Pharmacology, Biochemistry and Behavior 199 (2020) 173062 Contents lists available at ScienceDirect Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh mRNA and miRNA profiles in the nucleus accumbens are associated with psychological stress-induced susceptible and resilient mice Yanjun Lu a, Jiuyong Yang a, Jinyan Sun a, Wei Lu a,*, Jin-Hui Wang a,b,c,** a Qingdao University, School of Pharmacy, Qingdao, Shandong 266021, China b University of Chinese Academy of Sciences, Beijing 100049, China c Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China ARTICLE INFO ABSTRACT Keywords: Background: Stress may be one of the main causes of fear and anxiety. Previous studies have shown that the Psychological stress nucleus accumbens is involved in emotional responses. However, in the nucleus accumbens, the mRNA and Susceptibility miRNA profiles of stress susceptibility and resilience of psychological stress still need to be studied. Resilience Materials and methods: In this study, by observing the conspecific being attacked, the witness group experienced Anxiety psychological stress. After five days of psychological stress, the fear memory of mice was measured by social Nucleus accumbens interaction test, and the degree of anxiety was measured by elevated plus maze. mRNA and miRNA profilesin the nucleus accumbens tissue of control, susceptible and resilient mice were established by high-throughput sequencing. Results: In susceptible mice versus resilient mice, the Differentially expressed genes (DEGs) may be related to psychological stress-induced susceptibility. DEGs enriched in Cell adhesion molecules, Neuroactive ligand- receptor interaction, Gap junction, PI3K-Akt, VEGF, Jak-STAT, Ras, and Chemokine pathways were up- regulated. DEGs enriched in cGMP-PKG, B cell receptor, and NOD-like receptor pathways were down- regu­ lated. The sequencing results of mRNAs and miRNAs were verified by qRT-PCR and dual luciferase reporter assay. Conclusion: The imbalance of different synapses and pathways in the nucleus accumbens may be related to susceptibility and resilience caused by psychological stress. 1. Introduction nucleus accumbens also mediates a variety of stress responses by interacting with the limbic, cognitive, and motor circuits (Azogu and Stress is essential in the occurrence and expression of many mental Plamondon, 2017; Floresco, 2015). The changes of molecules in the illnesses. Stressful events in life are often associated with fear (Izquierdo nucleus accumbens may regulate various stress responses by changing et al., 2016; Makkar et al., 2010; Maren and Holmes, 2016). Excessive the function of neurons and then affecting the circuit. Structural ele­ stress is the cause of many mental illnesses, such as phobia, depression, ments and functions in neuronal circuits, including the nucleus accum­ generalized anxiety and PTSD (Coutellier and Usdin, 2011; Desmedt bens, are related to reward and fear memory. Cellular processes and et al., 2015; Dunsmoor and Paz, 2015; Orsini and Maren, 2012). Due to signaling molecules in the nucleus accumbens may be associated with their genetic within-strain variability, individuals respond differently to anxiety(Bosch-Bouju et al., 2016). In recent years, miRNAs have been stress exposure; some are susceptible to stress exposure, while some are used in the research of mental disorders such as anxiety and major capable of actively coping with stressors, being stress resilient (Khan depression. Some miRNAs play a crucial role in the stress response of et al., 2020). However, the molecular mechanism that mediates these animals (Haramati et al., 2011; Issler et al., 2014). Therefore, the stress resistance is still unclear. comprehensive study on mRNA and miRNA in nucleus accumbens is The nucleus accumbens is the linchpin structure of the reward cir­ more helpful to solve the comprehensive molecular problems, which is cuit, which is involved in the regulation of emotional response, and the related to fear memory and anxiety caused by stress. * Correspondence to: W. Lu, Qingdao University, School of Pharmacy, 38 Dengzhou road, Qingdao, Shandong 266021, China. ** Correspondence to: J.-H. Wang, University of Chinese Academy of Sciences, Institute of Biophysics, 15 Datun Road, Chaoyang District, Beijing 100101, China. E-mail addresses: [email protected] (W. Lu), [email protected] (J.-H. Wang). https://doi.org/10.1016/j.pbb.2020.173062 Received 25 September 2020; Received in revised form 15 October 2020; Accepted 18 October 2020 Available online 22 October 2020 0091-3057/© 2020 Elsevier Inc. All rights reserved. Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062 Fig. 1. Experimental process of psychological stress. (a) After seven days of acclimatization, the C57 mice were screened by EPM test and social interaction test before psychological stress. The witness mice were subjected to psychological stress for five days. EPM test and social interaction test were performed again. The nucleus accumbens were removed for transcriptome sequencing. (b) C57 mouse was placed in the side with CD1, and the mice in the witness group were placed on the other side of the cage. The witness group watched the same species under attack, and thus experienced psychological stress. (c) Social avoidance detection is performed in an interactive social area (50*50 cm), with 10 * 10 cm transparent and perforated small cages on the edges. The area around the small cage is definedas the communication area. The psychological and pathological effects of stress on specific in­ dividuals are significantlydifferent (Krishnan, 2014; Russo et al., 2012). After psychological stress, mice can be divided into susceptible and resilient based on their different effects on physical and mental states. Stress can induce a range of physiological responses (including nervous, endocrine and immune systems) and may be harmful in some cases. Susceptible mice showed fear, anxiety and other symptoms, while resilient mice did not show these symptoms(Berton et al., 2006; Krishnan et al., 2007). Therefore, it is necessary to study the mRNA and miRNA profiles related to susceptibility and resilience induced by psy­ chological stress, as it remains to be systemically studied. To study the relationship between the molecules of the nucleus accumbens and psychological stress response, our strategy is shown below. These mice were divided into control and witness groups. The control group were raised normally without any treatment. And the witness group experienced psychological stress by observing that conspecific mice being attacked. After psychological stress, the mice were tested for fear memory and anxiety-like behavior. According to the test results, the witness group were further divided into susceptible and resilient groups. After the behavior test, the nucleus accumbens tissues of the mice were taken out and their mRNA and miRNA profiles were sequenced. Through the combined comparison and analysis of mRNA and miRNA, we hoped to find molecular and pathways of susceptility and resilience caused by psychological stress in the nucleus accumbens, and then develop new treatment methods and strategies. 2. Materials and methods 2.1. Mice Fig. 2. Social avoidance test of mice after psychological stress treatment. Control n = 12, resilient n = 16, susceptible n = 12. (a) shows the social C57BL/6J mice and CD-1 mice were purchased from Beijing life behavior tracks of control, susceptible and resilient mice on day6 with and without CD1. (b) shows the ratio (%) of the stay time of the three groups of River Experimental Animal Technology Co., Ltd. and Qingdao Daren mice in the communication area on day 0 and day6 when there was a CD1 fortune Animal Technology Co., Ltd. The experimental protocols were attacker in the small container and no CD1 attacker. Two-way ANOVA was used approved by the Animal Use and Care Committee of Qingdao University. for the comparisons among control, resilient and susceptible mice, while four The mice were fed with free access to water and food. The ambient ◦ asterisks show p < 0.0001, two asterisks show p < 0.01, one asterisk show p temperature was 22 ± 2 C. Relative humidity was 55 ± 5%. The < 0.05. 2 Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062 Fig. 3. Test of EPM in mice after psychological stress treatment. Control n = 12, resilient n = 16, susceptible n = 12. (a) shows the susceptible, resilient, and control mice tracks on the EPM before (day 0) and after (day 6) psychological stress. (b) shows the time (%) of the control, resilient and susceptible mice in the open zone on day 0 and day 6. Two-way ANOVA was used for the comparisons among control, resilient and susceptible mice, while one asterisk show p < 0.05. circadian rhythm was 12 h in the dark (07:00–19:00), 12 h in the light 2.3. Psychological stress (19:00–07:00). The resident-intruder paradigm was used as the social pressure for 2.2. Timeline of the experimental process this experiment(Golden et al., 2011; Montagud-Romero et al., 2018), psychological stress in social interactions may be more close to real life Concerning Golden ‘s protocol, 15-week-old male CD1 mice were than stress caused by electrical stimulations. During the adaptation used as attackers(Golden et al., 2011). Based on previous research, male period, male CD1 mice were placed in a room that took up half of a 5-week-old C57BL/6 J mice were used as subjects. When male C57 mice normal cage (29 × 17.5 × 12.5 cm). A neighboring room (29 × 17.5 × enter the residence of CD1 mice, CD1 mice are more aggressive(Ham­ 12.5 cm) was separated by a transparent partition. After adaptation mels et al., 2015). Mice were allowed to have a seven-day adaptation period, the attacked C57 mice were placed in the same cage as the CD1 period before the screening (Fig. 1a). At the end of the acclimation mice, and the mice in the witness group were placed on the other side of period, EPM (see below) and social interaction test (see below) were the cage.