Pathological Changes And Regulation Of Motor And Non-motor Symptoms Of Zona Incerta Neurons In Parkinson’s Disease Model Mice

Parkinson’s disease（PD）,as a neurodegenerative disease commonly occurring in middle-aged and elderly people,is characterized by degeneration and loss of dopaminergic neurons（DA）in the substantia nigra pars compacta and decreased dopamine content in the striatum,resulting in abnormal function of basal ganglia circuits.PD patients show a variety of typical motor symptoms such as slowness of movement,static tremor and muscle rigidity,as well as a variety of non-motor symptoms such as pain sensation,abnormal cardiovascular function and sleep disturbance.Although levodopa has been regarded as the most effective drug in the treatment of PD in recent decades,drug resistance and gastrointestinal side effects caused by long-term use of patients are still thorny problems.Recent studies have shown that deep brain stimulation（DBS）of the zona incerta（ZI）improved the motor symptoms of PD patientswithout obvious side effects.The zona incerta is a nucleus cluster located in the subthalamus dominated by inhibitoryγ-aminobutyric acid（GABA）energetic neurons,with strong cellular heterogeneity.In addition to containing GABAergic neurons,more than 20neurotransmitters such as Glutamic acid（Glu）and DA were also distributed.In rodents,these neurons are located in four subregions:rostral zona incerta（ZIr）,dorsal zona incerta（ZId）,ventral zona incerta（ZIv）and caudal zona incerta（ZIc）.ZId and ZIv are the core regions of the zona incerta.There is extensive fiber projection between the zona incerta and basal ganglia and it is involved in the regulation of pain sensation.Although clinical studies have proved that zona incerta is a safe and effective target for DBS,the underlying mechanism is still in its infancy.The pathological changes of various types of neurons in the zona incerta in PD are still unclear,and how the regulation of the activity of neurons in the zona incerta affects the motor and non-motor symptoms of PD needs further discussion.Objective:1.To observe the pathological changes of GABAergic neurons in zona incerta in6-OHDA and MPTP-induced PD model mice.2.To investigate the effects of pharmacological activation or inhibition of zona incerta on the motor and non-motor symptoms in 6-OHDA-induced PD model mice.Methods:1.To make the PD mouse model,6-OHDA and MPTP were selected respectively.6-OHDA was injected into unilateral striatum by stereotactic technique;PD mouse model was prepared by MPTP was intraperitoneal injected for five consecutive days.2.Immunofluorescence technique was used to observe the expression changes ofGABA,parvalbumin（PV）,nitric oxide synthase（NOS）,tyrosine hydroxylase（TH）and other neurons in the zona incerta.3.GABA_Areceptor antagonist Bicuculline and/or agonist Etomidate were injected into the zona incerta;The effects of Bicuculline and/or Etomidate on the motor behavior of 6-OHDA-induced PD model mice were observed through balance beam test,pole climbing test and open field test.4.GABA_Areceptor antagonist Bicuculline and/or agonist Etomidate were injected into the zona incerta;The effects of Bicuculline and/or Etomidate on pain sensation were observed by Von Frey test and Hotplate test.Results:（1）Pathological changes of zona incerta’s neurons in 6-OHDA-induced PD model mice.After intraperitoneal injection of apomorphine,6-OHDA-induced PD model mice showed healthy side rotation behavior,while the normal control group did not show spontaneous rotation behavior,and the difference was statistically significant（P<0.001）.In 6-OHDA-induced PD model mice,immunofluorescence staining results showed that the number of TH-positive neurons in the substania nigra decreased,and the difference was statistically significant compared with the normal control group（P<0.001）.The numbers of GABA,NOS,and PV-positive neurons in the zona incerta were decreased,and the difference was statistically significant compared with the normal control group(P_GABA<0.001;P_PV<0.05;P_NOS<0.01).There was no significant difference in the number of TH-positive neurons in the zona incerta compared with the normal control group.（2）Pathological changes of zona incerta’s neurons in MPTP-induced PD model mice.In MPTP-induced PD model mice,immunofluorescence staining results showed that the number of TH-positive neurons in the substantia nigra decreased,and the difference was statistically significant compared with the normal control group（P<0.001）.The number of GABA,PV,and NOS-positive neurons in the zona incerta decreased,and the difference was statistically significant compared with the normal control group(P_GABA<0.01;P_PV<0.01;P_NOS<0.001).There was no significant difference in the number of TH-positive neurons in the zona incerta compared with the normal control group.（3）Effects of injection of Bicuculline or Etomidate into the zona incerta on motor symptoms in 6-OHDA-induced PD model mice.1.Compared with normal saline injection group,Etodimate injection significantly reduced the distance of movement and the speed of motion in the open field test（P<0.001）and did not show anxiety symptoms;In the pole climbing experiment,the time from the top to the bottom of the pole was significantly prolonged（P<0.001）,suggesting PD-like motor dysfunction in mice with zona incerta inhibition.2.The balance ability of PD model mice induced by 6-OHDA decreased significantly in the balance beam test,with an average score of 3.533,which was statistically significant compared with the normal control group（P<0.001）;After treatment with GABA_Areceptor antagonist Bicuculline,the average score was restored to4.133,and the difference was statistically significant compared with that in 6-OHDA group（P<0.05）;After the injection of Etomidate,the score of mice was further reduced to2.933 points,with a more serious decrease in balance ability,and the difference was statistically significant compared with the 6-OHDA group（P<0.05）.These results suggest that activation of zona incerta by Bicuculline can improve the balance ability of mice.3.The climbing time of PD model mice induced by 6-OHDA was significantly increased in the pole climbing test,and the climbing time from the top to the bottom of the pole was 8.222 seconds,which was statistically significant compared with the normal control group（P<0.001）;The climbing time of mice with Bicuculline injection was reduced to 6.935 seconds,which was statistically significant compared with the 6-OHDA group（P<0.05）;After the injection of Etomidate,the climbing time of mice was further extended to 10.21 seconds,which was statistically significant compared with the6-OHDA group（P<0.001）.These results suggest that activation of zona incerta by Bicuculline can improve coordination in mice.4.The crawling distance of PD model mice induced by 6-OHDA was shortened in the open field test,and the difference was statistically significant compared with the normal control group（P<0.001）;The movement distance and speed of mice with Etomidate injection was further reduced,with a statistically significant difference compared with the 6-OHDA group（P<0.01）;After Bicuculline injection,however,there was no significant difference in the distance and speed of the mice compared with the6-OHDA group.There was no difference in the percentage of the exercise time in the central area and the exercise time in the central area in the total exercise time among all groups,suggesting that the mice did not show anxiety symptoms.（4）Effects of injection of Bicuculline or Etomidate into the zona incerta on pain perception in 6-OHDA-induced PD model mice.Von Frey test and Hotplate test showed that 6-OHDA-induced PD model mice showed a decrease in the pain perception threshold of the damaged hind limb to mechanical and thermal stimulation,and the difference was statistically significant compared with the normal control group（P<0.001）;The threshold of nociception was further reduced after the injection of Etomidate(P_{Von Frey test}<0.05,P_{Hotplate test}<0.001),and Bicuculline injection will increase the threshold of nociception,with a statistically significant difference compared with the 6-OHDA group（P<0.05）.Conclusion:Compared with normal mice,the numbers of GABA,PV and NOS positive neurons in zona incerta were decreased in PD model mice induced by 6-OHDA and MPTP,while there was no significant difference in the number of TH positive neurons in ZIr.PD-like motor dysfunction can be induced in normal mice with injection of Etomidate,a GABA_Areceptor agonist.Injection of GABA_Areceptor antagonist Bicuculline into zona incerta could improve the motor function of mice and improve the threshold of pain perception to mechanical and thermal stimulation in 6-OHDA-induced PD model mice.The results of this study provide a theoretical basis for understanding the pathological changes of zona incerta in PD and the regulation of PD motor and non-motor symptoms.