The Molecular Mechanism And Electrophysiological Study Of MTOR Signaling Pathway In The Dyskinesia

Objective:To investigate The mechanism of molecular mechanisms and electrophysiology in levodopa induced dyskinesias in mTOR signaling pathway.Methods:Parkinson’s disease model was induced by intracranial injection of 6DOPA(n=54);Intracranial injection of normal saline as the sham operation group(n=8);After 2 weeks,the rats were injected apomorphine to induced rotational behavior,the number of rotations per minute is more than 7 times as a successful model of Parkinson’s disease,in the abdomen injection with Levodopa and benserazide 7 days making Baja dyskinesia model,the behavior of these rats scores were recorded by abnormal involuntary movement(AIM),AIM standards for more than 16 scores was recorded by the cumulative score four times a day,these rats were randomly divided into three groups,these groups were administered by L-DOPA as LID group(LID,group LID),intraperitoneal injection of mTOR inhibitor Rapamycin(rapamycin)as a treatment group that the intervention group(Rapamycin,group R)and rapamycin solvent control group(Conctol,group C);Group LID continues to be given by L-DOPA drug;Group R was given by L-DOPA and rapamycin drug;group C was given L-DOPA and rapamycin solvent.Behavior of the control group and the intervention group was recorded by AIM science scores,Using Western blot technology to detect the changes of AMP A receptor subunit GluRl and GluR2 loci and GluR1(Ser845),GluR2(Ser880)in phosphorylation sites and SAP97 site specific antibody and immunohistochemical detection of AMPA receptor subunit GluR1 and GluR2 technology GluR1(ser845),GluR2(Ser880)SAP97 in the striatum.Parkinson’s disease model was induced by intracranial injection of 6-DOPA(n=20);Intracranial injection of normal saline as the sham operation group(n=4);After 2 weeks,the rats were injected apomorphine to induced rotational behavior,the number of rotations per minute is more than 7 times as a successful model of Parkinson’s disease(n=10);A multi channel microfilament electrode array was implanted into the right striatum In the sham operated group and Parkinson’s disease group,NeuroStudio 128 channel data acquisition was used to collect data on the fifth day after operation,record 2 hours a day at the same time,lasting for 7 days.PD rats were given levodopa for 7 days,neuroStudio 128 channel data acquisition was used to collect data for 7 days,record 2 hours a day at the same time;behavior of these rats scores were recorded by abnormal involuntary movement(AIM),AIM standards for more than 16 scores was recorded by the cumulative score four times a day,LID rats were treated with rapamycin for 2 weeks,neuroStudio 128 channel data acquisition was used to collect data for 7 days,record 2 hours a day at the same time.The spontaneous firing frequency,the brust discharge and the local field potential of the lesioned striatum of SD rats in each group were analyzed by Offline Sorter and Neuroexplorer.Finally,In order to identify the location accuracy damaged the electrode and tissue embedded section.Multichannel electrode was not located in the striatum of SD rats(n=6).Results:1.There was significant difference in AIM scores between the intervention group and the control group(P<0.05).2.Western blot analysis showed that the expression level of SAP-97 was significantly different between the intervention group and the control group(_P<0.05).AMPA receptor subunit GluRl,GluR2 locus protein expression level changes between groups,no significant difference;GluR1(ser845),GluR2(ser880)expression levels were decreased significantly after the intervention,the intervention group and the control group had significant difference(P<0.05);3.The results of immunohistochemistry showed that SAP-97 in the intervention group was lower than that in the solvent group,and the difference was statistically significant(P<0.05).The expression of AMPA receptor subunit GluR1 and GluR2 in the intervention group had no change compared with the control group,and the difference was not statistically significant(P>0.05).4.The results show that the multi channel microarray is located in the striatum.5.12 rats has been successfullyimplanted multichannel electrode in This experiment.the The data selected from 4 normal rats group Collected a total of 30 neurons;6 rats in the experimental group,PD collected 72 neurons;LID 45 neurons,1W after rapam ycin treatment 39 neurons,2W after rapamycin treatment 78 neurons.6.Comparison of striatal neurons discharge:LID group was the highest,rapamycin after R1,the discharge frequency of R2 were dyskinesia group decreased,there was statistical significance with group and intervention group differences in discharge frequency(P<0.05).7.Statistical analysis of neuron burst discharge,:LID group is the highest,rapamycin intervention burst release decreased,R1 group than in LID group burst release was statistically significant(P<0.05);R2 group than in LID group burst release decreased,the difference was statistically significant(P<0.05);compared with R1 group R2 burst issued no obvious changes,no difference statistical significance(P>0.05).8.Spectrograms analysis of LFP in different treatment groups,below 10Hz frequency section,the lightest color in PD group(It has the lowest energy),the most vivid color in LID group(It has the hightest energy),Compar with the energy of LID group R1 group which after rapamycin treatment 1 w has been declined,Compar with the energy of LID group R2 group which after rapamycin treatment 2 w has been declined,the energy has no significantly between R1 group and R2 group;between 10Hz-20Hz frequency section,the lightest color in PD group,the energy of LID group significantly higher than PD group,the energy of R1 group(after rapamycin treatment 1w)lower than LID group,the energy of R2 group(after rapamycin treatment 2w)lower than LID group,between 10Hz-20Hz frequency section R1group and R2 group had no obvious change in each energy;between 20Hz-30Hz frequency section R1 group and N group had the lowest energy,LID group had the lowest color and the highest energy,The energy distribution can be visualized directly by the time-frequency diagram.Conclusions:1.mTOR signaling pathway is involved in the pathogenesis of LID,Its mechanism is to regulate the expression of SAP-97 and the phosphorylation of AMPAR isoforms make synaptic plasticity changesand ultimately generate and maintain cortical synapses "pathological LTP",and thus mediates occur of LID.2.The firing frequency,burst release and LFPs of neurons in the striatum of mTOR rats were changed in the activation and inhibition of the signal pathway,this indicates that there is a correlation between mTOR signaling pathway and LID,from the point of view of electrophysiology,expounds the mechanism of mTOR signaling pathway in a rat model of levodopa induced dyskinesias.