M₄ Receptor Modulates Cdk5/DARPP-32 Thr75 Involved In Balance Of Striatum Ach-DA In Isolated Medium-sized Spiny Neurons

Objective:To investigate the regulation of Cdk5/DARPP-32 Thr75 signaling pathway by M₄ in MSNs,and study M₄/Cdk5/DARPP-32 Thr75 signaling pathway on the dopaminergic neural pathway in MSNs.Methods:?1?Fluorescent staining was performed using GABAergic neuron marker molecules neurofilament?NF?and GAD.Five mAChRs subtypes of MSNs were were studied by laser confocal imaging;shRNA lentiviral particles were coated with Lipofectamine 3000,and viral GFP was observed at 24,48,72,96 h after transfection.and laser confocal Imaging techniques to study the GFP expression.?2?The Co-localization expression between M₄ receptor?M₁ receptor and Cdk5 was performed by laser confocal technique;Real-time calcium imaging was used to detect changes in neuronal cytosolic calcium concentration after treatment with different concentrations of Oxo-M;The M₁ receptor antagonist MT7(10^-7 mol/L)and the M₄ receptor antagonist MT3(10^-7 mol/L)were pretreated,western blotting and immunofluorescence were used to observe the changing of Cdk5 and p35/25.?3?Oxo-M(10^-6 mol/L)was used to treat KD-M₄ MSNs for 15 min,and the expression of Cdk5 and the phosphorylation status of DARPP-32 Thr75 were observed by western blot and immunofluorescence.Cdk5 inhibitor ROSC(10^-5 mol/L)was used to pretreat medium spinous neurons for 10 min,and Oxo-M was treated at different concentrations(10^-7,10^-6,10^-5 mol/L)for 15 min.The expression of DARPP-32 p-thr75 was observed by western blot and immunofluorescence.APO(10^-6 mol/L),a non-selective dopamine receptor agonist in intermediate spinous neurons,was treated for 10 min.The production of cAMP was detected by fluorescence resonance energy transfer.The expression of DARPP-32 p-Thr34 was detected by western blot and immunofluorescence.Oxo-M(10^-6 mol/L)was pretreated for5 min and incubated with APO(10^-6 mol/L)for 10 min.The phosphorylation level of DARPP-32 Thr34 and the expression of Cdk5,p35/25 and pp-1 were detected by confocal laser and western blot.Results:?1?The GAD positive cells in GABAergic neurons was higher.The expression of M₄receptor and M₁ receptor was mainly distributed in cell membrane and cytoplasm.After transfection for 96 h at MOI-20,the GFP expression of Chrm4-shRNA virus was higher,and the M4 receptor in MSNs was knocked down.?2?Laser confocal results showed that 10^-6 mol/L Oxo-M treatment of MSNs for 15 min was significantly increased the expression of Cdk5?p<0.01?and p35/25?p<0.01?;M4receptor and Cdk5 were totally co-localized in MSNs,while M1 receptor and Cdk5 were not co-localized,suggesting that There is a functional coupling of M4 and Cdk5;the results of calcium imaging of living cells show that the concentration of calcium ion can be observed only when the concentration of Oxo-M is 10^-4 and 10^-2 mol/L.No increase was observed for the ranges of 10^-88 and 10^-6 mol/L.In the MT7(10^-5 mol/L)pretreatment group,intracellular calcium concentration was significantly inhibited;Western blot results showed that compared with the control group,10^-6 mol/L Oxo-M can significantly up-regulated the expression of Cdk5?p<0.01?and p35/25?p<0.01?;compared with Oxo-M group,M4receptor antagonist MT3 pretreated MSNs,Cdk5 and p35/25 expression were inhibited?p<0.01?.There was no significant change in Cdk5 expression by pretreatment of the M1receptor antagonist MT7.?3?Oxo-M?10^-66 mol/L?treatment of KD-M4 MSNs for 15 min,compared with normal MSNs,Cdk5?p<0.001?and DARPP-32 Thr75 phosphorylation?p<0.01?were inhibited;Compared with Oxo-M group,Rosc(10^-5 mol/L)pretreated MSNs for 10 min,Significant inhibition of Oxo-M-induced Cdk5 expression?p<0.001?and DARPP-32 Thr75phosphorylation?p<0.001?;Administration of non-selective dopamine receptor agonist APO(10^-2 mol/L)at 1 min,the cAMP content was significantly increased.Pretreated with D₁ receptor blocker SKF23390,APO-induced cAMP production significant decrease in MSNs.Pretreated with Oxo-M(10^-6 mol/L)for 5 min,then incubated with APO(10^-6 mol/L)for 10 min,compared with the control group,the DARPP-32 p-Thr34 was significantly reduced?p<0.01?.Conclusion:M₄ receptor specifically regulates the expression of Cdk5 and p35/25 in MSNs,and activation of the M₄/Cdk5/DARPP-32 Thr75 signaling pathway inhibits APO-induced DARPP-32 Thr34 phosphorylation.