Movement Behavioral Effect Of Optogenetic Neuromodulation In The Pedunculopontine Nucleus Of The Parkinsonian Rat Model

Background:In recent years,there is growing evidence that deep brain stimulation the pedunculopontine nucleus can alleviate the motor symptom caused by advanced stages of Parkinson's disease.However,it is not clear whether the mechanisms underlying the therapeutic effects of deep brain stimulation can actives or inhibits target neurons,or other effects.Recently a new technology,optogenetic technology,can manipulate the activity of specific neurons with light in milliseconds.Therefore,in this study,we further explored the mechanism of PPN low frequency stimulation on the improvement of gait deficits with optogenetic technology,especially the locomotor activity.Methods:In this study,fifty-five adult male Sprague-Dawley rats with a weight of about 310g were used.In the light activation experiment,rats were randomly divided into three groups:(1)Unilateral medial forebrain bundle(MFB)sham lesion with physiological saline injection group;(2)Unilateral medial forebrain bundle(MFB)with 6-hydroxydopamine injection group;(3)Unilateral medial forebrain bundle(MFB)with 6-hydroxydopamine injection combined with PPTg optic fiber implantation.The SD rats in the light inhibition experiment were also randomly assigned to one of three following groups:(1)Unilateral medial forebrain bundle(MFB)sham lesion with physiological saline injection group;(2)Unilateral medial forebrain bundle(MFB)with 6-hydroxydopamine injection group;(3)Unilateral medial forebrain bundle(MFB)with 6-hydroxydopamine injection combined with PPTg optic fiber implantation.In the light activated study of all rats were received injections of 0.5 uL hSynapsin-CHR2-mCherry AAV targeted at the ipsilateral PPN;In the light inactive experiment of all rats were received injections of 0.5 uL hSynapsin-NpHR-mCherry AAV targeted at the ipsilateral PPN.AAV-hSynapsin-NpHR-mCherry and AAV-hSynapsin-CHR2-mCherry were two common types of adeno-associated viruses in optogenetics experiments.HSynapsin is a specific promoter of neuron.CHR2 is a non-selective cationic channel protein,and the expression of CHR2 in the neuron can induce the neuron to depolarise immediately and induce the action potential.NpHR is a kind of chloride ion pump,which with yellow light would inhibit action potential occurrence.We used the morphine rotation test,Catwalk and the open field test to assess the effect of light stimulation on the motor function of Parkinson's rats.After all the behavioral tests were completed,Substantial unilateral losses of TH-positive cells in the SNC was performed by the chemical staining of tyrosine hydroxylase(TH)immunohistochemistry.Serial coronal sections of the PPN were washed in 0.5%bovine serum albumin in PBS and stained with DAPI.Image were acquired with the Lecia Application Suite.Result:Three weeks after channelrhodopsin and halorhodopsins injection,open field test showed that rats with 6-OHDA lesions were showed a significance reduction in their locomotor activity compared with sham lesion group(p<0.05).According to the results of PD group,different viruses had no effect on the locomotor activity of rats(p>0.05).However,the 6-OHDA lesion rats with blue light stimulation(20Hz,5ms pulse width)showed significant improvement in movement distance compared with no light stimulation group(p<0.05).the 6-OHDA lesion rats with yellow light stimulation(20Hz,5ms pulse width)showed significant aggravation in movement distance compared with no light stimulation group(p<0.05).In rotation test,all rats in 6-OHDA lesion group showed an ipsilateral rotation after apomorphine injection.In optogenetic activation test,there was no significant change induced by blue light stimulation in these animals(P>0.05,n = 6 rats,t test with ?= 0).In the light inhibition experiment,the mean rotations of rats were also not affect by PPN inhibition((P>0.05,n = 6 rats,t test with ?= 0).Conclusion:The current data showed that the neurons in the PPTg with blue light could moderately increase the movement distance of PD rats and improve the gait deficts of PD rats.While the neurons in the PPTg with yellow light could significantly shorten the movement distance of PD rats.According to our results,it is reasonable to speculate that the mechanism of PPN-DBS is related to the activation of the neurons in PPTg.Future studies can move forward to investigate the relationship of PPN neurons with other related structures of PD and the neuronal activity of these related neurons with optical modulation.