| Objective: Alcohol addiction is a common mental illness that has serious consequences for individuals and society.Recently,the changes in synaptic plasticity are thought closely related to alcohol dependence.However,the molecular mechanism is complex and still unclear.In this study,we established a mice alcohol two-bottle choice model to observe the behavioral effects of alcohol dependence in mice after over-expression of MMP-9 in the hippocampus,and analyzed the plasticity changes of neurons during the process.This study exploring the molecular mechanism of alcohol dependence and provides a new clue.Methods: 1.Overexpression of MMP-9 in the mouse hippocampal brain region: AAV-MMP-9 was injected into the bilateral hippocampus of mice through brain stereotactic injection.MMP-9 protein expression were observed three weeks later,and the effect of overexpression of MMP-9 on alcohol metabolism of mice was detected.2.Forty-five 8-week-old C57BL/6J mice were randomly divided into three groups,fifteen in each group.They were the control group(Control),alcohol group(Alcohol),and alcohol + MMP-9 group(Alcohol + MMP-9).In the alcohol + MMP-9 group,1 ?l AAVMMP-9 was injected bilaterally in the hippocampus.The following experiments were performed after 3 weeks:(1)Two-bottle choice experiment: each mouse was fed separately,and two identical bottles were placed in each cage.Both bottles of the control group were filled with tap water.In alcohol group and alcohol + MMP-9 group,an alcohol bottle and a water bottle were placed.The mice gradient drank 2.5%,5%,10% and 20% alcohol,and alcohol consumption and alcohol preference of each mouse were detected.Meanwhile,the total liquid consumption and weight of the mice were observed.(2)The effects of MMP-9 on taste preference(saccharin and quinine)of mice were detected,and the amount of alcohol and alcohol preference were observed when the mice relapse after four weeks' withdrawal.(3)Golgi staining was used to observe the effect of MMP-9 on the dendritic spines of hippocampal neurons in each group.(4)The effects of MMP-9 on the synapse-associated proteins(Glu N1 and PSD-95)of mice in each group were detected by Western blot.(5)The effects of MMP-9 on the cytoskeletal protein F/G actin ratio in each group was detected by Western blot method.Results: 1.Overexpression of MMP-9 in the hippocampus of mice.Tissue fluorescence assay showed that the expression of MMP-9 significantly increased in hippocampus.Moreover,Western blot showed that compared with the control group,the expression of MMP-9 in AAV-MMP-9 group was significantly increased(P < 0.001).In the alcohol metabolism experiment,there was no statistical difference in blood alcohol concentration between the control and MMP-9 overexpressing mice;however,the blood alcohol concentration were significantly decreased at 2 h after injection compared with 1 h in two both groups(P < 0.05).2.Behavioral testing(1)Alcohol two-bottle choice experiment: When drinking alcohol at concentrations of 2.5% and 5%,there was no statistical difference in alcohol consumption between the alcohol and the alcohol + MMP-9 groups;meanwhile,the alcohol preference of the two groups was not significant difference.When drinking 10% and 20% alcohol,compared with the alcohol group,the alcohol consumption of the alcohol + MMP-9 group was significantly reduced(P < 0.05);and at the alcohol concentration of 10%(P < 0.05)and 20%(P < 0.01)alcohol,the alcohol preference in the alcohol + MMP-9 group was also significantly reduced.There was no significant difference in total fluid consumption and body weight changes among the three groups at all alcohol concentrations.(2)Saccharin and quinine experiments: In the saccharin drinking experiment,there was no difference between the three groups in the consumption of different concentrations of saccharin(0.04% and 0.08%);but as the concentration of saccharin increased,the amount of saccharin consumed in the three groups increased significantly(P<0.001);at the same time,there was no statistical significance in saccharin preference,total liquid consumption and body weight.In the quinine drinking experiment,there was no difference between the three groups in the consumption of different concentrations of quinine(30 ?M and 60 ?M),but quinine consumption in the control group(P < 0.001)and the alcohol + MMP-9 group(P < 0.05)were increased along with quinine concentration raised;meanwhile,there was no statistical difference in quinine preference,total fluid consumption,and body weight.(3)Relapse experiment after withdrawal: Relapse alcohol by two-bottle choice after four weeks of withdrawal.Compared with the alcohol group,the alcohol consumption of the alcohol + MMP-9 group was significantly reduced(P < 0.01),and the alcohol preference was also decreased(P < 0.05);there was no significant difference in total liquid and body weight between the three groups.3.Synaptic-associated protein detection: Compared with the control group,the expression of Glu N1 protein in alcohol group was significantly reduced(P < 0.01);and the expression of Glu N1 protein in the alcohol + MMP-9 group was increased compared with the alcohol group(P < 0.01).Furthermore,there was no statistical difference of PSD-95 protein expression between the three groups.4.Golgi staining experiment: Compared with the control group,the density of dendritic spines in hippocampal CA1 area of the alcohol group increased(P < 0.05),but the density of dendritic spines decreased after overexpression of MMP-9(P < 0.01).5.Analysis of F/G actin ratio: Western blot showed that compared with the control group,the ratio of F/G actin in the hippocampus of the alcohol group increased(P < 0.01),but the F/G actin ratio were decreased in alcohol + MMP-9 group,the F / G-actin ratio decreased(P < 0.001).Conclusion: 1.MMP-9 can specifically and efficiently express in mouse hippocampal neurons,and MMP-9 didn't affect the rate of alcohol metabolism in mice.2.Overexpression of MMP-9 can reduce alcohol consumption and alcohol preference in mice,and this preference was not related to taste preference;MMP-9 can still reduce alcohol consumption and alcohol preference significantly when replase after withdrawal.3.MMP-9-induced behavioral changes in mice were closely related to hippocampal neuron plasticity such as synaptic transmission-related proteins and cytoskeleton proteins. |
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