The Effect Of NMDA Receptor On Spontaneous Activity Of Dopaminergic Neurons In Substantia Nigra Of Mice With Parkinson’s Disease And Its Underlying Mechanism

The main pathological change of Parkinson’s disease(PD)is the progressive loss of dopaminergic neurons in the substantia nigra(SN)of the midbrain and the discovery of misfolded α-Synuclein,α-Syn)to form Lewy body(LB).It has been found that the firing frequency of dopaminergic(DAergic)neurons in SN of PD patients increases.Our previous research showed that the firing frequency of DAergic neurons in SN of PD transgenic mice increased with age,but the specific mechanism is still unclear.Under the pathological condition of PD,the excitatory innervation provided by the subthalamic nucleus(STN)to neurons containing glutamate receptors in the substantia nigra pars compacta(SNpc)is increased,which may be related to the change of the firing frequency of DAergic neurons in SN.However,it has not been reported how glutamate acts on its receptors and how it affects the discharge of DAergic neurons.In recent years,it has been found that the activity of N-methyl-D-aspartate acid(NMDA)receptor at the synaptic site can regulate the intracellular signal transduction pathway.NMDA receptor is one of the ionic glutamate receptors,which is composed of four subunits and has high Ca2+ permeability.NMDA receptors are divided into seven subtypes: Glu N1,Glu N2A-D,Glu N3A/B.Among them,Glu N2 A and Glu N2 B play a major role in the content of synaptic sites.Non receptor tyrosine kinase Fyn can affect the receptor activity by regulating the phosphorylation level of NMDA receptor.However,in PD,how NMDA receptor participates in the change of spontaneous activity of DAergic neurons in SN and its possible mechanism have not been clarified.Therefore,in order to explore the effect of NMDA receptor on the spontaneous activity of DAergic neurons in SN of PD mice and its possible mechanism,C57 BL/6 mice treated with rotenone were used as the main research object in this experiment.The open field experiment and rod rotation experiment were used to detect the autonomous movement and balance ability of mice.The number of TH+ neurons in SN of mice and the expression of NMDA receptor were observed with immunofluorescence technology,In vivo electrophysiological techniques were used to record the spontaneous activity of DAergic neurons in the SN of mice.Western blotting techniques were used to observe the protein expression of tyrosine hydroxylase(TH)in the SN of mice α-Syn phosphorylated(p-α-Syn)level,observe the main pathological changes in SN,detect the expression and phosphorylation level of Glu N2 A,Glu N2 B,Fyn by western blotting,and observe the change of NMDA receptor activity.The results are as follows:1.The body weight of mice in the rotenone treated group decreased by 27.04%(P<0.001).The results of the open-field experiment showed that the total distance and reduction in locomotion of mice in the rotenone treated group was 20.34%(P<0.01),the mean locomotion speed was reduced by 20.95%(P<0.01),and the total resting time was prolonged by 43.91%(P<0.01),all of which were statistically significant compared with the control mice.However,the time point of the first exploration of the intermediate did not change significantly.The results of the rotating bar experiment showed that the time of movement on the rotating bar was significantly reduced by 49.60%(P<0.01)in the rotenone treated group of mice.Compared with the control group,the difference was statistically significant.2.Immunofluorescence staining of TH+ neurons in the SN of the midbrain of mice showed that the number of TH+ neurons in the mice in the rotenone treated group was reduced.Western blotting experiments showed that the content of TH protein which located in the SN was reduced by 21.93%(P<0.05)and p-α-Syn was increased by 52.55%(P<0.01).Compared with the control group,the difference was statistically significant.3.To detect the effect of DAergic neurons in the SN of control mice to NMDA receptor agonist,after recording the stable firing DAergic neurons,the firing frequency of neurons increased by 53.22 ± 15.94%(P<0.05)after micropressure injection of 50 μM NMDA into the SN.Compared with the artificial cerebrospinal fluid(ACSF)alone,the difference was statistically significant.4.Extracellular recording of spontaneous firing frequency of DAergic neurons in the SN of mice showed that the spontaneous firing frequency of DAergic neurons in the rotenone treated group increased from 2.99 ± 0.99 Hz to 4.98 ± 1.65 Hz,with a mean increase of 66.50%(P<0.05).Compared with the control group,the difference was statistically significant.5.To detect the effect of DAergic neurons in the SN of mice in the rotenone treated group to NMDA receptor antagonist,after recording the stable firing DAergic neurons,the firing frequency of neurons decreased by 39.03 ± 9.251%(P<0.05)after injecting 250 μM of NMDA receptor antagonist D-AP5 into the SN.Compared with the artificial cerebrospinal fluid(ACSF)alone,the difference was statistically significant.6.D-AP5 was given in the presence of α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA)receptors’ antagonist NBQX and gamma-amino butyric acid(GABA)receptors’ antagonist SR95531,the firing frequency of DAergic neurons in the SN of the rotenone treated group was significantly reduced by 56.04 ± 10.07%(P<0.001),Compared with SR95531+NBQX group,the difference was statistically significant.7.Protein expression and phosphorylation levels of the NMDA receptor GluN2 A and Glu N2 B subunit in the SN were examined by immunofluorescence staining and western blotting.Western blotting showed that the phosphorylation level of Glu N2 A and Glu N2 B respectively increased by 50.36% and 25.84%(P<0.05).Compared with the control group,the difference was statistically significant.However,immunofluorescence staining and western blotting showed that the total protein expression of the Glu N2 A and Glu N2 B subunit in the SN was not significantly changed(P>0.05).8.Western blotting showed that the total protein expression of Fyn in the SN of rotenone treated mice did not change significantly,but its phosphorylation level increased significantly by 440.40%(P<0.001).Compared with the control group,the difference was statistically significant.In conclusion,the increased phosphorylation levels of Glu N2 A and Glu N2 B subunits mediated by Fyn in PD may increase NMDA receptor activity.The abnormal activation of NMDA receptor may induce glutamate excitotoxicity,which may be an important reason for the change of spontaneous activity of DAergic neurons and even degeneration and death.This study provides new ideas and directions for the mechanism and treatment of PD.