Selective Cholinergic Depletion Of Pedunculopontine Tegmental Nucleus Aggravates Freezing Of Gait In Parkinsonian Rats

BACKGROUND:When PD progresses to advanced stage,however,about half of the patients experience axial symptoms,such as severe gait and postural impairments(more specifically,freezing of gait and falls)that are not ameliorated by levodopa or STN-DBS.In recent years,there is growing evidence that the pedunculopontine nucleus(PPN)plays a vital role in the occurrence of axial symptoms that can be alleviated by PPN-DBS.Research on the functions of the PPN and the dysfunction of it in PD will contribute to the understanding of the pathological substrates of axial symptoms and the mechanism of PPN-DBS.Consequently,it is reasonable to assume that the cholinergic degeneration may be more relevant to intractable axial symptoms than dopaminergic degeneration.In the present study,we further investigate the role of the PPN cholinergic cells on motor deficits,especially the axial motor impairments.MethodsForty-two adult male Sprague-Dawley rats were used in this study,Rats were randomly assigned to one of four following groups:(1)sham lesion with saline vehicle;(2)unilateral SNc dopaminergic lesion with 6-hydroxydopamine(6-OHDA);(3)unilateral PPTg cholinergic lesion with anti-ChAT-SAP;and(4)unilateral SNc dopaminergic lesion combined with PPTg cholinergic lesion.The anti-ChAT-SAP is a tool for eliminating cells that express choline acetyltransferase(ChAT)in multiple species;targeted via the affinity-purified rabbit polyclonal antibody to ChAT,eliminated via saporin(the ribosome-inactivating protein from the seeds of the plant,Saponaria officinalis).Unilateral SNc dopaminergic lesion were achieved via injection of 4 il of 6-OHDA into the right medial forebrain bundle(MFB).For unilateral PPTg cholinergic lesion,the IgG immunotoxin-targeting choline acetyltransferase(anti-ChAT-SAP)was infused into the right PPTg.Sham lesion was obtained by injecting the same dose of saline into the right MFB and PPTg.Gait testing was conducted on the CatWalk XT(Noldus information Technology,Wageningen,Netherlands)that was used in our previous studies to analyze gait of unforced moving rats.During behavioral re-evaluations after lesion,rats showed moderate to severe gait freezing(start hesitation)during testing except the sham lesion group.leading to the disability of finishing the required three runs per testing session.The number of unaccomplished sessions(less than three runs)resulted from gait freezing(start hesitation)were counted as the number of FOG,and the percentage of FOG in the total number of testing sessions was calculated for each group.After the last behavior test,Immunohistochemical staining for tyrosine hydroxylase(TH)in the SNc and was performed as previously described.The same procedures were taken for the immunohistochemical staining of ChAT in the PPTg.Images were obtained with the Leica Application Suite.Assessment of the number of TH+ neurons in the SNc or ChAT+ neurons in the PPTg was made according to the optical fractionator principle.Every 6th section covering the entire extent of the SNc or PPTg was included in the counting procedure.The data are expressed as a percentage of the corresponding area from the intact side.ResultsRats in the SNc lesion group showed a mean loss of 90.19%of DA neurons.There was no group difference between SNc lesion and combined lesion rats.Cholinergic cell loss in the PPTg lesion group reached 77.62%,in the combined lesion group reached 81.75%,which both reduced significantly compared with sham lesion rats.No significant difference of cholinergic cell loss was found between PPTg lesion and combined lesion group.The CatWalk system captures both dynamic and static gait parameters.After lesion,run duration,stand and step cycle significantly increased while stride length and swing speed significantly decreased in SNc lesion and combined lesion rats compared with sham lesion and PPTg leison rats.No significant difference was found between SNc lesion and combined lesion rats.Max contact area and mean intensity only presented interactions in the left side paws(contralateral to the brain lesion).Compared with sham lesion and PPTg leison group,these two gait parameters dramatically decreased in the left front and hind paws of SNc lesion and combined lesion group.There was no group difference between SNc lesion rats and combined lesion rats.The parameter of base of support(BOS)for hind limbs increased substantially in the SNc leison and combined lesion group,while increased faintly for fore limbs in these two groups compared with sham and PPTg lesion groups.The terminal dual stance of all paws significantly increased in SNc dopaminergic lesion and combined lesion rats.In addition,the left side in combined lesion group increased higher than SNc lesion group.The SNc and PPTg combined lesion group showed the most severe freezing among four groups(57.58%,p<0.05).To some degree,independent lesion of SNc dopaminergic neurons also led to FOG compared with sham lesion rats(26.66%,p<0.05).There was no significant difference between sham lesion and PPTg lesion rats(0.00%vs.13.88%).In the PPTg lesion group.In the combined lesion group,the deficit in the terminal dual stance for the left limbs only correlated with cholinergic survival(partial correlation coefficient:left forelimb:-0.77,p = 0.04;left hindlimb:-0.79,p = 0.02),but no other parameters showed significant correlation.conclusionThis study compares the gait dysfunction in three different types of lesion of parkinsonian related rats.The results indicate that rats with independent loss of cholinergic neurons in the PPTg did not induce gait disturbance.However,6-OHDA lesion combined with PPTg cholinergic lesion aggravates the FOG in parkinsonian rats.Our data suggest that the PPN cholinergic neurons play a vital role in the occurrence of FOG in PD.Future studies can move forward to investigate the relationship of PPN cholinergic neurons with other related structures of PD and the involvement of these neurons in the treatment of PPN-DBS.