| Objective:Major depressive disorder is a common psychiatric disorder with high levels of morbidity,recurrence,disability and suicide,causing a serious social burden.Electroconvulsive therapy(ECT)has become the best choice for the clinical treatment of major depressive disorder because of its fast onset and high efficiency.At present,the only research results have not satisfactorily explained the efficacy and mechanism of electroconvulsive therapy.More importantly,there is a lack of data on the neural circuits of electroconvulsive antidepressant,which limits the development of precise local neural regulation strategies for depression.The early clinical imaging data suggest that the change of functional connection from medial prefrontal cortex to subcortical region may be related to the efficacy of electroconvulsive therapy.However,this conclusion is only a speculation based on the results of f MRI detection of metabolic activity in various brain regions.The specific brain regions and neuronal types directly affected by electroconvulsive shock cannot be determined.Therefore,this study intends to establish a mouse model of depression induced by chronic restraint stress(CRS).Behavioral experiments and immunofluorescence techniques were used to observe the improvement of depression-like behavior of CRS mice by electroconvulsive shock treatment and to analyze the effects of electroconvulsive shock on various brain regions of CRS mice,including medial prefrontal cortex(m PFC),nucleus accumbens(NAc),anterior cingulate cortex(ACC),amygdala(AMY)hippocampus(HPC)and hypothalamus(HY)(c-Fos protein),the specific region location of electroconvulsive therapy(medial prefrontal cortex)was identified.On this basis,immunofluorescence technique was used to further clarify the specific cell types of medial prefrontal cortex involved in antidepressant effect of electroconvulsive shock.This study will provide new evidence for revealing the neural mechanism of rapid and efficient antidepressant in electroconvulsive shock,and provide new ideas for the design of more targeted local neural regulation techniques in the future.Methods:The experiment was randomly divided into four groups:(1)normal control group(CON group):C57BL/6 mice aged 6-8 weeks were selected and raised normally.(2)Chronic restraint stress group(CRS group):C57 mice were given chronic restraint stress for 21days.(3)Chronic restraint stress+pseudo-electroshock group(CRS+sham ECT group):the depressed mice were selected and given a pseudo-electroshock every other day,that is,the ears were clamped without corresponding electric current for 2 weeks for a total of 7times.(4)Chronic restraint stress+electroconvulsive shock treatment group(CRS+ECT group):the depressed mice were selected and given electroconvulsive stimulation every other day for 2 weeks for a total of 7 times.Forced swimming test was performed 90minutes after the seventh electroconvulsive stimulation,and the mice in the electroconvulsive therapy group were selected to improve their depression-like behavior.The following experiments were carried out in each group:the first part of the experiment:(1)Behavioral experiment:the depression-like mouse model induced by chronic restraint stress was established.The behavioral indexes of depression in each group were detected.(2)Immunofluorescence experiment:immunofluorescence staining technique was used to detect the expression of neuronal activity markers c-Fos in different brain regions(m PFC,NAc,ACC,AMY,HPC and HY)of mice in order to screen the specific brain regions of electroconvulsive antidepressant effect.The second part of the experiment:the protein expression levels of glutamatergic neuron marker GLUD1 andγ-aminobutyric neuron marker GAD1 in medial prefrontal cortex were detected by immunofluorescence staining to identify the specific types of neurons in medial prefrontal cortex affected by electroconvulsive shock.Immunofluorescence staining was used to detect the protein expression of glutamatergic neuron marker GLUD1 andγ-aminobutyric neuron marker GAD1 in m PFC brain area,in order to identify the specific types of neurons in medial prefrontal cortex affected by electroconvulsive shock.Results:1.Compared with the normal control group,the mice in the CRS depression group showed significant depressive behavior,including an obvious decrease in the percentage of sucrose preference(P<0.01),a significant decrease in the total exercise distance and central area residence time(P<0.05,P<0.01)and the percentage of stay time in the central area decreased significantly(P<0.01)in the open field experiment,and a significant increase in the immobility time of forced swimming(P<0.01).Compared with the CRS depression group,the percentage of sucrose preference of mice in the effective group of electroconvulsive shock treatment significantly increased(P<0.05),the total exercise distance and central area residence time(P<0.05,P<0.01)and the percentage of stay time in the central area increased(P<0.01)in the open field experiment,and the immobility time significantly decreased in the forced swimming experiment(P<0.05).Compared with the CRS depression group,the behavior of the mice in the pseudo-electroshock group had no significant changes(P>0.05).2.Compared with the normal control group,the number of c-Fos~+cells s in the m PFC,ACC and HY(P<0.001,P<0.01,P<0.05)regions of the CRS depression group mice increased,while the number of c-Fos~+cells in the NAc and HPC(P<0.05,P<0.05)regions decreased.There was no statistical difference in the number of c-Fos~+cells in the AMY brain region(P>0.05).Compared with the CRS depression group,the number of c-Fos~+cells in the AMY,NAc,HPC,ACC and HY(P<0.001,P<0.01,P<0.01,P<0.05,P<0.05)regions in the electroconvulsive shock treatment group increased,while the number of c-Fos~+cells in the m PFC brain region decreased.Compared with the normal control group,the number of c-Fos~+cells in the ACC,AMY,HY,NAc and HPC(P<0.001,P<0.001,P<0.01,P<0.05,P<0.05)regions of the CRS depression group mice increased,while the number of c-Fos~+cells in the m PFC was no statistical difference.There was no significant difference in the number of c-Fos~+cells in all brain regions between the pseudo-electroshock group and the CRS depression group mice(P>0.05).3.Compared with the normal control group,the relative average fluorescence intensity of GLUD1 in the m PFC brain region of mice in the CRS depression group increased(P<0.05)and the relative average fluorescence intensity of GAD1 significantly decreased(P<0.05),and the ratio of GLUD1~+/GAD1~+cells per unit area increased(P<0.05).Compared with the CRS depression group,the relative average fluorescence intensity of GLUD1 in the m PFC brain region in the electroconvulsive shock treatment group decreased(P<0.05)and the relative average fluorescence intensity of GAD1increased(P<0.05),and the ratio of GLUD1~+/GAD1~+cells per unit area decreased(P<0.05)Compared with the normal control group,there was no significant difference between the relative average fluorescence intensity of GLUD1 and the relative average fluorescence intensity of GAD1 in m PFC brain regions in the electroconvulsive group(P>0.05),and there was no significant difference in the ratio of GLUD1~+/GAD1~+cells per unit area(P>0.05).Compared with the CRS depression group,there was no statistically significant difference in the relative average fluorescence intensity of GLUD1 and GAD1in the brain region of m PFC in the pseudo-electroshock group mice,and the ratio of GLUD1~+/GAD1~+cells per unit area(P>0.05,P>0.05,P>0.05).Conclusion:1.Combining the results of three behavioral experiments,namely,CRS mice showed obviously depressive behavior and electroconvulsive therapy was helpful to improve the depressive behavior of mice.2.According to the results of c-Fos immunofluorescence staining in different brain regions(m PFC,NAc,ACC,AMY,HPC,HY)of mice in each group,m PFC was the only brain region where the expression of c-Fos increased after chronic restraint stress,but returned to normal after electroconvulsive therapy,so m PFC may be the key brain region for electroconvulsive improvement of depression-like behavior in CRS mice.3.Electroconvulsive therapy can reverse the changes of GLUD1 protein,GAD1protein and the ratio of GLUD1~+/GAD1~+cells per unit area in m PFC brain of mice induced by chronic restraint stress,indicating that electroconvulsive therapy may improve the depressive behavior of CRS mice by reversing the imbalance between glutamatergic neurons andγ-aminobutyric neurons. |
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