c mice: Analysis of corticosterone response and glucocorticoid gene expression in cortex and hippocampus

Journal Pre-proof Glycine transporter type 1 (GlyT1) inhibition improves conspecific-provoked immobility in Balb/c mice: Analysis of corticosterone response and glucocorticoid gene expression in cortex and hippocampus

Jessica A. Burket, Jerrah C. Pickle, Allison M. Rusk, Bronson A. Haynes, Julia A. Sharp, Stephen I. Deutsch PII:

S0278-5846(19)30833-4

DOI:

https://doi.org/10.1016/j.pnpbp.2020.109869

Reference:

PNP 109869

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date:

4 October 2019

Accepted date:

15 January 2020

Please cite this article as: J.A. Burket, J.C. Pickle, A.M. Rusk, et al., Glycine transporter type 1 (GlyT1) inhibition improves conspecific-provoked immobility in Balb/c mice: Analysis of corticosterone response and glucocorticoid gene expression in cortex and hippocampus, Progress in Neuropsychopharmacology & Biological Psychiatry(2019), https://doi.org/10.1016/j.pnpbp.2020.109869

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© 2019 Published by Elsevier.

Journal Pre-proof Glycine Transporter Type 1 (GlyT1) Inhibition Improves Conspecific-Provoked Immobility in Balb/c Mice: Analysis of Corticosterone Response and Glucocorticoid Gene Expression in Cortex and Hippocampus Jessica A. Burket, Ph.D.1, 2, Jerrah C. Pickle, M.S.1, Allison M. Rusk, B.S.1, Bronson A. Haynes, Ph.D. 2, Julia A. Sharp, Ph.D.3, Stephen I. Deutsch, M.D., Ph.D. 1,4 Affiliations: 1

Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, Norfolk, VA

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Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, United States. 3

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Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, United States

Corresponding Author: 4

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Stephen I. Deutsch, M.D., Ph.D. Anne Armistead Robinson Endowed Chair in Psychiatry Professor and Chairman, Department of Psychiatry and Behavioral Sciences 825 Fairfax Avenue, Suite 710 Norfolk, Virginia 23507 757 446 5888 (office) 757 446 5918 (facsimile) [email protected]

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Running Title: GlyT1 inhibition and immobility in Balb/c mice

Journal Pre-proof Abstract

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Stress reactivity and glucocorticoid signaling alterations are reported in mouse models of autism spectrum disorder (ASD). Balb/c mice display decreased locomotor activity in the presence of stimulus mice and spend less time exploring enclosed stimulus mice; this mouse strain has been validated as an ASD model. VU0410120, a glycine type 1 transporter (GlyT1) inhibitor, improved sociability in Balb/c mice, consistent with data that NMDA Receptor (NMDAR) activation regulates sociability, and the endogenous tone of NMDAR-mediated neurotransmission is altered in this strain. Effects of a prosocial dose of VU0410120 on conspecific-provoked immobility, and relationships between conspecific-provoked immobility and corticosterone response were explored. VU0410120-treated Balb/c mice showed reduced immobility in the presence of conspecifics and increased the conspecific-provoked corticosterone response. However, the intensity of conspecific-provoked immobility in VU0410120-treated Balb/c mice did not differ as a function of corticosterone response. Expression profiles of 88 glucocorticoid signaling associated genes within frontal cortex and hippocampus were examined. Balb/c mice resistant to prosocial effects of VU0410120 had increased mRNA expression of Ddit4, a negative regulator of mTOR signaling. Dysregulated mTOR signaling activity is a convergent finding in several monogenic syndromic forms of ASD. Prosocial effects of VU0410120 in the Balb/c strain may be related to regulatory influences of NMDAR-activation on mTOR signaling activity. Because corticosterone response is a marker of social stress, the current data suggest that the stressfulness of a social encounter alone may not be the sole determinant of increased immobility in Balb/c mice; this strain may also display an element of social disinterest.

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Keywords: Balb/c; GlyT1 inhibition; Autism Spectrum Disorder; NMDA receptor, Immobility; Glucocorticoid Signaling

Journal Pre-proof Introduction Impaired social communication is a debilitating core symptom domain of autism spectrum disorder (ASD). High levels of comorbid anxiety (~40-50%) are often seen in children and adults with ASD [1–5]. Data from recent imaging and physiological studies show altered neurobiological responses to stressors in children with ASD who experience anxiety, such as decreased amygdala volume and differences in skin conductance activity in response to threatening situations, compared to childen with ASD-alone and/or neurotypical individuals [6–9]. mRNA levels of the glucocorticoid and mineralocorticoid receptors within the frontal gyrus of ASD brains were altered compared to age-matched control brains, suggesting these pathways may play a role in ASD pathophysiology [10].

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Supporting these recent clinical findings, alterations in stress reactivity and glucocorticoid signaling have been documented in relevant mouse models of ASD, with anxiety-like behaviors observed in several of these well-characterized models [11–15]. For example, hypothalamic-pituitary-adrenal (HPA) activity was measured in genetically-inbred BTBR T+ tf/J (BTBR) mice by assessing peripheral corticosterone, and expression of corticotropin-releasing factor (CRF), glucocorticoid receptor and oxytocin peptide levels within the hippocampus and paraventricular nucleus of the hypothalamus (PVN), compared to the C57Bl/6J (B6) mouse strain [15]. In addition to showing increased plasma corticosterone, BTBR mice displayed increased basal mRNA levels of glucocorticoid receptor only in the CA1 region of the hippocampus. Within the PVN, levels of oxytocin peptide were elevated in BTBR mice compared to the C57Bl/6J (B6) mouse strain. Interestingly, both CRF mRNA and peptide expression did not differ between strains in this area. BTBR mice did not exhibit anxiety-like behaviors, as measured in the elevated-plus maze or light-dark task, compared to the B6 mice; they displayed low stress reactivity measured as decreased response during the hot plate test. However, BTBR mice did show decreased immobility on tail suspension and forced swim tasks [15]. Moreover, in another syndromic model of ASD, Ube3a-maternal-deficient mice (a model of Angelman syndrome), downregulation of several glucocorticoid signaling genes within multiple brain regions was associated with increases in stress and anxiety-like behavior; these mice also displayed increased peripheral corticosterone levels and reduced glucocorticoid receptor expression, as well as a reduced number of hippocampal parvalbumin-containing GABA inhibitory interneurons [12]. Furthermore, enhanced anxiety behavior and involvement of glucocorticoid signaling abnormalities were observed in two different mouse models of Rett syndrome [14,16]. Taken together these data provide a compelling rationale to study relationships between glucocorticoid response and social stressors in other relevant mouse models of ASD.

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The NMDA Receptor (NMDAR) is a heterotetrametric receptor composed of four polypeptide subunits (2 GluN1 and 2 GluN2A-D), whose activation requires the cooperative binding of glutamate and its co-agonist (D-serine or glycine) [17–21]. The NMDAR has emerged as a viable therapeutic target in ASD [19,22–26]. Recently, corticosterone, was shown to modulate NMDAR-mediated neurotransmission via regulation of GluN2B synaptic surface dynamics and mineralocorticoid signaling [27]. Specifically, acute exposure of cultured rat embryonic (E18) hippocampal neurons to corticosterone increased the synaptic content of the GluN2B subunit, relative to the GluN2A subunit, and increased NMDAR-mediated miniature EPSCs (mEPSCs) [27]. The corticosterone-evoked increase of NMDAR-mediated mEPSCs was related to reduced surface dynamics caused by increased anchoring and retention of the GluN2B-NMDAR within the glutamate synapse (i.e., the surface diffusion of the GluN2B-NMDAR was significantly decreased, whereas there was no change in the surface diffusion of the GluN2A-NMDAR); data suggest that this rapid effect of corticosterone to reduce synaptic diffusion of the GluN2B-NMDAR was mediated at the cell membrane by a “mineralocorticoid-”like signaling pathway (i.e., a non-permeate active analogue of corticosterone could not mimic this effect). The effect of corticosterone on the surface dynamics and redistribution of the synaptic ratio of the GluN2B (increased)- to GluN2A (decreased)-NMDARs favored induction of long-term potentiation (LTP) [27]. Further, transgenic mice with diminished expression of the NR1 subunit or reduced affinity of the NMDAR for the obligatory glycine co-agonist show quantitative deficits of sociability, providing direct evidence consistent with a regulatory role of the NMDAR in normal mouse sociability [28,29]. Moreover, mice with genetically-engineered loss-of-function mutations of Shank scaffolding proteins, which are critical for maintaining the postsynaptic architecture of the excitatory synapse, also show quantitative deficits of sociability that are attenuated by D-cycloserine, a partial glycineB agonist [30,31]. The latter data are also consistent with a critical prosocial role for efficient transduction of the extracellular

Journal Pre-proof glutamate/glycine signal by the NMDAR. Interestingly, the inbred Balb/c mouse strain, which has been validated as a model of ASD [32–34], shows a functional alteration of the endogenous tone of NMDARmediated neurotransmission [35–37]. Balb/c mice display decreased locomotor activity in the presence of enclosed and freely-behaving salient social stimulus mice; also, they spend less time exploring and in the vicinity of an enclosed stimulus mouse, and less time engaged in anogenital sniffing, mounting and social approach when interacting with freely-behaving conspecifics, among other impaired social behaviors [32– 34,38]. However, their exploratory behavior toward an inanimate object does not differ and they have similar performance in the elevated plus maze, a behavioral measure of anxiety, to Swiss Webster mice [39]. DCycloserine, an NMDAR partial glycineB agonist, and VU0410120, a novel glycine type 1 transporter (GlyT1) inhibitor, improved the sociability of the Balb/c mouse, consistent with both the functional impairment of NMDAR-mediated neurotransmission and the ability of NMDAR activation to improve sociability in this mouse strain [19,35–37,40–43].

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The current study explored differences in conspecific-provoked immobility (CPI) between the geneticallyinbred Balb/c and outbred Swiss Webster mouse strains, and the relationship between CPI and the corticosterone (CORT) response, a measure of acute stress, in these strains. Moreover, the effect of a prosocial dose of VU0410120 on the CPI and CORT response of the Balb/c mouse was also explored. Finally, because brain regions such as cortex and hippocampus have important roles in stress and glucocorticoid receptor regulation, strain differences within these areas were investigated by targeted expression profiling of common glucocorticoid signaling genes known to be implicated in the social stress and anxiety phenotypes. Ideally, assessment of immobility behavior and corticosterone secretion would clarify relationships between diminished locomotor activity of Balb/c mice in the presence of an enclosed or freely-moving salient stimulus mouse, intensity of immobility behavior, and the corticosterone response, a marker of stress exposure. In any event, the ASD “phenotype” of the Balb/c mouse will be better characterized by exploring these relationships, which may also help to address the complex question of whether a same-aged conspecific stimulus mouse elicits social interest or serves as an aversive anxiogenic stimulus for the Balb/c strain.

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Methods Animals Test mice were experimentally-naïve, 4-week old male, outbred Swiss Webster and genetically-inbred Balb/c mice (BALB/cAnNHsd) (Envigo Laboratories, Fredrick, MD, USA), housed 2 mice per cage. Stimulus mice were 4-week old ICR mice (Charles River Laboratories, Wilmington, MA, USA), housed 4 per cage. All animals were housed in hanging clear Plexiglas cages with free access to food and water and maintained on a 12-h light/dark cycle. Test mice were individually weighed prior to drug administration (N ≥ 21/condition). Immediately following behavioral testing, mice were sacrificed for collection of trunk blood and brain tissue. Whole hippocampal and cortical tissues were micro-dissected on ice, immediately snap-frozen in liquid nitrogen and stored at -80°C until future analysis. All animal procedures were approved by the Eastern Virginia Medical School Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Drug VU0410120 (2,4-dichloro-N-((4-(cyclopropylmethyl)-1-(ethylsulfonyl)piperidin-4-yl)methyl)benzamide) (synthesized by Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN, USA), a highly-selective, highly potent GlyT1 inhibitor, is a non-sarcosine derived derivative of a 4,4-disubstituted piperidine GlyT1 inhibitor. VU0410120 has high selectivity for the GlyT1 site; the IC50 for the GlyT1 site is 26 nM, whereas its IC50 for the GlyT2 “taurine transporter” is greater than 30 µM [44]. VU0410120 was dissolved in 20% hydroxypropyl-β-cyclodextrin (Cyclodextrin Technologies Development Inc., Alachua, FL, USA), and drug or vehicle was injected intraperitoneally (i.p.) in a volume of 0.01 mL/g of body weight, 20 minutes prior to beginning behavioral testing.

Journal Pre-proof Assessment of Immobility Behavior Immobility behavior was measured in a standard three-compartment sociability apparatus, which consisted of a black Plexiglas rectangular box (52.07 cm x 25.40 cm x 22.86 cm), without a top or bottom. The center compartment was slightly smaller (12.07 cm x 25.40 cm) than the two end compartments that were of equal size (19.05 cm x 25.40 cm). Inverted wire cups (Galaxy Cup, Kitchen Plus) were placed in each side of the end compartments during Sessions I and II (discussed below) and housed the stimulus mouse. Weighted dark bottles were placed on top of the inverted wire cups to prevent climbing during testing. The apparatuses, wire cups and bottles were thoroughly cleaned with Quatricide PV solution after each test mouse was studied. All Sessions were conducted in dim lighting (< 3.5 lux) and videotaped using Sony HD Video Cameras (Sony Corp., Tokyo, Japan) in nightshot mode with infrared lighting for future viewing and data collection. Immobility was measured as discrete episodes, total seconds per 10-min session, and seconds/discrete episode of immobility in sessions I (acclimation), II (enclosed stimulus) and III (freely-behaving stimulus). Immobility behavior was defined as stationary while not engaged in social/stereotypic behavior.

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Measurement of Corticosterone levels Trunk blood from all subjects was collected ≤30 min after the end of session III and allowed to clot for ~30 minutes. Serum was obtained by centrifugation for 10 minutes at 3,000 rpm and then stored at -80°C until assayed. Samples were diluted 1:70 and corticosterone levels were measured in duplicate using a commercially available ELISA kit, following the manufacturer's protocol (ab108821, Abcam). Only mean values with CVs below 12 were used in data analysis.

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Determine Groups for Microarray Experiment and Transcriptome Analysis A brief overview of the study design and workflow for the microarray experiments and transcriptomic analysis is provided (Figure 1). Previously published sociability measures obtained in session III were ranked within each treatment group (e.g. vehicle-treated Balb/c or Swiss Webster; and VU0410120-treated [18mg/kg] Balb/c or Swiss Webster) [41]. Importantly, the dose selection of VU0410120 (18 mg/kg) was chosen due to its prosocial effects in Balb/c mice and absence of spontaneously emerging stereotypic behaviors within the Swiss Webster mice. Rank scores were used to designate the global prosocial responses of individual mice to VU0410120 and assign them as members of “High”, “Medium” and “Low” prosocial response categories. Specifically, the rated social measures from session III that were used to determine social rank score included discrete episodes of the following: social approach, social avoidance, anogenital sniffing and pursuit, and time spent in pursuit. Each social measure was ranked individually for each mouse and then all social measures were combined to give a total ranked social score for each animal. This total ranked score was used to characterize vehicle and VU0410120-treated groups of mice according to their low, medium or high total ratings of prosocial behaviors. In the vehicle- and VU0410120-treated groups of Balb/c mice, the samples designated with the highest and lowest total ratings of prosocial behaviors were used for microarray analysis (n=4/condition); groups of Swiss Webster mice with a “medium” social ranking score were chosen for comparisons. Specifically, the following groups of mice were used for the microarray experiments: vehicle-treated Balb/c-low sociability; vehicle-treated Balb/c-high sociability; vehicle-treated Swiss Webster-medium sociability; VU0410120-treated Balb/c-low (i.e., “non-responders”) sociability; VU0410120-treated Balb/c-high (i.e., “responders”) sociability; and VU0410120-treated Swiss Webstermedium sociability. The response categories informed interrogation of the large repository of regionallyselective transcriptomic data obtained in frontal cortex and hippocampus; comparisons were made between VU0410120-treated Balb/c mice in the “High” and “Low” response categories, VU0410120-treated Swiss Webster mice in the “Medium” response category, and vehicle-treated Balb/c and Swiss Webster mice, whose total rank scores placed them in the “High” and “Low” prosocial response categories (Balb/c) and “Medium” response category (Swiss Webster), respectively. Analysis of Glucocorticoid Signaling and Immediate Early Gene Expression Cortical and hippocampal RNA from vehicle and drug-treated Balb/c and Swiss Webster mice (n = 4/group) was extracted using RNAeasy Mini Kit (QIAGEN). RNA quality was analyzed by microfluidic gel electrophoresis (Agilent 2100 Bioanalyzer); only samples with a high RNA integrity number (RIN > 8) were selected for analysis. Transcriptome profiling was performed using the Affymetrix GeneChip Mouse Gene 2.1 ST array (ThermoFisher Scientific, Waltham, MA). Briefly, 100 ng of total input RNA was used to

Journal Pre-proof generate cDNA and samples were hybridized to the GeneChip. Preliminary evaluation of differences in gene expression was determined using Affymetrix Transcriptome Analysis Console (TAC) software; fold changes (-1.25 ≤ or ≥1.25, p<0.05) and False Discovery Rate (FDR, p<0.05). Differences in expression of 88 glucocorticoid signaling genes in particular, and other exploratory comparisons between groups were performed (e.g., differential gene expression of immediate early genes and gene ontology analysis [fold change -1.20 ≤ or ≥1.20, p<0.05 and FDR, p<0.05]) using Affymetrix TAC, DAVID (the database for annotation, visualization and integrated discovery) version 6.8 and/or Kyoto Encyclopedia of Genes and Genomes (KEGG) [45]. Heatmaps displaying a clustering analysis of related groups of genes based on their expression were created using R-studio [46].

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Statistical Approach One-way ANOVA using the Kruskal-Wallis nonparametric test was used to determine differences in corticosterone response, measures of immobility behavior (e.g., episodes of immobility, total time spent immobile, and time spent immobile per episode of immobility) observed in Session II and III and the relationship between CPI and corticosterone response between untreated Balb/c and Swiss Webster mice and VU0410120-treated Balb/c mice. Where appropriate, post-hoc comparisons using Dunn’s multiple comparison test were applied.

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Results Effect of VU0410120 on Immobility Behavior in Response to a Social Stimulus To determine whether Balb/c and Swiss Webster mouse strains differed in terms of their immobility behavior in the presence of enclosed and freely-behaving stimulus mice, we rated the number of episodes and duration of immobility behavior in Balb/c and Swiss Webster mice over three-10 minute sessions (e.g. acclimation [session I], enclosed stimulus mouse [session II], and free interaction between test and stimulus mouse [session III]) within the 3-chambered sociability apparatus (Figure 2). On all immobility measures in session II and III, Balb/c mice were significantly more immobile than Swiss Webster mice. Immobility behavior rated in session I was only present in Balb/c (7 out of 21 mice) compared to Swiss Webster mice (0 out of 21); however, intensity of immobility behaviors seen in Balb/c mice during session I was significantly less compared to their immobility rated in sessions II and III (Figure 2).

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Relationship between Immobility Behavior and Corticosterone Levels in Response to a Social Stimulus To determine differences in conspecific-provoked corticosterone response, strain-selective relationships between CPI and corticosterone response, and effects of VU0410120 on the conspecific-provoked corticosterone response, serum corticosterone levels drawn ≤30 min after the end of session III from VU0410120- and vehicle-treated Balb/c and vehicle-treated Swiss Webster mice were analyzed (KruskalWallis statistic, 22.57; p<0.0001, Figure 3A). Importantly, Swiss Webster mice treated with VU0410120 did not display any observed immobility behavior and, thus, could not be included in these analyses. There was no significant difference in serum corticosterone levels between vehicle-treated Swiss Webster and vehicletreated Balb/c mice (Figure 3A). Session III data were used to separate each drug group into “high” immobility (> 73 sec/10 min) or “low” immobility (< 73 seconds/10 min) in order to examine relationships between observed immobility and corticosterone levels (Figure 2D). A cutoff of 73 seconds was chosen based on the mean total time (sec) that Swiss Webster mice engaged in immobility during session III (dotted line, Figure 2F). This further analysis revealed that regardless of high or low immobility, corticosterone levels did not differ between any of the groups. Importantly, corticosterone levels did not differ between the groups of vehicle-treated Balb/c mice with high immobility (N=8) versus those with low immobility (N=9) scores (total seconds, 600 sec epoch) in session III (Figure 3B). These data suggest that the intensity of conspecific-provoked immobility in Balb/c mice may not be related to the corticosterone response elicited by their exposure to enclosed and freely-behaving ICR stimulus mice. Relative to its vehicle-treated condition, Balb/c mice treated with VU0410120 showed a significant increase in serum corticosterone (Dunn’s multiple comparisons test, p<0.01, Figure 3A). However, when comparisons were made between low or high immobility, corticosterone levels of VU0410120-treated Balb/c mice (N=11) with high (N=3) and low (N=8) immobility scores (total sec) in session III did not differ between groups (Figure 3B).

Journal Pre-proof In summary, a regulatory role of NMDAR activation in the corticosterone response is consistent with the observed elevation of serum corticosterone in the VU0410120-treated Balb/c mice [27,47]. However, although vehicle-treated Balb/c mice were significantly more immobile in sessions II and III than the vehicletreated comparator Swiss Webster mice, their serum corticosterone response following exposure to an enclosed and freely-behaving stimulus mouse did not differ. Further, Balb/c mice treated with VU0410120 (18 mg/kg), a highly-selectively, high-potency indirectly-activating glycineB agonist, showed diminished immobility in the presence of a salient stimulus mouse, compared to vehicle-treated Balb/c mice; however, the VU0410120-induced reduction of immobility was apparently independent of its effect on the serum corticosterone response. The data on corticosterone elevation in VU0410120-treated Balb/c mice show that this strain can mount a corticosterone response. However, the fact the corticosterone response after exposure to the ICR stimulus mouse was not significantly elevated in vehicle-treated Balb/c mice over levels observed in the vehicle-treated Swiss Webster comparator strain suggests that “immobility” may differ from “freezing”, the latter being a behavioral response to threatening social stimuli. Moreover, the data suggest that the corticosterone response in the Balb/c strain may be insensitive to a mild social stressor.

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Transcriptomic analysis of glucocorticoid signaling and immediate early gene expression Because glucocorticoid signaling may reflect or underlie social stress and anxiety phenotypes, we conducted a targeted “hypothesis-driven” comparison of gene expression profiles of 88 glucocorticoid signaling associated genes within frontal cortex and hippocampus using the Affymetrix TAC software. Differences between Swiss Webster and Balb/c mouse strains and their prosocial response categories were determined by foldchange difference (-1.25 < or > 1.25, p ≤ 0.05; FDR p ≤ 0.05; Table 1-3Table , Figure 4 and 5). Interestingly, results from these analyses showed that expression of the gene encoding Ddit4, a known negative regulator of mTORC1 signaling, was significantly increased within frontal cortex in VU0410120treated Balb/c “non-responders”, compared to both VU0410120-treated Balb/c “responders” and the VU0410120-treated Swiss Webster “medium” social response group (Table and Figure 4).

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Independently of this hypothesis-driven analysis that focused on finding differences in the expression of genes related to glucocorticoid signaling, a preliminary unbiased, “atheoretical” approach to exploring between-group comparisons of the entire frontal cortical transcriptome was conducted using DAVID and KEGG bioinformatic tools. Specifically, gene ontology (GO) analysis was performed using DAVID for biological processes of significant upregulated genes of VU0410120-treated Balb/c “non-responders”, compared to the VU0410120-treated Balb/c “responders” group. DAVID GO analysis revealed that the ranking of “negative regulation of mTOR signaling” was within the “top 10” of gene ontology for functional biological processes, with a significant fold-enrichment of 5.4 (EASE score, p = 0.013; Error! Reference source not found.). Importantly, expression of five genes involved in negative regulation of mTOR signaling was upregulated in frontal cortex of the VU0410120-treated Balb/c “non-responders”: Akt1s1, Ddit4, Epm2a, Gsk3a, and Tmem127. As will be discussed, the non-phosphorylated form of PRAS40, the protein product of Akt1s1, associates with the mTORC1 complex serving as an inhibitor of its serine/threonine kinase activity. Moreover, KEGG analysis also confirmed differences in six genes involved in mTOR signaling within VU0410120-treated Balb/c “non-responders” compared to the Balb/c “responders” group (Error! Reference source not found.). Importantly, vehicle-treated Balb/c and vehicle-treated Swiss Webster mice did not differ in their expression of Akt1s1 and Ddit4 in frontal cortex and hippocampus; moreover, when the vehicletreated Balb/c mice were assigned to either “High” or “Low” sociability subgroups, expression of Akt1s1 and Ddit4 in frontal cortex and hippocampus did not differ between these subgroups (Table 5). Although preliminary, these gene expression data further encourage earlier work exploring therapeutic strategies targeting mTORC1 over-activation and are consistent with a therapeutic role for NMDAR activation in the regulation (especially dampening) of the mTOR signaling pathway, which is disturbed in several paradigmatic models of ASD [19,22]. Immediate early genes (IEGs) are activity-regulated genes, which include many transcription factors, that have important roles in regulating the properties of the neuronal membrane and synapse dynamics, as well as, refining neural circuits [48]. As discussed, VU0410120, a high-affinity, highly-selective inhibitor of the Glycine type 1 transporter (GlyT1), was shown to have prosocial and procognitive effects in the Balb/c mouse model of ASD (Burket et al., 2015). These effects were observed 30 minutes after its intraperitoneal injection

Journal Pre-proof during sessions II and III in the 3-chamber sociability apparatus. Immediate early gene expression was examined in frontal cortex and hippocampus to confirm that NMDAR-activation was implicated in these prosocial and procognitive effects (Table 6). Importantly, long-term depression (LTD) and dampening of gene expression in Fragile X Syndrome has stimulated thinking about novel therapeutic strategies and complex cross-talk and interaction between subtype-selective NMDA and mGluR5 receptors, regulation of LTD, and ASD [49]. Thus, strain differences in the regionally-selective “relative” suppression of immediate early gene expression in response to GlyT1 inhibition observed in this gene expression analysis may be “surprisingly” relevant to proposed mechanisms of prosocial effects of VU0410120.

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As shown in Table 6, relative expression of a panel of IEGs did not differ in frontal cortex or hippocampus between vehicle-treated Balb/c (N = 8) and vehicle-treated Swiss Webster mice (N = 4). A relative increase of expression of IEGs was selectively observed in the frontal cortex of VU0410120-treated Balb/c mice (N = 8) compared to VU0410120-treated Swiss Webster mice (N = 4). Interestingly, consistent with the proposed complex relationship between NMDAR “dysregulation” and ASD symptomatology, relative expression of several IEGs was increased in vehicle-treated Balb/c mice (N = 8) compared to VU0410120-treated Balb/c mice (N = 8) [19,49–52]. Treatment of the Swiss Webster comparator with VU0410120 was also associated with relative suppression of several IEGs, but this lacked the anatomic selectivity observed in the Balb/c strain as suppression of expression was also observed in the hippocampus. Exhibited sociability and strain differences did not appear to affect the relative expression of IEGs in either frontal cortex or hippocampus; these data suggest that observed differences in relative expression of IEGs were due to treatment with the high-affinity, highly selective GlyT1 inhibitor (Table 6). Further, increased Jun expression in frontal cortex was observed in VU0410120-treated Balb/c mice when compared to VU0410120-treated Swiss Webster mice, irrespective of whether the treated Balb/c mice were assigned to the “Low” or “High” sociability group (Table 6). Moreover, relatively increased Jun expression in frontal cortex of VU0410120-treated Balb/c mice distinguished mice belonging to the “Low” sociability group (N = 4) from VU0410120-treated Balb/c mice belonging to the “High” sociability group (N = 4; Table 6). Effects of VU0410120 treatment on relative expression of other IEGs in frontal cortex of Balb/c mice as a function of sociability group membership and comparisons to the VU0410120-treated Swiss Webster strain are also shown (Figure 6 and Table ).

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Discussion To determine whether social impairments in the Balb/c mice reflect decreased social interest or a stressful anxious response, the current study evaluated differences in conspecific-provoked immobility (CPI) between Balb/c and Swiss Webster mouse strains, and the relationship between CPI and the corticosterone (CORT) response. Balb/c and Swiss Webster mice differed in terms of their immobility in the presence of enclosed and freely-behaving stimulus mice; however, the conspecific-provoked corticosterone response did not mirror immobility behavior. Cortisol reactivity was shown to be a valid measure of social stress in humans, consistent with preclinical studies measuring corticosterone reactivity as a biomarker of social stress in rodents [53,54]. However, in addition to greater variability in the reactivity of their HPA axis to stress in general, data suggest that changes in cortisol output may be a less reliable marker of social stress in persons with ASD [1,3]. NMDAR activation influences the corticosterone response and diminishes immobility in the presence of a social stimulus in Balb/c mice; however, the relationship between these two “outputs” is complex. For example, data support subtle and complex modulatory effects of the NMDAR on CRFexpressing neuronal projections involved in behavioral responses of mice to social stress and their sociability; these modulatory effects were influenced by the nature of the stress paradigm and gender of the mouse [47]. For example, the social behavior of male mice with both homozygous deletion of the NR1 subunit in CRFexpressing cells and prior histories of social stress is more sensitive to disruption than similarly sociallystressed male controls [47]. NMDAR activation contributes to the regulation of CRF-signaling in anatomic nodes of socio-cognitive circuits (e.g., amygdala), as well as the glucocorticoid response to social stress [47,55]. CRF-Expression is enriched in the central nucleus of the mouse amygdala; consistent with this enriched expression, CRF antagonism in the central nucleus of the amygdala and the bed nucleus of the stria terminalis (BNST) attenuate behavioral expression of social defeat. Importantly, the amygdala participates in processing

Journal Pre-proof facially expressed emotions in humans, especially threatening facial stimuli [56]. Activation of the amygdala in response to threatening social stimuli promotes the “glucocorticoid stress response” that is initiated by release of corticotropin-releasing hormone (CRH) by parvocellular neurosecretory neurons located in the hypothalamic paraventricular nucleus [55]. The hippocampus, an area of the brain containing glucocorticoid receptors, provides feedback inhibition of the hypothalamic release of CRH. Chronic stress can lead to hippocampal cell atrophy and death with loss of this important counter-balancing inhibitory influence on hypothalamic CRH release, which is reflected in chronic elevation of blood levels of cortisol in the human and corticosterone in the mouse. Importantly, projections from the central nucleus of the amygdala to the BNST co-express CRF and NMDARs.

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Another study explored the relationship between exposure to social stress, learned fear responses, and NMDAR expression. Specifically, exposure of male adolescent (postnatal day [PD] 35) rats to social defeat stress resulted in their altered expression of NMDARs in key nodes of the social brain and altered fear conditioning in young adulthood (PD56) [57]. Using quantitative autoradiography, 3H-MK-801 binding was altered in the infralimbic region of the medial prefrontal cortex (decreased), CA3 region of the hippocampus (increased), and the central nucleus of the amygdala (decreased). The directional changes in 3H-MK-801 binding may be influenced by the nature of the social stressor, its timing in adolescence, and differences in the pushing (amygdala) and pulling (hippocampus) nature of the nodes on hypothalamic CRF expression. A prosocial dose of VU0410120 significantly reduced the CPI of Balb/c mice and significantly increased the conspecific-provoked corticosterone response, compared to vehicle-treated Balb/c mice. This increase in corticosterone levels in VU0410120-treated Balb/c mice was expected due to the role of the NMDAR in regulating corticosterone signaling [27,58]. However, the corticosterone response did not differ as a function of the intensity of CPI in the VU0410120-treated Balb/c mice, suggesting that immobility in the presence of a social stimulus may not index fearfulness, and is distinguishable from freezing behavior.

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Transcriptomic analysis investigating fold-change differences between VU0410120-treated Balb/c “High” and “Low” responders and VU0410120-treated Swiss Webster mice displaying a “Medium” level of prosocial response revealed increased mRNA expression of Ddit4 (gene coding ‘DNA damage inducible transcript 4’) in VU0410120-treated Balb/c “Low” responders compared to both VU0410120-treated Balb/c “High” responders and VU0410120-treated Swiss Webster mice displaying “Medium” levels of prosocial response. Ddit4 inhibits mTORC1 activity by disrupting signaling between AKT and TSC2 [22,45,59]. Importantly, NMDAR activation is a known negative regulator of mTOR signaling activity, which is overactive in several monogenic syndromic forms of ASD, including tuberous sclerosis complex (TSC), neurofibromatosis I (NF1), fragile X syndrome (FXS), and tensin homolog deleted on chromosome 10 (PTEN) [22,60]. These latter observations of mTOR signaling overactivation support development of targeted NMDAR agonist interventions for the treatment of ASD to dampen this signaling activity [19,22]. The elevated expression of Ddit4, a gene coding a negative regulator of mTOR signaling activity, in VU0410120-treated Balb/c ‘Low” responders led to a hypothesis-driven preliminary ‘gene set enrichment analysis’ of specific genes involved in the mTORC1 complex [45,59]. mTOR is a serine/threonine kinase that is complexed with a variety of proteins, including PRAS40, the latter is both a component of mTORC1 and substrate for its phosphorylation by Akt and mTORC1 itself [61]. PRAS40 exerts an inhibitory constraint on mTORC1 activity and its phosphorylation leads to its dissociation from mTORC1 and relief of this inhibitory influence [61]. Thus, the finding that expression of Akt1s1, the gene coding PRAS40 in mice, was elevated in a preliminary ‘gene set enrichment analysis’ conducted in frontal cortex in VU0410120-treated Balb/c non-responders compared to VU0410120-treated Balb/c responders suggests that dampening of mTOR signaling may be a compensatory response in VU0410120-treated Balb/c non-responders. This preliminary finding encourages future studies exploring the effect of NMDAR activation on expression changes in mTOR signaling pathway-associated genes. In this study, we interrogated a number of IEGs known to have different regulatory roles in the brain [48,62,63]. Expression of IEGs is modulated by acute and chronic exposure to antipsychotic medications, which supports the value of measuring their expression to show target engagement, the role of medicationinduced changes of gene expression in their therapeutic mechanisms of action, and the temporal and spatial selectivity of engaging these therapeutic targets [62]. Our preliminary findings suggest that, irrespective of

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strain, treatment of either Balb/c or Swiss Webster mice with VU0410120 decreases their relative expression of IEGs in frontal cortex when compared to expression in frontal cortex in either vehicle-treated Balb/c or vehicle-treated Swiss Webster mice, respectively. Importantly, vehicle-treated and VU0410120-treated mice from both strains were exposed to ICR stimulus mice in sessions II and III, which serves as a mild social stressor for many test strains. Based on our own behavioral data, data from knockout mice with absent expression of the NR1 NMDAR subunit, and data on genetically-engineered mice with diminished affinity of their NR1 subunits for the glycine co-agonist, NMDAR hypofunction was posited to have a role in the pathogenesis of impaired sociability [28,29,35–37,64]. Thus, it is interesting that an immunohistological study reported that administration of MK-801, an uncompetitive antagonist of the NMDAR causing NMDAR hypofunction, increased c-fos expression in rat cortical slices, an effect that was partially attenuated by administration of D-cycloserine, a partial glycineB agonist [65]. These latter data support exploration of therapeutic targeting of the NMDAR for the treatment of ASD, whose therapeutic mechanisms will likely include selective effects on gene expression [22,66]. In order to discover candidate genes that may “contribute” to the sociability deficits Balb/c mice display in the presence of enclosed and freely-behaving ICR stimulus mice, future “subtraction” experiments should compare expression of IEGs between Balb/c mice taken directly from their home cages for RNA extraction with Balb/c mice exposed to ICR stimulus mice in the 3-chambered apparatus prior to RNA extraction. Additionally, future bioinformatics inquiries will examine comparisons of vehicle- and VU0410120-treated Balb/c mice with respect to expression of genes in the glucocorticoid signaling pathway.

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Clearly, relationships between NMDAR activation, stress, social behavior, and the glucocorticoid response are complex and may be confounded by many factors including, gender, prior stress history and developmental age. Recently, it has been shown that there is relationship between corticosterone reactivity and mild social stress in adult mice that is impacted by a post-weaning enrichment environment [67]. Rearing with same-age peers in an enriched environment attenuated conspecific-provoked corticosterone reactivity, which may have relevance to reports of “peer socialization” having prosocial effects in young BTBR mice, a mouse model of ASD [68]. Based on the current data, the stressfulness of a social encounter alone may not be the sole determinant of increased immobility in Balb/c mice; perhaps, this strain also displays an element of social “disinterest.” ASD is a chronic, most commonly lifelong, neurodevelopmental disorder, whose initial insult most probably occurs during fetal brain development, which can be a major determinant of directional and regional changes in NMDAR expression [30].

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Disclosures The authors have nothing to disclose.

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Acknowledgement The authors acknowledge the support they received from the Office of the Dean of Eastern Virginia Medical School.

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Journal Pre-proof Figure Legends Figure 1: Study design and workflow for microarray experiment and transcriptomic analysis. Behavioral Assessment of Sociability: The 3-compartment sociability apparatus was used to obtain sociability measurements in session III. Isolate and Check RNA Integrity: Cortical and hippocampal RNA from vehicle and drug-treated Balb/c and Swiss Webster mice (n = 4/group) was extracted and checked for high integrity. Individual Ranking of Social Behaviors: The individually identified mice were given a “rank score”, reflecting the ranked contributions of discrete prosocial behaviors to this total “rank score”. Determine Groups for Microarray Experiment and Transcriptome Analysis: Rank scores were used to designate global prosocial responses of individual mice injected with VU0410120 or vehicle and assign them as members of “High”, “Medium” and “Low” prosocial response categories. The response categories informed interrogation of the large repository of regionally-selective transcriptomic data obtained in frontal cortex and hippocampus.

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Figure 2: Immobility behavior of Balb/c mice is increased in the presence of enclosed (Session II) and freely-behaving (Session III) stimulus mice. Scatter plots show the episodes of immobility (A-C), total time spent immobile (D-F) and time spent immobile per episode of immobility (G-I) made by 4-week old Swiss Webster (blue circles) and Balb/c (red circles) mice. Treatment of Balb/c mice with VU0410120 (18 mg/kg, ip, green circles) is shown. **p<0.01 and ***p<0.001 within Balb/c strain and ### p<.001 and #### p<.0001 between vehicle-treated strain comparisons.

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Figure 3: Relationship of CORT levels to measures of immobility in session III. (A) Scatter plot shows serum CORT levels (ng/mL) of VU0410120- and vehicle-treated Balb/c and vehicletreated Swiss Webster mice <30 min after a 10-min session of social interaction. (B) Scatter plots show serum CORT levels between Swiss Webster and Balb/c mice displaying low or high immobility behavior. Low and high immobility (seconds) was operationally-defined as “above” or “below” the dashed line depicting the immobility of vehicle-treated Swiss Webster mice in session III: low immobility < 73 sec, and high immobility > 73 sec in a 600 sec epoch (Figure 1: Study design and workflow for microarray experiment and transcriptomic analysis. Behavioral Assessment of Sociability: The 3-compartment sociability apparatus was used to obtain sociability measurements in session III. Isolate and Check RNA Integrity: Cortical and hippocampal RNA from vehicle and drug-treated Balb/c and Swiss Webster mice (n = 4/group) was extracted and checked for high integrity. Individual Ranking of Social Behaviors: The individually identified mice were given a “rank score”, reflecting the ranked contributions of discrete prosocial behaviors to this total “rank score”. Determine Groups for Microarray Experiment and Transcriptome Analysis: Rank scores were used to designate global prosocial responses of individual mice injected with VU0410120 or vehicle and assign them as members of “High”, “Medium” and “Low” prosocial response categories. The response categories informed interrogation of the large repository of regionally-selective transcriptomic data obtained in frontal cortex and hippocampus. FigureD). CORT levels analyzed by ELISA. **p<0.01 within group comparison. Figure 4: Gene expression profiles of glucocorticoid signaling genes in frontal cortex Venn Diagrams show significant fold-change differences in comparisons of strains, prosocial response categories within frontal cortex of (A) vehicle-treated and (B) VU0410120-treated Balb/c and Swiss Webster mice. Heat map displays the “clustering” of normalized expression intensities of 88 glucocorticoid signaling genes (C). Figure 5: Gene expression profiles of glucocorticoid signaling genes in hippocampus Venn Diagrams show significant fold-change differences in comparisons of strains, prosocial response categories within hippocampus of (A) vehicle-treated and (B) VU0410120-treated Balb/c and Swiss Webster mice. Heat map displays the “clustering” of normalized expression intensities of 88 glucocorticoid signaling genes (C).

Journal Pre-proof Figure 6: Gene expression profiles of immediate early genes in frontal cortex and hippocampus

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Venn Diagrams show significant fold-change differences in comparisons of strains, prosocial response categories within frontal cortex (A and C) and hippocampus (B and D) of vehicle-treated (A and B) and VU0410120-treated (C and D) Balb/c and Swiss Webster mice.

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Table 1: Significant expression changes in frontal cortex and hippocampus of glucocorticoid signaling genes in vehicle-treated Balb/c and Swiss Webster mice

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Vehicle-treated Balb/c Low Sociability vs Swiss Webster Frontal Cortex Gene

Fold Change

p-value

FDR

Gene Function

Arid5b

-1.36

0.0002

0.0182

Transcription Factor

Nfkbia

1.28

0.0006

0.0379

Stress Response

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Gene

Vehicle-treated Balb/c High Sociability vs Swiss Webster

l a

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e r P

Fold Change

Hippocampus p-value

FDR

Gene Function

No Significant Changes

Frontal Cortex cortex of glucocorticoid signaling genes in VU0410120-treatedHippocampus Table 2: Significant expression changes in frontal Balb/c and Swiss Webster mice Gene

Fold Change

p-value

FDR

Arid5b

-1.61

2.46E-05

0.0034

Xdh

-1.32

0.0009

0.0276

Creb3

1.35

0.0001

0.0106

Pdgfrb

1.33

0.0009

0.0275

Crhr1

1.33

0.001

0.0302

Gene Function

Gene

Fold Change

p-value

FDR

Transcription Factor

Xdh

-1.39

0.0005

0.0207

Nucleotide Metabolism

Nucleotide Metabolism

Crh

1.35

0.001

0.0301

Other GR Target Genes

rn

u o

J

Gene Function

GR & Co-transcription Factors Cell Surface Receptor Other GR Target Genes

Vehicle-treated Balb/c Low Sociability vs Balb/c High Sociability Frontal Cortex Gene

Fold Change

p-value

FDR

No Significant Changes

Hippocampus Gene Function

Gene

Fold Change

p-value

FDR

No Significant Changes

Gene Function

Journal Pre-proof VU0410120-treated Balb/c Low Sociability (Non-responders) vs Swiss Webster Gene

Fold Change

p-value

FDR

Pdgfrb

2.15

7.80E-05

0.0012

Slc19a2

2.82

4.33E-07

5.86E-05

Slc22a5

1.38

0.003

0.0121

Gene

Fold Change

p-value

FDR

Errfi1

1.21

0.0082

0.0248

Other GR Target Genes

Channels & Transporters

Spsb1

1.53

0.0002

0.002

Other GR Target Genes

Channels & Transporters

Usp54

1.25

0.003

0.0121

Other GR Target Genes

Gene Function Cell Surface Receptor

Gene Function

Per1

1.32

0.0029

0.0119

Circadian Rhythm

Adarb1

1.25

0.0091

0.0266

RNA Processing

Edn1

1.42

0.0005

0.0036

Cytokines & Chemokines

Aff1

1.38

0.0082

0.0247

RNA Processing

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Il6

-1.93

2.53E-05

0.0006

Cytokines & Chemokines

Hnrnpll

1.51

9.38E-05

0.0013

RNA Processing

Il6ra

1.23

0.0332

0.0704

Cytokines & Chemokines

Bmper

1.31

0.0043

0.0156

Signal Transduction

Snta1

1.37

0.0033

0.0129

Cytoskeleton Regulators

Diras2

1.65

0.0001

0.0014

Signal Transduction

Ehd3

1.65

0.0001

0.0015

Endocytosis & Exocytotsis

0.0141

0.0368

Signal Transduction

Cebpa

1.48

0.0009

0.0055

GR & Cotranscription Factors

1.47

0.0002

0.0021

Signal Transduction

Creb1

1.42

0.0004

0.003

GR & Cotranscription Factors

1.52

3.34E-05

0.0007

Signal Transduction

Stat5b

1.67

0.0002

0.0018

GR & Cotranscription Factors

Usp2

1.39

0.0174

0.0431

Signal Transduction

Asph

1.22

0.0212

0.05

Glucose & Fatty Acid Metabolism

e

1.21

Bcl6

1.22

0.0024

0.0104

Stress Response

Gdpd1

1.27

0.0011

0.0061

Glucose & Fatty Acid Metabolism

Ddit4

2.39

0.0004

0.0034

Stress Response

Got1

1.35

0.0031

0.0124

Glucose & Fatty Acid Metabolism

Nfkbia

1.3

0.0184

0.0448

Stress Response

H6pd

1.24

0.0053

0.018

Glucose & Fatty Acid Metabolism

Pdcd7

1.56

0.0002

0.0023

Stress Response

Pdp1

1.47

0.0003

0.0028

Glucose & Fatty Acid Metabolism

Sesn1

1.52

0.0003

0.0025

Stress Response

Metallothioneins

Fosl2

1.25

0.009

0.0265

Transcription Factor

Nucleotide Metabolism

Klf13

1.73

3.14E-05

0.0007

Transcription Factor

Nucleotide Metabolism

Klf9

1.55

0.0016

0.0079

Transcription Factor

Other GR Target Gene

Tbl1xr1

1.38

0.0047

0.0167

Transcription Factor

Other GR Target Gene

Tnfaip3

1.62

0.0002

0.0018

Transcription Factor

Other GR Target Gene

Zfp36

1.61

0.0035

0.0136

Transcription Factor

Mt1

1.26

0.0022

0.01

Ak2

1.47

0.0001

0.0017

Ampd3

1.78

4.89E-06

0.0002

Crh

2.02

0.0002

0.0022

Crhbp

1.57

0.0032

0.0127

Crhr1

1.33

0.0073

0.0226

Table 2 continued:

l a

rn

u o

J

Pik3r1 Pld1

r P

Rasa3

o r p

Journal Pre-proof VU0410120-treated Balb/c High Sociability (Responders) vs Swiss Webster Gene Function

Fold Change

p-value

FDR

Ghrhr

1.28

0.0091

0.0315

Cell Surface Receptor

Pdp1

1.32

0.0022

0.012

Glucose & FA Metabolism

Pdgfrb

1.4

0.0008

0.0062

Cell Surface Receptor

Got1

1.24

0.0076

0.0279

Glucose & FA Metabolism

Slc22a5

1.33

0.0067

0.0254

Channels & Transporters

Ampd3

1.53

3.78E-05

0.0009

Nucleotide Metabolism

Slc19a2

2.12

1.99E-05

0.0006

Channels & Transporters

Usp54

1.19

0.0153

0.0461

Other GR Target Gene

Il1rn

1.21

0.0037

0.0169

Cytokines & Chemokines

Crh

1.87

0.0004

0.0039

Other GR Target Gene

Il6

-2.01

9.66E-06

0.0004

Cytokines & Chemokines

Hnrnpll

1.21

0.0083

0.0295

RNA Processing

Edn1

1.25

0.0039

0.0175

Cytokines & Chemokines

Aff1

0.0003

0.0035

RNA Processing

Rhoj

1.51

0.0006

0.0053

Cytoskeleton Regulators

Pik3r1

Anxa4

1.3

0.0038

0.0173

Endocytosis & Exocytotsis

Pld1

Ehd3

1.27

0.0013

0.0087

Endocytosis & Exocytotsis

Pdcd7

Stat5a

1.34

0.0037

0.0169

GR & Cotranscription Factors

Nr3c1

1.22

0.0048

0.0201

GR & Cotranscription Factors

Creb1

1.21

0.011

0.0362

GR & Cotranscription Factors

Stat5b

1.29

0.0155

0.0464

GR & Cotranscription Factors

l a

Gene

Fold Change

Gene

f o

o r p 1.57

FDR

Gene Function

0.0038

0.0172

Signal Transduction

1.46

0.0004

0.004

Signal Transduction

1.46

4.02E-05

0.0009

Stress Response

1.62

0.0018

0.0104

Stress Response

Arid5b

1.2

0.0125

0.0398

Transcription Factor

Tbl1xr1

1.42

0.0105

0.0349

Transcription Factor

Tnfaip3

1.39

0.0129

0.0406

Transcription Factor

e

r P

Sesn1

1.25

p-value

Table 3: Significant expression changes in hippocampus of glucocorticoid signaling genes in VU0410120-treated Balb/c and Swiss Webster mice

n r u

VU0410120-treated Balb/c Non-responders vs Balb/c Responders Gene

Fold Change

p-value

FDR

Fosl2

1.53

0.0009

0.0252

Ak2

1.22

0.0013

0.0295

Slc22a5

1.3

0.001

0.0273

Pdgfrb

1.26

0.0023

0.0395

Rasa3

1.22

0.0017

0.0336

Ddit4

1.45

0.0024

0.04

Gene Function

Jo

Transcription Factor

Nucleotide Metabolism Channels and Transporters Cell Surface Receptors Signal Transduction Stress Response/Regulator of mTOR Signaling

Journal Pre-proof VU0410120-treated Balb/c Low Sociability (Non-responders) vs Swiss Webster Gene

Fold Change

p-value

FDR

Gene Function

Tbl1xr1

-1.27

3.00E-04

0.0325

Transcription Factor

Anxa4

-1.23

3.00E-04

3.30E-02

Crhbp

1.44

0.0002

0.0246

f o

VU0410120-treated Balb/c High Sociability (Responders) vs Swiss Webster Gene

Fold Change

p-value

Rgs2

1.21

0.0008

Il10

-1.22

0.0015

Ehd3

1.2

0.0003

Angptl4

1.22

0.0008

Crhbp

1.51

4.34E-06

Rasa3

1.19

Gene

a n

r u

Fold Change

Jo .

P l 0.001

VU0410120-treated Balb/c Non-responders vs Balb/c Responders

re

-p

p-value

No Significant Changes

ro

FDR

Endocytosis & Exocytotsis Other GR Target Gene

Gene Function

0.0327

Signal Transduction

0.0449

Cytokines & Chemokines

0.0206

Endocytosis & Exocytotsis

0.0333

Growth Factors

0.0012

Other GR Target Genes

0.0367

Signal Transduction

FDR

Gene Function

Journal Pre-proof

f o

Table 4: Functional bioinformatic analyses of upregulated genes in frontal cortex of VU0410120treated Balb/c mice (non-responders vs responders) DAVID Analysis (Functional Annotation Tool) GO Term (Biological Process)

5

l a

Gene Report (mTOR Signaling Pathway)

rn

DAVID (Negative Regulation of TOR Signaling) Akt1s1

u o Ddit4

Epm2a

J

e

Gene Count

Negative Regulation TOR Signaling

o r p

r P

p-value

Fold Enrichment

0.013

5.4

KEGG (mTOR Signaling Pathway) Akt1s1 Ddit4 Pik3cg

Gsk3a

Rps6kb2

Tmem127

Stk11 Ulk3

Journal Pre-proof

Table 5: Ddit4 and Akt1s1 gene expression in vehicle-treated Balb/c and vehicle-treated Swiss Webster mice

of

Vehicle-treated Balb/c (8) vs Vehicle-treated Swiss Webster (4)

p-value

FDR

Ddit4

1.42

0.0059

0.0699

Akt1s1

1.05

0.4379

0.7117

Gene

Fold Change

pvalue

FDR

Ddit4

-1.09

0.0678

0.3801

Not Significant

-1.07

0.3309

0.6925

Not Significant

Not Significant

ro

Fold Change

Not Significant

Akt1s1

re

Gene

Hippocampus

-p

Frontal Cortex

Frontal Cortex p-value

Ddit4 Akt1s1

1.08 -1.02

0.3557 0.3772

FDR

na

Fold Change

0.9048 0.9080

Not Significant Not Significant

Hippocampus Gene

Fold Change

pvalue

FDR

Ddit4 Akt1s1

1.10 1.02

0.0097 0.7061

0.1117 0.8603

ur

Gene

lP

Vehicle-treated Balb/c Low Sociability (4) vs Vehicle-treated Balb/c High Sociability (4)

Not Significant Not Significant

Vehicle-treated Balb/c Low Sociability (4) vs Vehicle-treated Swiss Webster (4)

Jo

Frontal Cortex

Gene

Fold Change

p-value

FDR

Ddit4

1.58

0.0088

0.1643

Akt1s1

1.10

0.1571

0.6839

Hippocampus

Not Significant Not Significant

Gene

Fold Change

pvalue

FDR

Ddit4

-1.03

0.4204

0.8363

Akt1s1

-1.06

0.7199

0.9395

Not Significant Not Significant

Vehicle-treated Balb/c High Sociability (4) vs Vehicle-treated Swiss Webster (4) Frontal Cortex

Hippocampus

Gene

Fold Change

p-value

FDR

Gene Function

Ddit4 Akt1s1

1.28 1.20

0.0167 0.0179

0.1357 0.1415

Not Significant Not Significant

Gene

Fold Change

pvalue

FDR

Ddit4

-1.20

0.0059

0.0789

Akt1s1

-1.06

0.1456

0.3893

Gene Function Not Significant Not Significant

Journal Pre-proof

Table 6: Complex Suppressive Effects of VU0410120 on Immediate Early Gene Expression in Balb/c and Swiss Webster Mice Vehicle-treated Balb/c (N=8) vs Vehicle-treated Swiss Webster (N=4) Frontal Cortex

Hippocampus

Fold Change

p-value

FDR

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.05 -1.01 -1.11 1.08 1.04 -1.06 1.12

0.838 0.3764 0.0616 0.3172 0.5361 0.9512 0.2304

0.9389 0.6631 0.2643 0.6124 0.7801 0.9831 0.5254

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

-1.01 -1.02 -1.04 -1.07 1.17 1.29 -1.07

0.961 0.7899 0.7995 0.6614 0.0159 0.3514 0.2119

0.989 0.9331 0.9362 0.8809 0.2048 0.7054 0.5861

of

Gene

p-value

FDR

1.14 1.39 1.12 1.45 1.23 -1.14 1.48

0.0769 5.00E-03 0.1564 0.0081 0.0002 0.7454 3.00E-04

0.1303 0.0144 0.2327 0.0213 0.0013 0.806 0.0017

Gene

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

re

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

Fold Change

lP

Gene

-p

Frontal Cortex

ro

VU0410120-treated Balb/c (N=8) vs VU0410120-treated Swiss Webster (N=4)

Hippocampus

Fold Change

1.1 1.05 1.25 1.29 1.13 1.07 1.27

p-value 0.2696 8.19E-01 0.0337 0.0181 0.02 0.7628 5.42E-02

FDR 0.561 0.9277 0.2061 0.1502 0.1583 0.9008 0.26

na

Vehicle-treated Balb/c (N=8) vs VU0410120-treated Balb/c (N=8) Frontal Cortex

1.3 1.56 1.38 1.42 1.04 1.24 1.41

p-value

FDR

0.0037 0.0286 0.0001 9.04E-05 0.3367 0.0537 0.0067

0.0427 0.1275 0.0098 0.0091 0.5368 0.1812 0.058

ur

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

Fold Change

Jo

Gene

Hippocampus Gene Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

Fold Change 1.48 1.21 1.2 1.5 1.06 1.26 1.09

p-value 0.0006 0.003 0.0592 4.00E-04 0.0655 0.1519 0.5248

FDR 0.0895 0.1499 0.354 0.0755 0.3668 0.4946 0.7911

Vehicle-treated Swiss Webster (N=4) vs VU0410120-treated Swiss Webster (N=4) Frontal Cortex Gene Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

Hippocampus

Fold Change

p-value

FDR

1.25 2.23 1.64 2.24 1.16 1.16 1.97

0.0208 1.85E-06 0.0002 0.0011 0.0337 0.1518 0.0005

0.0529 0.0002 0.0024 0.0071 0.0755 0.2382 0.0046

Gene Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

Fold Change 1.51 1.46 1.51 1.93 1.08 -1.01 1.3

p-value 0.0003 2.00E-04 0.000077 1.01E-05 0.2324 0.5863 0.0344

FDR 0.0127 0.0103 0.0078 0.0038 0.4239 0.7415 0.1351

Journal Pre-proof

Table 6 continued: Vehicle-treated Balb/c Low Sociability (N=4) vs Vehicle-treated Swiss Webster (N=4) Frontal Cortex

Hippocampus

Fold Change

p-value

FDR

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

-1.04 -1 -1.09 1.01 1.04 1.01 1.08

0.9991 0.5294 0.1409 0.8504 0.4221 0.7991 0.3148

0.9996 0.8183 0.5045 0.956 0.7575 0.9371 0.6775

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

-1.02 1.01 -1.01 1.06 1.12 1.42 1.02

0.9806 0.403 0.6742 0.7128 0.0682 0.135 0.6194

0.9956 0.8281 0.9263 0.9377 0.5337 0.639 0.9114

of

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.11 -1.07 -1.14 1.17 1 -1.08 1.16

0.569 0.2962 0.0882 0.2218 0.8723 0.8425 0.0561

0.798 0.5954 0.3309 0.522 0.9525 0.9392 0.2602

lP

Hippocampus

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.02 -1.11 -1.01 -1.13 1.16 1.21 -1.01

0.8599 0.5063 0.8544 0.0752 0.014 0.7069 0.1935

0.9412 0.7348 0.9386 0.2783 0.1205 0.8644 0.4504

re

Gene

-p

Frontal Cortex

ro

Vehicle-treated Balb/c High Sociability (N=4) vs Vehicle-treated Swiss Webster (N=4)

na

Vehicle-treated Balb/c Low Sociability (N=4) vs Vehicle-treated Balb/c High Sociability (N=4) Frontal Cortex

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

-1.11 -1.05 1.03 -1.11 1.04 1.04 -1.13

p-value

ur

Fold Change

0.1575 0.8242 0.9806 0.0987 0.3503 0.6488 0.2184

Jo

Gene

Hippocampus

FDR

Gene

Fold Change

p-value

FDR

0.8652 0.9816 0.9975 0.8587 0.9043 0.9545 0.8746

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

-1.02 1.11 -1.04 1.19 -1.1 1.16 1.13

0.9588 0.1396 0.7773 0.0716 0.0353 0.4545 0.0022

0.9834 0.3884 0.8972 0.2808 0.1999 0.6938 0.0568

VU0410120-treated Balb/c Low Sociability (N=4) vs VU0410120-treated Swiss Webster (N=4) Frontal Cortex

Hippocampus

Gene

Fold Change

p-value

FDR

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.26 2.2 1.55 2.29 1.42 1.19 2.17

0.0211 4.44E-05 0.0005 0.0006 0.0004 0.0789 9.03E-06

0.0499 0.0008 0.0037 0.004 0.003 0.1382 0.0003

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.26 1.12 1.33 1.53 1.12 1.13 1.37

0.1058 9.71E-01 0.0079 0.001 0.0517 0.5932 2.82E-02

0.4636 0.9907 0.1753 0.0686 0.3611 0.8458 0.2896

Journal Pre-proof

26

Table 6 continued: VU0410120-treated Balb/c High Sociability (N=4) vs VU0410120-treated Swiss Webster (N=4) Frontal Cortex

Hippocampus

Fold Change

p-value

FDR

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.15 1.31 1.06 1.31 1.21 -1.27 1.39

0.2122 0.0059 0.1481 0.0418 0.011 0.7513 0.0009

0.3195 0.0234 0.2435 0.0949 0.0362 0.8186 0.0068

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.06 1.06 1.09 1.18 1.11 -1.1 1.18

0.899 0.2797 0.0702 0.1028 0.031 0.6899 0.2223

0.96 0.5613 0.2939 0.351 0.2001 0.8553 0.5033

of

Gene

Frontal Cortex

ro

VU0410120-treated Balb/c Low Sociability (N=4) vs VU0410120-treated Balb/c High Sociability (N=4) Hippocampus

Fold Change

p-value

FDR

Gene

Fold Change

p-value

FDR

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.08 1.5 1.23 1.28 -1.02 1.27 1.27

0.6894 0.0018 0.2942 0.0208 0.4279 0.2336 0.0601

0.8313 0.0348 0.5246 0.1222 0.6435 0.4617 0.2175

Fos Jun Egr1 Arc Creb1 Npas4 Nptx2

1.21 1.1 1.23 1.32 -1 1.23 1.13

0.0961 0.6419 0.0269 0.0019 0.5536 0.4048 0.1151

0.7991 0.9352 0.759 0.7343 0.9151 0.8757 0.8061

re

lP

na

Ethical Statement

-p

Gene

Jo

ur

The authors have read and agree with the journal's ethical standards. The authors declare that this manuscript has not been previously published and is not under consideration with any other journals. All animal procedures were approved by the Eastern Virginia Medical School Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6