| Parkinson’s disease(PD)is a common neurodegenerative disease that mainly occurs in the elderly.The neuropathological basis of PD is the selective loss of dopamine(DA)ergic neurons in the substantia nigra pars compacta and the reduction of DA content in striatal axonal terminals.Imbalance between direct and indirect pathways in the basal ganglia leads to a range of motor symptoms such as bradykinesia,stiffness,resting tremor,and postural instability.Deep brain stimulation(DBS)is surgical treatment for motor symptoms of PD.In addition to the subthalamic nucleus and globuspallidus internus being the most classical stimulation targets,recent studies have reported that DBS of zona incerta(ZI)can also effectively relieve the motor symptoms of PD,and its therapeutic effect is similar to that of traditional subthalamic nucleus stimulation without obvious side effects.However,the underlying mechanism is still unclear.DBS is considered to regulate the function of brain regions mainly by interfering with the electrical activity of stimulated nucleus neurons and affecting the information transmission of related nerve conduction pathways.There were many types of neurons in the zona incerta,mainly inhibitory γ-aminobutyric acid(GABA)ergic neurons.Therefore,systematic study of the electrophysiological characteristics of the zona incerta in normal and PD disease states will provide beneficial evidence for understanding the possible mechanism of the zona incerta in the treatment of PD.Objective:To observe the electrophysiological activity and pathological changes of GABAergic neurons in zona incerta of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced subacute and chronic PD mice.Methods:1.C57BL/6 mice were injected intraperitoneally with MPTP for five days to establish a subacute PD model,and chronic PD model was established by injecting MPTP twice a week for a total of 5 weeks.2.Patch clamp technique was used to observe the spontaneous and evoked firing of GABAergic neurons in the zona incerta of normal and PD mice.3.Immunofluorescence technique was used to observe the expression of GABA,c-fos and substantia nigra tyrosine hydroxylase(TH)in the zona incerta.4.Open field was used to observe the changes in motor behavior of mice.Results:(1)Spontaneous and evoked discharges of GABAergic neurons in zona incerta of normal miceThe spontaneous firing frequency of GABAergic neurons in 10-20 d mice was5.60±2.70 Hz,and that in 2-month-old mice was 17.54±3.17 Hz,and the difference was statistically significant(P<0.001,n=13-27).The firing frequency of GABAergic neurons in zona incerta of 10-20 d mice was less than that of adult mice,and the difference was statistically significant(P<0.05,n=28-31).(2)The electrophysiological changes of GABAergic neurons in zona incerta of subacute PD mice induced by MPTPMPTP was used to establish a subacute PD mouse model.The results showed that the number of TH positive neurons in the substantia nigra was decreased,and the total distance of movement in the open field mice was decreased,and the difference was statistically significant compared with the normal control group(P<0.05,n=7).The spontaneous discharge frequency of PD model mice was 4.96±0.88 Hz,which was significantly lower than that of normal control mice 18.43±2.72Hz(P<0.001,n=21-22).The evoked firing frequency of GABAergic neurons in PD model mice.was statistically significant(P<0.05,n=10).Taking 50 p A depolarizing current stimulation as an example,the changes of other indications of action potential(AP)were analyzed.The interspike interval(ISI),after hyperpolarization duration(AHP duration),AP duration,and AP initiation were increased,and the difference was statistically significant(P<0.05,n=6).There was no significant change in AP amplitude and AHP amplitude(n=6).In MPTP-induced subacute PD mice,the number of c-fos positive neurons in the zona incerta decreased,and the difference was statistically significant compared with the normal control group(P<0.05,n=7).(3)The electrophysiological changes of GABAergic neurons in zona incerta of chronic PD mice induced by MPTP1.A chronic PD mouse model was established by intraperitoneal injection of MPTP for 5 weeks.The number of TH positive neurons in the substantia nigra was significantly decreased at 1-5 weeks after MPTP injection compared with the normal control group(P<0.001,n=6).2.One week after MPTP injection,the evoked firing frequency of GABAergic neurons in the zona incerta increased,and the difference was statistically significant(P<0.05,n=10-11).Taking 150 p A depolarizing current stimulation as an example,the onset time of AP decreased,and the difference was statistically significant(P<0.05,n=10).There were no significant changes in ISI,AHP duration,AP duration,AP amplitude and AHP amplitude did not change significantly(n=10).3.After 2-4 weeks of MPTP injection,there was no significant change in the firing frequency of GABAergic neurons in zona incerta(n=15-21).Taking 150 p A depolarizing current stimulation as an example,there were no significant changes in ISI,AHP duration,AP duration,AP amplitude and AHP amplitude,and AP initiation(n=13-14).4.5 weeks after MPTP injection,the number of evoked discharges of GABAergic neurons in zona incerta decreased,and the difference was statistically significant(P<0.05,n=19-23).Taking 150 p A depolarizing current stimulation as an example,the onset time of AP increased,and the difference was statistically significant(P<0.05,n=19).There were no significant changes in ISI,AHP duration,AP duration,AP amplitude and AHP amplitude(n=19).5.There was no significant difference in the number of c-fos positive neurons in the zona incerta between 1-3 weeks after MPTP injection(n=6),but the number of c-fos positive neurons in the zona incerta decreased significantly between 4-5 weeks after MPTP injection(P<0.05,n=6).(4)Pathological changes of GABAergic neurons in zona incerta in MPTP-induced PD miceThe number of GABAergic neurons in zona incerta did not change significantly after1-4 weeks of MPTP injection(n=6),but decreased after 5 weeks of MPTP injection(P<0.01,n=6).Conclusion:In subacute and chronic PD mice induced by MPTP,the firing frequency of GABAergic neurons in the zona incerta decreased,and the number of c-fos positive neurons decreased,suggesting that there are pathological damage and changes in electrical activity of GABAergic neurons in the zona incerta in PD.In the chronic PD model mice,the pathological damage and changes in the electrical activity of the neurons in the zona incerta showed a time-dependent manner with the progression of PD.The number of evoked action potential bursts increased at week 1 after MPTP injection,but did not change at week 2,3and 4 after MPTP 2 injection.After 5 weeks of MPTP injection,the number of evoked action potential was significantly reduced.The results of this study provide a theoretical basis for understanding the pathological and electrophysiological changes of zona incerta neurons in PD. |
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