Regulation Of Zona Incerta GABAergic Neurons On Motor Behavior In Parkinson’s Disease Model Mice

Parkinson’s disease（PD）is a common neurodegenerative disease characterized by the selective loss of dopaminergic（DA）neurons in the substantia nigra pars compacta and reduced DA content in the striatum.PD patients classically display static tremor,muscle rigidity,bradykinesia,and postural instability.In recent years,several clinical studies have proved that deep brain stimulation in zona incerta（ZI）can effectively relieve motor symptoms of PD patients without obvious side effects.ZI is a largely inhibitory subthalamic region connecting with multiple brain areas.The contribution of ZI GABAergic neurons to PD therapy needs to be further investigated.Objective:Chemogenetics and optogenetics were used to activate or inhibit the ZI GABAergic neurons,and the changes of motor behavior and nigrostriatal system function in 6-OHDA-induced PD model mice were observed.Methods:1.PD model mice were prepared by unilateral or bilateral injection of 6-OHDA into the striatum by stereolocation technique.Behavioral tests including open field,pole climbing,balance beam and gait analysis were performed to observe the changes in motor behavior.Changes in the expression of tyrosine hydroxylase（TH）in the substantia nigra and striatum of PD mice were measured by immunofluorescence.2.6-OHDA-induced PD mice received injections of chemogenetic or optogenetic virus.Open field test,pole climbing test,balance beam test and gait analysis were performed after activation or inhibition of GABAergic neurons of ZI.High-performance liquid chromatography with electrochemical detection was used to detect the changes of DA content in the striatum.3.We also used Vgat-Cre mice to further confirm the role of GABAergic neurons in the regulation of PD motor behavior.Results:1.6-OHDA induced motor dysfunction and substantia nigra dopaminergic neurons injury in miceDegeneration of nigra-striatum system was induced unilaterally by 6-OHDA injection into the striatum.Two weeks after 6-OHDA injection,intraperitoneal injection of apomorphine induced asymmetric rotation to the healthy side of model mice（P<0.001）.TH immunofluorescence staining showed that the number of TH-positive neurons in the substantia nigra and TH-positive fibers in the striatum in the 6-OHDA group were significantly decreased（P<0.001）.The results of open field test showed that the total moving distance and moving speed of 6-OHDA group decreased significantly（P<0.05）.The results of the balance beam test showed that the score of 6-OHDA group mice on the balance beam decreased significantly（P<0.001）.The results of the pole test and the rotarod test showed that the time from the top to the bottom of the pole was significantly increased and the time spent on the rod was significantly decreased in the 6-OHDA group（P<0.01,P<0.05）.2.Chemogenetic stimulation or inhibition ZI GABAergic neurons affects motor behavior in 6-OHDA-lesioned mice2.1 C57BL/6 mice were injected with p AAV-GAD67-h M4D（Gi）-m Cherry-WPRE virus.The balance beam test showed that the scores of mice in h M4D group were significantly reduced after unilateral and/or bilateral inhibition(P_unilateral<0.001,P_bilateral<0.05).The results of the pole test after bilateral injection of virus showed that the time from the top to the bottom of the rod was significantly increased in h M4D group（P<0.05）.These results suggested that chemogenetic inhibition of GABAergic neurons in ZI induces motor symptoms in na?ve mice.2.2 PD mice induced by 6-OHDA were injected with activated chemogenetic virus p AAV-GAD67-h M3D（Gq）-m Cherry-WPRE.The changes in motor behavior of the mice were detected after three weeks of successful virus expression.2.2.1 Effects of single chemogenetic activation of ZI GABAergic neurons on motor behavior of PD mice:The results of open field test showed that the total travel distance and speed of mice in h M3D virus group were significantly increased（P<0.05）.The scores of h M3D virus group mice on balance beam increased（P<0.05）.In the gait analysis test,the minimum strength of the left forelimb contact increased significantly and the transverse distance between the forelimbs and feet decreased significantly（P<0.05）.Therefore,chemogenetic activation of ZI GABAergic neurons improved the motor behavior of PD mice.2.2.2 Effects of chemogenetic continuous activation of ZI GABAergic neurons on motor behavior of PD mice:Three weeks after the successful expression of the virus,CNO was injected intraperitoneally for seven consecutive days to detect the behavior.（1）Low dose of CNO（1 mg/kg）:The total distance and speed of movement of mice in h M3D virus group increased in open field（P<0.05）and improved scores on the balance beam（P<0.01）,the time spent in pole climbing decreased（P<0.05）.HPLC showed that the contents of DA and its metabolites in the striatum of h M3D virus group mice were significantly increased（P<0.05）.It is suggested that the continuous activation of ZI GABAergic neurons with low dose of CNO can improve the motor behavior and increase the release of DA in the striatum of PD mice.（2）High dose of CNO（5 mg/kg）:Open field test showed that the total distance and speed of movement of mice in h M3D virus group increased（P<0.05）and significantly increased scores on the balance beam（P<0.01）,the time spent in pole climbing decreased（P<0.01）.Gait analysis showed that the maximum contact area,print length,print width and print area of h M3D virus group mice increased（P<0.05）.It is suggested that continuous activation of undetermined GABAergic neurons by high dose of CNO can improve the movement disorder of PD mice.3.Effects of optogenetic stimulation or inhibition of ZI GABAergic neurons on motor behavior of 6-OHDA-leisoned mice3.1 C57BL/6 mice were injected with the suppressed optogenetic virus p AAV-GAD67-e Np HR 3.0-EGFP-3x FLAG-WPRE.The results of open field test showed that the total distance and speed of movement of mice in Np HR group decreased during light stimulation（P<0.05）,decreased scores in the balance beam test（P<0.01）.Bilateral inhibition results showed that the total distance and velocity of Np HR group mice in open field decreased during light stimulation（P<0.05）,decreased scores in the balance beam test（P<0.01）,increased the time of pole test（P<0.05）.In addition,Vgat-Cre mice were injected with the suppressed optogenetic virus p AAV-EF1-DIO-e Np HR 3.0-m Cherry-WPRE.The results of open field test showed that the total distance and speed of movement of mice in Np HR group during light stimulation were significantly reduced compared with those in m Cherry control group（P<0.05）.It is suggested that optogenetic inhibition of ZI GABAergic neurons can cause motor dysfunction in normal mice.3.2 PD mice were injected with activated optogenetic virus p AAV-GAD67-h Ch R2（H134R）-EGFP-3x FLAG-WPRE.The results of open field test showed that the total distance and speed of Ch R2 group mice in open field increased during both unilateral and bilateral light stimulation（P<0.05）.It is suggested that light activation of ZI GABAergic neurons can improve motor retardation in PD mice.3.3 Vgat-Cre mice were injected with the activated optogenetic virus p AAV-EF1a-DIO-h Ch R2（H134R）-m Cherry,and 6-OHDA was injected into the striatum to produce PD model.The results of open field test showed that the total distance and speed of Ch R2 group mice in open field increased during light stimulation（P<0.05）.It is suggested that light activation of ZI GABAergic neurons in Vgat-Cre mice can improve motor retardation in PD mice.Conclusion:Striatum injection of 6-OHDA can cause dopaminergic neuron injury in substantia nigra and motor dysfunction in mice.Chemogenetics and optogenetics inhibition of ZI GABAergic neurons caused dyskinesia in na?ve mice.The motor behavior of 6-OHDA-induced PD mice could be improved and the content of DA and its metabolites in striatum could be increased after single or continuous activation of ZI GABAergic neurons by chemogenetics.The motor behavior of 6-OHDA-induced PD mice was improved by unilateral or bilateral optogenetic activation of ZI GABAergic neurons.Finally,Vgat-Cre mice were used to verify that unilateral optogenetic activation of ZI GABAergic neurons could also improve the motor behavior of PD mice.The results of this study provide a theoretical basis for understanding the role of ZI GABAergic neurons on PD movement therapy.