The Mechanism Of Memory Impairment Induced By Orexin-A Through Orexin 1 Receptor And Orexin 2 Receptor In Post Traumatic Stress Disorder Rats

Objective:Post-traumatic stress disorder(PTSD)is a stress-accociated mental disorder that occurs as a result of exposure to a traumatic event.Memory impairment and hypothalamus-pituitary-adrenal axis(HPA)dysfunction associated with stress were found in patients with PTSD.Medial prefrontal cortex(mPFC)and hippocampus are important components of the limbic system of the brain,and play an important role in learning and memory,emotion regulation,etc.Current studies have found that abnormal function and structure of mPFC and hippocampus are associated with many mental disorders.mPFC regulates the function of hippocampus and controls the activity of HPA axis together with hippocampus.Imaging studies revealed changes in hippocampal and mPFC volumes in patients with PTSD.The neuronal apoptosis and endoplasmic reticulum(ER)stress pathway and mitochondrial pathway resulted in programmed neuronal death in the hippocampus of PTSD rats.mPFC regulates the activity of hippocampus and HPA axis in its downstream,and plays an important role in regulating memory and some related functions,so the abnormality of mPFC,hippocampus and hypothalamus may be closely related to the disorder of memory,anxiety,depression,avoidance and numbness in patients with PTSD.Orexins,also known as hypocretins,are a pair of peptides produced by hypothalamic neurons,including orexin-A and orexin-B(hypocretin-1 and hypocretin-2).Orexin neurons project extensively into brain tissue and play roles by orexin 1 receptor(OX1R)and orexin 2 receptor(OX2R).The studies found that orexins is closely related to the brain feeding center and can promote feeding behavior.OX1R\OX2R was expressed in hippocampus and mPFC,and orexin regulated memory learning by regulating hypothalamus-pituitary-adrenal axis.In this study,the three behavioral experiments,Morris water maze(MWM),elevated plus maze(EPM)and open field test(OFT)were used to detect spatial learning and memory disorders,anxiety and spontaneous act of exploration in PTSD rat.We detected neuronal ultrastructure changes by transmission electron microscopy.This study explores the changes of orexin-A,OX1 R and OX2 R in the hypothalamus,hippocampus and mPFC in PTSD rats to reveal the pathophysiological process of stress response,memory and affective disorder in PTSD.Orexin-A was injected into the lateral ventricle of PTSD rats.The changes of learning and memory were observed by Morris water maze.The detection of OX1 R and OX2 R at the protein level provides a theoretical basis for the treatment of PTSD by orexin-A and its therapeutic mechanism.Methods: 1.In the first and second part of the experiment,a total of 120 healthy male Wistar rats,were randomly divided into five groups,including control group,1-day,4-day,7-day and 14-day group after SPS stimulation.2.The three behavioral experiments,MWM,EPM and OFT were used to detect spatial learning and memory disorders and anxiety in PTSD rats.We explored neuronal ultrastructure changes by transmission electron microscopy.3.The expression of orexin-A,OX1 R and OX2 R in hypothalamus,hippocampus and mPFC of PTSD rats was detected by immunohistochemistry,western bloting and real time PCR method.4.In the third part of the experiment,32 male Wistar rats were randomly divided into 4 groups,including SPS+vehicle,SPS+orexin-A,non-SPS+vehicle,non-SPS+orexinA.Each group was given intracerebroventricular injection of solution or orexin-A.The changes of learning and memory were observed by Morris water maze.OX1 R and OX2 R was detected by western bloting in hypothalamus,hippocampus and mPFC.Results:1.In the first part of the experiment,in MWM spatial probe test,the percentage of distance in the target quadrant/total distance and the target crossing in the SPS group were significantly decreased.In EPM,compared with the control group,the PTSD rats showed a decrease of the distance,entries,time in the open arm,the distance,entries in the closed arm,and a increase of the time in the closed arm;In OFT,after SPS stimulation,the distance,entries,max speed in outer zone,the distance,entries,max speed,time in inner zone,and the number of rearing reduced,but the time in outer zone increased.2.In the second part of the experiment,the expression levels of orexin-A protein and mRNA in hypothalamus were significantly decreased.The expression level of OX1 R protein and mRNA in hypothalamus and mPFC increased,and OX2 R dose not change.The expression levels of OX1R/OX2 R protein and mRNA in hippocampus were increased.3.In the third part of the experiment,after intracerebroventricular injection of orexin-A in SPS rats,the percentage of swimming distance/total distance in target quadrant and the frequency of crossing target quadrant in SPS+orexin-A group were higher than those in SPS+ vehicle group.The results showed that the symptoms of decreased spatial memory were significantly improved after orexin-A administration in SPS rats.After orexin-A administration,in hypothalamus and mPFC,the level of OX1 R protein was decreased,but the level of OX2 R was not changed;the level of OX1 R and OX2 R protein in hippocampus decreased.Conclusion: 1.The PTSD rats had decreased food intake,slow weight gain,decreased spatial memory,increased fear,reduced spontaneous exploration and anxiety.The ultrastructure of neurons in hypothalamus,hippocampus and medial prefrontal cortex was abnormal.2.The perturbation of the orexin system may play a critical role in memory impairment,appetite disturbance and anxiety in PTSD.Also,the changes in behavior and orexin receptor expression in SPS rats were partly reversed by orexin-A administration;these findings suggest that orexin-A has therapeutic potential for the treatment of PTSD.