Regulation Of Tricyclic Antidepressants On The Expression Of Potassium Channel In The Ventral Tegmental Area And Underlying Mechanism

Objective:Classic tricyclic antidepressants(TCAs)such as amitriptyline and imipramine play an important role in the treatment of major depressive disorder(MDD),but its mechanism is unclear.In recent years,it has been found that the potassium channels are closely related to mood disorders such as depression by regulating the excitability of dopaminergic neurons in the ventral tegmental area(VTA),and the potassium channels in the VTA are also associated with the resistance of individuals to social defeat stress.In recent years,activation of the resistance mechanism to stress has been proposed as a new antidepressant therapy,and some antidepressants have been found to enhance the resistance.However,the relationship between individual stress resistance and the pharmacology of classic antidepressants remains unclear.The current study mainly explores the regulation of TCAs on potassium channels and the related mechanisms.Methods:The depressive-like behaviors were induced by chronic social defeat stress(CSDS)in mice.The social interaction test(SIT),sucrose preference test(SPT),and tail suspension test(TST)were conducted to evaluate depression-like behavior of mice.Quantitative real-time polymerase chain reaction(q PCR)and western blotting were used to detect the expression of target genes and proteins.Changes in m RNA expressions of potassium channels were detected after stereotaxic injection of virus in the VTA to specificly overexpress the m~6A demethylases known as fat mass and obesity associated protein(FTO).The depressive-like behaviors of mice with TCAs was detected after specific blocking potassium channel by 4-aminopyridine(4-AP)in the VTA.Immunofluorescence(IF)was conducted to detect the expression of m~6A in N2a cells.Results:(1)The sucrose preference ratio of susceptible mice decreased significantly than that of control(Control:87.42±1.483%,Susceptible:48.42±6.012%,Resilient:89.06±1.570%,n=16-18,P<0.001).The social interaction ratio(Control:1.864±0.1019,Susceptible:0.6125±0.07320,Resilient:1.817±0.1126,n=16-18,P<0.001)and social interaction time was significantly reduced in the susceptible mice(Control:73.10±2.946 sec,Susceptible:31.66±4.573 sec,Resilient:65.53±3.954 sec,n=16-18,P<0.001).(2)Both imipramine and amitriptyline increased the social interaction ratio of susceptible mice(CSDS+Vehicle:0.8111±0.1626,CSDS+Imipramine:1.311±0.1397,n=10-11,P<0.05;CSDS+Vehicle:0.7688±0.1566,CSDS+Amitriptyline:1.394±0.1870,n=14-15,P<0.05).(3)After intraperitoneal injection of amitriptyline(10 mg/kg)for two weeks,the m RNA expression of KCNG1(Control:1.000±0.1712,CSDS+Vehicle:1.426±0.2365,CSDS+Amitriptyline:2.216±0.2157,n=5-6,P<0.05),KCNF1(Control:1.000±0.08817,CSDS+Vehicle:0.6674±0.1163,CSDS+Amitriptyline:1.185±0.1864,n=5,P<0.05),KCNQ3(Control:1.000±0.03591,CSDS+Vehicle:0.8456±0.05149,CSDS+Amitriptyline:1.088±0.06044,n=5-6,P<0.05),KCNQ2(Control:1.000±0.03321,CSDS+Vehicle:0.7358±0.08597,CSDS+Amitriptyline:1.108±0.05406,n=5-6,P<0.01)significantly increased in the VTA of susceptible mice.(4)Intraperitoneal injection of amitriptyline(10 mg/kg,once daily,two weeks)reduced immobility time of control mice in TST(Veh+PBS:155.7±16.03 sec,Ami+PBS:107.0±16.43 sec,n=9-10,P<0.05)and FST(Veh+PBS:85.71±16.89 sec,Ami+PBS:43.50±9.051 sec,n=7-10,P<0.05),and treatment with 4-AP in the VTA eliminated the effect of amitriptyline in TST(Ami+PBS:107.0±16.43 sec,Ami+4-AP:179.7±21.50 sec,n=10,P<0.05)and FST(Ami+PBS:43.50±9.051 sec,Ami+4-AP:110.2±21.46 sec,n=10,P<0.05).(5)Imipramine(10μmol/L)and amitriptyline(10μmol/L)significantly reduced the m~6A level in N2a cells(Control:1.000±0.04443,Imipramine:0.8172±0.04961,Amitriptyline:0.8346±0.03300,n=9-13,P<0.05).(6)Compared with the control group,imipramine(10μmol/L)and amitriptyline(10μmol/L)significantly increased FTO protein expression in the N2a cells(Control:1.000±0.06922,Imipramine:1.258±0.09743,Amitriptyline:1.207±0.05763,n=6-8,P<0.05).However,there was little effect on the expression of METTL3 and METTL14protein.(7)Imipramine significantly increased the protein expression of FTO in the VTA of control mice(Vehicle:1.000±0.06363,Imipramine:1.182±0.04621,Amitriptyline:0.9344±0.08221,n=5,P<0.05),but did not affect FTO expression in m PFC,NAc and Hip.(8)After FTO overexpression in the VTA of susceptible mice,the m RNA expressions of KCNG1(AAV-GFP:0.8642±0.1051,AAV-FTO:1.302±0.1590,n=7,P<0.05),KCNF1(AAV-GFP:0.8991±0.1096,AAV-FTO:1.476±0.1571,n=7-8,P<0.05)and KCNQ2(AAV-GFP:1.044±0.1786,AAV-FTO:1.823±0.1753,n=4-5,P<0.05)were significantly increased,but the m RNA expression of KCNA6(AAV-GFP:1.000±0.03238,AAV-FTO:0.6132±0.1431,n=4,P<0.05)significantly decreased.The m RNA expressions of KCNQ3,KCNG4,KCNH3,KCNK9,KCNMA1 and KCNAB1 showed little changes.Conclusion:TCAs increases m RNA expressions of potassium channel in the VTA of susceptible mice,and blockade of potassium channels in the VTA prevents the antidepressant effect of TCAs,suggesting that potassium channels may contribute to the antidepressant effect of TCAs.