Stress Inhibits The Expression Of GABA_B Receptor And Membrane Transport In Amygdala And The Role Of CHOP In The Amygdala Of Rats

Objective:In clinical work and forensic practice,victims often suffer mental injury after illegal restriction of personal freedom such as binding and bondage.Because of the unclear mechanism of injury,there are great difficulties in clinical diagnosis,treatment and forensic diagnosis.Excessive mental stress is regarded as a common characteristic in the background of the above cases.Whether this kind of stress can cause mental injury or not,there is still a lack of scientific basis.Amygdala plays a core role in the regulation of advanced brain activity and is a highly sensitive area of stress.The researches confirmed that the removement of high inhibitory state of amygdala in stress can produce a series of mental injury symptoms such as anxiety.GABA is an important inhibitory neurotransmitter,which plays an important role in maintaining the high inhibitory state of amygdala.After gene knockout of GABA_B receptor,the animals showed a long period of anxiety symptoms,indicating that the GABA_B receptor may be involved in mental injury.The changes in the expression and function of the amygdala GABA_B receptor are not clear during stress.Endoplasmic reticulum stress(ERS)is one of the subcellular mechanisms of stress-induced damage,therefore,the present study intends to establish stress model rats,to investigate the expression and membrane transport of GABA_B receptor in amygdala.The research results will provide the experimental basis for clarifying the mechanism of relieving of high inhibitory state of amygdala,which is important for revealing the relationship between stress and mental injuries.Methods:The rat model of binding stress combined with ice water swimming was established.The experiments were divided into control group,binding stress model group(model),CHOP inhibitor group(inhibitor)and DMSO(as an solvent control)group.The model was evaluated by measuring the changes of body weight and the concentrations of serum catecholamine.The elevated cross maze test and spontaneous activity test were used to observe the changes of anxiety behavior in rats.The expression of CHOP,GABA_B1 and GABA_B2protein was detected by immunohistochemical technique and Western blot technique.Co-immunoprecipitation technique was used to observe the interaction between CHOP and GABA_B1 and GABA_B2,respectively.Results:1.The changes of rat body weights:compared with control group,the body weight were significantly decreased in model group.2.Catecholamine concentration changes in plasma:compared with control group,the concentrations of adrenalin,norepinephrine and dopamine increased rapidly after first days of stress,and then decreased gradually,but those were still higher than that of control group.3.The behavioral research results:There was no significant difference in the spontaneous locomotion ability of each group.The results of elevated cross maze test showed that compared with control group,both the Open-arms entries percentage and the Open-arms times percentage were significantly reduced in model group,and that in inhibitor group was higher than that in model group,suggesting that inhibition of ERS can alleviate anxiety like behavior in rats.4.The immunohistochemistry staining results:CHOP staining results showed that the protein expression level in group model was significantly higher than that in control group,while the protein expression level in inhibitor group was lower than that in model group.The variation tendency of protein expression levels of GABA_B1 and GABA_B2 was consistent,that is,compared with the control group,that of the model group was significantly lower,and the protein expression levels in inhibitor group was higher than that of model group.5.Western blot detection results:(1)First,the expression levels of CHOP,GABA_B1 receptor and GABA_B2receptor were detected in total protein not including nuclear protein.The detection results of CHOP showed that that of group model was significantly higher than that of control group,and inhibitor group was lower than model.Both the protein levels of GABA_B1 receptor and GABA_B2 receptor were significantly lower in group model than in control group,while that in inhibitor group was higher than that of model group.The results suggest that inhibition of ERS can reverse the decreasing of GABA_B1 and GABA_B2protein expression induced by stress.(2)After the total protein was further divided into membrane protein and cytoplasmic protein,the expression levels of GABA_B1 receptor and GABA_B2receptor were detected.In membrane protein,the expression levels of GABA_B1 receptor and GABA_B2 receptor in model group were significantly lower than that in control group(P<0.05),and that in inhibitor group was significantly higher than that in model group(P<0.05).In cytoplasm protein,the expression levels of GABA_B1 protein were consistent with that of membrane protein,but GABA_B2 protein levels showed no significant difference in each group(P>0.05).These results suggested that ERS activation has an effect on the protein expression and membrane transport of both receptors.6 Immunoprecipitation detection results:After the total protein in control group was removed nuclear protein,the expression levels of CHOP were detected inGABA_B1 and GABA_B2 receptors precipitates respectively.The results showed that the expression level of CHOP in GABA_B2 receptor precipitate was significantly higher than that in GABA_B1 receptor precipitate,suggesting that CHOP was mainly bound to GABA_B2(P<0.05).Furthermore,immunoprecipitation experiments were carried out with cytoplasmic protein and membrane protein.In cytoplasmic protein,variation tendency ofthe expression levels of CHOP in GABA_B1 receptor sediment and GABA_B2 receptor sediment was consistent,that is,compared with control group,that of model group was significantly increased(P<0.05),and expression level in inhibitor group was significantly decreased(P<0.05)compared with that of model group.The detection results of cell membrane protein showed that CHOP expression was not ovserved in both GABA_B1 and GABA_B2 receptor precipitates These results suggested that the interaction between CHOP and GABA_B1 or GABA_B2 was increased during stress.Inhibition of ERS can decrease the interaction between CHOP and two receptor proteins,and the interaction between proteins occurs only in cytoplasm.Above results all showed that there was no significant difference between DMSO group and control group.Conclusions:1.Restriction stress combined with ice water swimming can reduce amygdala GABA_B1 and GABA_B2 receptor expression in rats and inhibit membrane transport of GABA_B1 and GABA_B2,which was connected with ERS.2.CHOP,as a ERS marker,mainly binds to GABA_B2 receptor in cytoplasm,which hinders GABA_B1 and GABA_B2 from forming dimer and affects GABA_B receptor membrane expression.