| Part Ⅰ The role of gephyrin s-nitrosylation in the basal lateral amygdala in anxiety-like behavior of ratsObjective:The inhibitory neurotransmitter gamma-aminobutyric acid(GABA)and its receptor-mediated inhibitory synapses transmission in amygdala are involved in the development of anxious disorder.Gephyrin is one of important anchor proteins that regulate inhibitory postsynaptic function,which enhances the function of inhibitory synapses by increasing the membrane stability of GABA receptors.Since gephyrin could be subjected to s-nitrosylation with its functional alterations,the post-translational modification of gephyrin may be involved in the induction of anxiety-like behavior.However,it remains to be elucidated that the role and molecular mechanism of gephyrin s-nitrosylation in psychiatric diseases such as anxiety.In this study,we used the natural anxiety model of rats and the anxiety model induced by chronic corticosterone exposure to investigate s-nitrosylation of gephyrin in basolateral amygdala(BLA)in anxiety-like behavior and its effect in the anxiety-like behavior.Methods:The rat model of anxiety included both the natural anxiety and anxiety model induced by chronic corticosterone exposure.The elevated plus maze(EPM)and open field test(OFT)were used to evaluate the anxious state of rats.Western blot,Acyl-biotinyl Exchange(ABE),biotin method and other molecular biological methods were used to detect the membrane expression of GABA_A receptor subunits and s-nitrosylation level of gephyrin in BLA of rats in either high anxious state or low anxious state.Results:High-anxiety(HA)rats and low-anxiety(LA)rats,were screened by EPM.HA rats had significantly lower open-arm duration(LA group:140.4±4.15 s,n=10;HA groups:88.35±6.07s,n=12,P<0.0001)and open-arm distance(LA group:6.72±0.27 m,n=10;HA group:4.42±0.39 m,n=12,P<0.001).There was no significant difference in total distance(LA group:12.64±0.52 m,n=10;HA group:11.98±1.08 m,n=12).Western blotting experiments showed that the expression of GABA_ARγ2 membrane in BLA of high anxiety rats was decreased(LA group:1.05±0.08,HA group:0.76±0.09,n=6,P<0.05),and the level of gephyrin s-nitrosylation in BLA of high anxiety rats was increased(LA group:0.74±0.09,HA group:0.98±0.05,n=6,P<0.05).An anxiety model induced by chronic corticosterone exposure was also introduced.The open-arm duration was significantly reduced in the EPM test of for corticosterone(cort)group compared to vehicle group(veh)(Veh group:121.60±6.19 s,n=6;Cort group:51.94±9.58 s,n=8,P<0.001);central zone duration was significantly reduced in the OFT test(Veh group:134.10±21.02 s,n=6;Cort group:23.64±3.45 s,n=8,P<0.0001);there was no significant difference in total distance in the OFT test(Veh group:13.17±2.31 m,n=6;Cort group:14.00±1.68 m,n=8).ABE test showed an increase in gephyrin s-nitrosylation in BLA of high-anxiety rats(Veh group:0.94±0.10,n=6;Cort group:1.30±0.10,n=8,P<0.05).Conclusion:The membrane expression of GABA_ARγ2 in BLA of high-anxiety rat was decreased,with no changed total expression of gephyrin,and the level s-nitrosylation of gephyrin was increased.It suggests that s-nitrosylation modification of gephyrin may be involved in the anxiety-like behavior induced by s-nitrosilation of gephytin in rats.Part Ⅱ The mechanism of gephyrin s-nitrosylation in basal lateral amygdala in anxiety-like behavior of rats Objective: As an important enzyme in the central nervous system,neuronal nitric-oxide synthase(nNOS)can produce nitric oxide(NO),a signaling molecule to regulate neuronal activity in the central nervous system.nNOS can also affect downstream signals by regulating s-nitrosylation of proteins.Studies have shown that nNOS has been involved in anxiety-like behavior in rats.In part one,we found that s-nitrosylation of gephyrin was increased in high anxiety mice,and it has been reported that gephyrin can undergo s-nitrosylation modification,suggesting that nNOS may mediate nitrosylation of gephyrin and induce anxiety-like behavior.Thus in the second part,both pharmacological and molecular biological methods were used to regulate nNOS to explore its role in anxiety-like behavior induced by gephyrin nitrosylation.Methods: Co-Immunoprecipitation(CO-IP)assay was used to detect the interaction between nNOS and gephyrin.gephyrin nitrosylation was detected by ABE method.7-Nitroindazole(7-NI),the selective pharmacological inhibitor of nNOS and tetrodotoxin(TTX),a specific blocker of sodium ion channel were used to inhibited nNOS activity.Then effects of 7-NI and TTX on nitrosylation of gephyrin were detected by ABE.After administration of lentivirus that overexpress nNOS in BLA or systemic administration of 7-NI,EPM and OFT of rats were evaluated to reveal the involvement of nNOS and anxiety-like behavior induced by snitrosylation of gephyrin.Results: CO-IP experiments revealed that nNOS and gephyrin interacted in BLA of rats.SNAP increased s-nitrosylation of gephyrin(Veh group: 1.01 ± 0.12,SNAP group: 2.25 ± 0.22,n = 4,P < 0.01).TTX(30 uM)to inhibit nNOS activity could reduce gephyrin s-nitrosylation(Veh group: 1.65 ± 0.08,TTX group: 0.75 ± 0.08,n = 4,P < 0.001).Glycine(Gly,200 μM)to increase the activity of nNOS elevated gephyrin s-nitrosylation(Veh group: 1.00 ± 0.26,Gly group: 1.65 ± 0.18,n = 4,P < 0.05).This elevation could be abolished by 7-NI(100 uM),a nNOS specific inhibitor(Gly group: 1.65 ± 0.18,Gly+7-NI group: 1.07 ± 0.17,n = 4,P < 0.05).Over-expression of nNOS in BLA(LV-GFP group: 0.48 ± 0.02;LV-nNOS group: 0.57 ± 0.01,n=8,P<0.01),decreased open arm duration(LV-GFP group: 146.30 ± 20.78 s,LV-nNOS group: 84.48 ± 4.68 s,n = 8,P < 0.05)and pen arm distance in EPM test(LV-GFP group: 4.20 ± 0.55 m,LV-nNOS group: 2.56 ± 0.22 m,n = 8,P < 0.05);This overexpression had no effect on total distance(LVGFP group: 7.6 ±0.54 m,LV-nNOS group: 9.09 ± 0.58 m,n = 8)and mean motor velocity(LVGFP group: 0.025 ± 0.001 m/s,LV-nNOS group: 0.030 ± 0.001 m/s,n = 8).Similarly,OFT showed that overexpression of nNOS significantly reduced central zone duration(LV-GFP group:47.21 ± 8.34 s,LV-nNOS group: 17.99 ± 3.27 s,n = 8,P < 0.05),but had no effect on total distance(LV-GFP group: 14.27 ± 0.87 m,LV-nNOS group: 13.51 ± 1.83 m,n = 8),indicating that overexpression of nNOS induced anxiety-like behavior in rats;After inhibition of nNOS activity by 7-NI,open arm entries(Veh group: 4.88 ± 0.63,7-NI group: 7.77 ± 0.69,n = 10,P < 0.05),and open arm duration(Veh group: 60.56 ± 13.05 s,7-NI group: 99.37 ± 9.69 s,n = 10,P < 0.05)were significantly increased in OFT.7-NI also significantly increased central zone shuttles(Veh group: 7.66 ± 0.97,7-NI group: 12.40 ± 1.74,n = 10,P < 0.05)in OFT.Conclusion: nNOS and gephyrin interact with each other in BLA,and nNOS in BLA is involved in anxiety-like behavior induced by s-nitrosilation of geohyrin of rats. |
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