Igf2, Clock, administration has revealed impairments in fear memory Grm2, Trhr1) that have been previously associated with stress- extinction during adulthood (58), in line with our observation related phenotypes. Interestingly, Igf2 methylation has been that a prior history of stress results alterations in inflammation, found to associate with PTSD (45), fear extinction (30), and might suggest that prolonged inflammatory responses contribute more classically in chronic stress response (46). Among other to impaired fear extinction. Further, it is of interest that differentially expressed genes in Immo+FC vs. FC, Clock, inhibition of pro-inflammatory cytokines has been suggested a gene involved in the circadian rhythms, is particularly to facilitate fear memory extinction (59). Taken together, these interesting as mice with mutations of this gene have altered findings suggest that a closer examination of the induction anxiety behaviors (31), and as sleep disturbances are commonly of inflammation and cytokine pathways in stress and trauma Frontiers in Psychiatry | www.frontiersin.org 9 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression TABLE 4 | Overlap in amygdala and blood in Immo+FC vs. FC. Gene Amygdala Blood Fold change p-value q-value Fold change p-value q-value Dmxl2 0.39 5E-05 0.003 0.99 5E-05 0.002 Erdr1 −0.59 5E-05 0.003 −1.07 5E-05 0.002 Lcn2 3.36 5E-05 0.003 0.95 5E-05 0.002 Pde7b 0.33 4E-04 0.015 1.15 5E-05 0.002 Trps1 0.40 2E-04 0.007 0.81 5E-05 0.002 Sdf2l1 0.39 7E-04 0.024 0.64 2E-04 0.007 Soat1 0.34 5E-04 0.019 0.55 5E-04 0.015 Thbd 0.69 5E-05 0.003 0.69 7E-04 0.019 Fgd4 0.78 5E-05 0.003 0.84 8E-04 0.021 Lbp 0.83 5E-05 0.003 0.94 2E-03 0.047 Gh −4.49 5E-04 0.019 −1.84 3E-03 0.058 Cers6 0.44 5E-05 0.003 0.55 4E-03 0.072 Rbm3 −0.30 1E-03 0.032 −0.46 4E-03 0.075 Stxbp6 −0.27 8E-04 0.025 0.74 7E-03 0.107 Lyst 0.53 5E-05 0.003 0.42 7E-03 0.110 Jhdm1d 0.35 5E-05 0.003 0.40 1E-02 0.154 Lnpep 3.10 5E-05 0.003 0.41 1E-02 0.178 Endou 1.01 5E-05 0.003 −0.76 2E-02 0.212 Cdk5rap2 0.72 5E-05 0.003 −0.44 2E-02 0.242 Polr2a −0.26 7E-04 0.023 −0.36 3E-02 0.270 F5 0.60 3E-04 0.012 −0.38 3E-02 0.282 Prkdc 0.29 1E-03 0.031 −0.37 3E-02 0.310 Hpcal1 −0.31 5E-05 0.003 0.33 3E-02 0.311 Nr4a1 0.31 1E-04 0.005 −0.33 3E-02 0.312 Dnajb14 1.55 5E-05 0.003 0.54 4E-02 0.342 Pcnx 0.26 5E-04 0.017 0.33 5E-02 0.368 Pisd_ps3 −0.40 5E-05 0.003 −0.31 5E-02 0.373 Gene associated in both blood and amygdala. Gene Name: Dmxl2, Dmx-like 2; Erdr1, erythroid differentiation regulator 1; Fgd4, FYVE, RhoGEF, and PH domain containing 4; Lcn2, lipocalin 2; Lbp, lipopolysaccharide binding protein; Pde7b, phosphodiesterase 7B; Soat1, sterol O-acyltransferase 1; Sdf2l1, stromal cell-derived factor 2-like 1; Thbd, thrombomodulin; Trps1, trichorhinophalangeal syndrome I. responses may yield new strategies for alleviating the deleterious anxiety processes using this paradigm, the altered transcriptional consequences of stress and trauma. processes observed in this current study suggest that even While our data reveal transcriptional alterations in the relatively brief exposures to stress prior to a threating situation, amygdala and peripheral blood that accompany fear conditioning such as auditory fear conditioning, can be useful in revealing the with and without prior stress history, it is important to note lasting imprint of stress history on molecular events associated that other recent studies employing alternative models of rodent with traumatic memory formation. While it would be unwise stress have examined gene expression in blood and brain (38, to intensively interpret our data with relevance to those which 60, 61). Our use of immobilization restraint stress is predicated have utilized different paradigms, it is important to highlight that on our prior experience with this paradigm resulting in altered many of these studies have also revealed alterations of genes that fear memory processes, anxiety behaviors and HPA axis function are associated with immune response, dopamine function, and (4, 15); as our primary goal was to examine the consequences glucocorticoid related signaling (38, 60, 61), which suggest that of stress history on subsequent molecular alterations that shared pathways altered by stress are emerging across a variety of accompany fear conditioning, we utilized this model to conduct behavioral models. this genetic discovery study. Importantly, while recent studies We acknowledge that this study has some limitations. First, employing different stress procedures, 21-day variable stress, although blood cell counts varied in the different groups, we chronic restraint stress, repeated shock administration, and were not able to account for such variation in the analysis but social defeat stress have been used to examine transcriptional were able to capture the information related to that variation. processes in the amygdala, this single-session immobilization Alternatively, as white blood cell counts change following stress procedure is relatively acute in comparison (38, 61, 62). immobilization, covarying for cell composition may obscure Coupled with our previous demonstration of altered fear and biological differences that accompany gene expression changes. Frontiers in Psychiatry | www.frontiersin.org 10 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression FIGURE 5 | Common Signals in Amygdala and Blood prior stress sensitization in representative genes. Box and Whiskers plots depict gene expression (y-axis) in FC and Immo+FC animals. Representative genes that associate with prior immobilization in both amygdala and blood are shown. On the y axis, gene expression values from the different animal group. The box represents their quartiles; whiskers the variability outside the upper, and lower quartiles; the horizontal line represents the median; outliers are indicated by single dots. Second, we focused our analyses on the amygdala, because of individual genes. Finally, we are aware that different strains of its role in fear and threat response behaviors. As such, we mice may generate different patterns of expression in response utilized auditory fear conditioning as it is well established to to FC; a cumulative analysis in different strains of mice could rely on amygdala function. As a result, we cannot extrapolate eventually increase the power to detect additional genes relevant these data to other brain regions that contribute to stress to PTSD-related phenotypes (66). and traumatic memory (e.g., hippocampus, insula, cingulate, In conclusion, this study provides a translational framework and prefrontal cortex). Additionally, this study was conducted for mouse and human studies aimed at examining the molecular exclusively in male rodents, which showed a higher response correlates that inform studies of psychiatric disorders. We were to shock-induced contextual fear conditioning than females in able to identify, in the amygdala, several genes that specifically terms of behavioral phenotypes (freezing and rearing activity) respond to the stress immobilization paradigm, some of which (63). However, given the higher prevalence and heritability have been traditionally associated with PTSD, and anxiety of PTSD in females in human population (64, 65), and the disorders. Future studies will be needed to evaluate if these genes emerging role of circulating estrogen levels in relation to fear and associate with PTSD or stress-related traits in human clinical anxiety behaviors, additional studies should specifically examine studies. transcriptional changes in females with full regard to the estrus phase. Next, in order to get sufficient RNA for a comprehensive AUTHOR CONTRIBUTIONS survey of the transcriptome, we pooled two animals per sample for each group. Though this does not limit our ability to identify AL performed statistical analyses and contributed to writing overall similarities and differences in different tissues of the same the manuscript. SM performed the mouse experiments and animals in response to threat exposure or to interpret pathway contributed to study design, and manuscript writing. SS and RA analysis, caution should be taken in interpreting the results of helped with the mouse experiments and manuscript editing. KR Frontiers in Psychiatry | www.frontiersin.org 11 January 2019 | Volume 9 | Article 778 Lori et al. Threat-Associated Gene Expression helped to design the experiment, interpret the data, and write ACKNOWLEDGMENTS the manuscript. AS helped to design the experiment, analyze and interpret the data, and write the manuscript. We would like to thank the Yerkes Genomics Core at Emory University, and in particular Dr. Gregory Tharp and Dr. Nirav FUNDING Patel for their help with RNA sequencing. This research was supported by NARSAD Young Investigator SUPPLEMENTARY MATERIAL Grants (#19233 and #22434) from the Brain Behavior Foundation. Additional support for RA provided by The Supplementary Material for this article can be found Ramón y Cajal program RYC2014-15784, RETOS-MINECO online at: https://www.frontiersin.org/articles/10.3389/fpsyt. SAF2016-76565-R, and FEDER funds. 2018.00778/full#supplementary-material REFERENCES amygdala and hippocampus. Proc Natl Acad Sci USA. (2014) 111:3158–63. doi: 10.1073/pnas.1318954111 1. Daviss WB. The child trauma screening questionnaire predicts ptsd onset 14. Kessler RC, Aguilar-Gaxiola S, Alonso J, Benjet C, Bromet EJ, Cardoso 6 months after traumatic accident better than the children’s impact of G, et al. Trauma and PTSD in the WHO world mental health surveys. events scale. Evid Based Ment Health (2007) 10:44. doi: 10.1136/ebmh. Eur J Psychotraumatol. (2017) 8:1353383. doi: 10.1080/20008198.2017.13 10.2.44 53383 2. Powers A, Fani N, Cross D, Ressler KJ, Bradley B. Childhood trauma, 15. 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Sex differences in the behavioural Conflict of Interest Statement: The authors declare that the research was and hypothalamic-pituitary-adrenal response to contextual fear conditioning conducted in the absence of any commercial or financial relationships that could in rats. Horm Behav. (2014) 66:713–23. doi: 10.1016/j.yhbeh.2014. be construed as a potential conflict of interest. 09.015 64. Kessler RC, Sonnega A, Bromet E, Hughes M, Nelson CB. Posttraumatic Copyright © 2019 Lori, Maddox, Sharma, Andero, Ressler and Smith. This is an stress disorder in the national comorbidity survey. Arch Gen Psychiatry (1995) open-access article distributed under the terms of the Creative Commons Attribution 52:1048–60. doi: 10.1001/archpsyc.1995.03950240066012 License (CC BY). The use, distribution or reproduction in other forums is permitted, 65. Duncan LE, Ratanatharathorn A, Aiello AE, Almli LM, Amstadter AB, provided the original author(s) and the copyright owner(s) are credited and that the Ashley-Koch AE, et al. Largest GWAS of PTSD (N=20 070) yields genetic original publication in this journal is cited, in accordance with accepted academic overlap with schizophrenia and sex differences in heritability. Mol Psychiatry practice. No use, distribution or reproduction is permitted which does not comply (2018) 23:666–73. doi: 10.1038/mp.2017.77 with these terms. Frontiers in Psychiatry | www.frontiersin.org 14 January 2019 | Volume 9 | Article 778