The Effect And Mechanism Of CORT On GABA_BR Membrane Expression Of Neurons In Hippocampus And Nucleus Accumbens

Background:Since the outbreak of novel coronavirus-19（COVID-19）,the incidence of depression in the world has increased."Lack of pleasure"is the common symptom of depression.Nucleus accumens（NAc）,as a signal regulation region of the reward loop,can control and regulate pleasure motivation.Hippocampus（HIPP）is a memory-related brain region,and the signal projection to NAc brain region is very important role in drivingneural activity.The signal projection of HIPP to NAc plays a role in the appropriate regulation of goal-oriented behavior,and their activity intensity may be very important to situational reward behavior.In addition,the occurrence of depression will destroy the function of GABAergic system,but the mechanism of GABA_Breceptor（GABA_BR）on the occurrence and development of depression is not clear.Objective:On the basis of previous in vivo experiments,we want to construct the in vitro depression model of HIPP and NAc primary neurons induced by corticosterone（CORT）,explore the expression of cell membrane GABA_BR and its related mechanism in CORT-induced HIPP and NAc primary neuronal depression model,and determine it as the key target,so as to provide new ideas and theoretical basis for clinical treatment of depression.Methods:1.CORT induced-a cell model of depression was established.（1）According to the location and shape of HIPP and NAc brain atlas,primary neurons were extracted and cultured for 14 days.The neuron types were identified by immunofluorescence（IF）experiment.（2）Determination of appropriate concentration and time of the treatment with CORT.The cells were treated with different concentrations of CORT（50μM/100μM/200μM）for different times（24h and 48h）.The cell viability after CORT treatment was measured by CCK-8 kit,and the concentration and time of about 50%cell viability were chosen.（3）The model of neuronal depression induced by CORT was established in vitro.The primary neurons of HIPP and NAc were induced by CORT to establish the depression model in vitro.Green fluorescent protein（GFP）tagged empty plasmids were transfected into HIPP and NAc primary neurons.The number of dendritic spines in HIPP and NAc primary neurons was observed after CORT induction.2.To explore the expression of GABA_BR cell membrane and effect in the process of CORT-induced neurons（1）In the model of cell depression induced by CORT,the expression of GABA_BR on the membrane of HIPP and NAc neurons was observed by Western blot（WB）combined with cell membrane protein extraction kit and IF to determine the expression of GABA_BR in the model of cell depression induced by CORT.（2）On the basis of the above experiments,using GABA_BR agonist（Baclofen）,CCK-8 assay was used to detect cell viability,and WB and IF techniques were used to observe the membrane expression of GABA_BR.3.Molecular mechanism of membrane expression of neuronal GABA_BR induced by CORT.（1）In the depression model of CORT cells,Fluo 4 fluorescence probe technique was used to detect the influx of Ca²⁺,and WB combined with cell membrane extraction kit and IF experiment were used to detect the membrane expression of Ca MKIIα（2）In the depression model of CORT-treated cells,the interaction of p-Ca MKII-GABA_B1R and the level of phosphorylation of GABA_B1R were detected by co-immunoprecipitation（Co-IP）.（3）On the basis of the above experiments,combined with Baclofen,Fluo 4fluorescence probe technique,WB combined with cell membrane extraction kit and IF were used to detect the influx of Ca²⁺and the expression of Ca MKIIα.Co-IP assay was used to detect the interaction of p-Ca MKII-GABA_B1R and the phosphorylation level of GABA_B1R.Results:1.The more than 90%of HIPP primary neurons were excitatory neurons and more than 90%of NAc primary neurons were GABA neurons.The cell viability of HIPP and NAc primary neurons was decreased to about 50%after the treatment with 100μM CORT for 48 hours.CORT reduced the number of dendritic spines in HIPP primary neurons.2.CORT decreased the membrane expression of GABA_BR of HIPP and NAc primary neurons,and restored the cell viability and cell membrane expression of GABA_BR in HIPP and NAc primary neurons after the use of Baclofen.3.CORT decreased the membrane expression of GABA_BR cells in HIPP and NAc primary neurons is associated with the increase of the intracellular Ca²⁺concentration,Ca MKIIαexpression,the interaction of p-Ca MKII-GABA_B1R,and the phosphorylation level of GABA_B1R.When Baclofen was used to restore Ca²⁺influx,and Ca MKIIαto the normal level,which reduced the interaction of p-Ca MKII-GABA_B1R and the level of phosphorylation of GABA_B1R.It was further confirmed that the mechanism of membrane expression of GABA_BR was related to Ca²⁺/Ca MKII.Conclusion:In the CORT-induced depression cell model,the expression of GABA_BR in the primary neurons of HIPP and NAc decreased,and the molecular mechanism was closely related to the increase in the activity of Ca²⁺/Ca MKIIα,interaction of p-Ca MKII-GABA_B1R and the level of GABA_B1R phosphorylation.