MRNA And MiRNA Profiles In The Amygdala Are Relevant To Fear Memory Induced By Physical And/or Psychological Stress

Objective:Stress has many effects on the brain.Appropriate stress can not only preparing an individual for the acute consequences of dangerous or threatening situations and the return to homeostasis,but also inducing long-term adaptive responses.Excessive stress can significantly impact brain function and increase the risk for developing various psychiatric disorders.For example,stress-induced fear memory can further drive mood toward anxiety or depression,and even their secondary disorders.Many of the brain regions that are implicated in psychiatric disorders and are vulnerable to the effects of stress are also involved in mediating emotional learning.The vast majority of studies have focused on the mechanisms by which stress affects the amygdala and amygdala-dependent fear memories.But when forming and evoking the symptoms of fear memory,there is relatively little research on the changes of molecular expression profile in amygdala.Comprehensive molecular profiles underlying fear memory by different types of stresses need to be addressed.Therefore,in the present study,we focus on analyzing mRNA and miRNA molecular profiles in the amygdala from the mice treated by physical/psychological stresses(PPS)or psychological stress(PS).Methods:Five days of stress training,PPS to an intruder with receiving physical attacks as well as pre-/post-attack from male CD-1~?(ICR)IGS residents.PS to an observer was given by seeing aggressor attacks.The elevated plus-maze,interaction zone test was used to determine the behavior of fear memory and anxiety in mice.Amygdala tissues from these mice of showing fear memory and anxiety versus control were harvested to analyze mRNA and miRNA with high throughput sequencing to quantify their expression levels,and investigated molecular mechanisms in the amygdala underlying fear memory in these mice induced by physical/psychological stress versus psychological stress.Verification of some differentially expressed mRNAs and miRNAs by qRT-PCR experiment,the prediction results of target genes were verified by dual luciferase report assay.Results:Fear memory and anxiety in mice was induced by social defeat.By comparing the sequencing data of the intruder,observer and control group,the mRNA and miRNA differentially expressed genes in the intruder and control group,intruder and observer group were screened,the multiple of the differentially expressed genes should be more than 1.5 times.In the amygdala of intruders and observers with fear memory,the genes of encoding 5-HTR1b,5-HTR2a,DAR2,AChRM3 and IP3R1 are upregulated,whereas genes of encoding GP?11,GP?13,GP?T2,RasC3 and P450 are downregulated,indicating that these molecules are involved in fear memory induced by physical/psychological stress.In the comparison of intruders with observes,the upregulation of 5-HTR6,GP?8,P2R7,NF?2,CREB3/1 and Itg?9 as well as the downregulation of DAR5,5-HTR1a and HSP1a are involved in fear memory by physical stress.The upregulation of DAR1,5-HTR5a and SSR2/3 as well as the downregulation of AdR?1,CREB3/1,GP?13 and GP?8 are involved in fear memory by psychological stress.Some differentially expressed mRNAs and miRNAs in the high throughput sequencing have been confirmed by qRT-PCR analysis,the change direction of the gene in the verification results is consistent with the sequencing results.Furthermore,the direct interactions between mRNA Htr2a and miRNA-34b-5p as well as between Nfkb2 and miRNA-344e-5p are confirmed by dual luciferase report analysis,which further proved the accuracy of the sequencing results.Conclusions:Physical and/or psychological stresses in social interaction is more realistic in the life,the analysis of molecular profiles in the amygdala from fear memory mice should help to figure out the role of the amygdala in fear memory and anxiety based on molecules analyzed.Fear memories and anxiety by physical/psychological stress versus psychological stress are caused by the imbalanced regulation of different synapses and signaling pathways in the amygdala.The upregulation and downregulation among multiple signaling pathways as well as among genes in intra-signaling pathway make molecular networks in the amygdala to be imbalance,which lead to neuronal dysfunction in the amygdala for stress-induced fear memory and anxiety.