The Mechanism Of Subthalamic Nucleus-Anterior Thalamic Nucleus Plasticity In Motor Deficits Of Parkinson’s Disease Rodents Model

Parkinson’s disease is a neurodegenerative disease that characterized as dopaminergic neurons loss in substantia nigra pars compacta,which lead to aberrant activity in basal ganglia.It has been reported that aberrant activity in basal ganglia is associated with motor deficits in PD.Manipulating this circuit by deep brain stimulation(DBS)is an effective method to treat motor symptoms of both PD patients and nonhuman primates(NHPs).STN is a main DBS targets to treat PD.What’s more,GPi,a downstream nucleus of STN,is another DBS target to treat PD.The two targets showed some differences in treat effects and side effects.These observations suggest that other circuit,in addition to STN-GPi circuit,also plays a role in motor control.What’s more,it has been reported that long-term nucleus activity increase leads to plasticity changes.It’s unclear whether plasticity changes are involved in PD motor deficits.Therefore,we design the following experiments to investigate the questions.ObjectiveInvestigating whether there exist other downstream nuclei of STN participating in PD motor deficits except STN-GPi pathway,and whether plasticity changes between STN and the newly identified nuclei contribute to PD motor deficits.Methods1.We used virus tracing methods to verify the anatomical connection between STN and new identified downstream nucleus,and functional connection was verified by optogenetics coupling with electrophysiology method.2.We established hemi-parkinsonian rodent model by 6-OHDA injection.ANT activity was assessed by c-fos staining and in vivo recordings in PD model rodents.3.What’s more,in terms of verifying the role of ANT in PD model mice,we manipulated ANT activity or STN-ANT circuit by pharmacological and optogenetics methods and accessed motor deficits by behavioral tests such as balance beam test and apomorphine induced rotation test.4.STN-ANT circuit plasticity was measured by ratio of AMPAR and NMDAR evoked currents in vitro whole-cell recordings.The change of AMPA-Glu R1phosphorylation site was observed by Western blot,and the role of STN-ANT plasticity on motor dysfunction in PD mice was verified by accessing motor deficits after blocking PKA or S845 phosphorylation site.Results1.STN-ANT circuit is a newly identified pathwayWe mapped STN-projected nuclei with a series of viral tracing studies.EYFP-positive fibers were evident in ANT after anterograde tracing virus injected in mouse STN,which suggested ANT was a new downstream nucleus of STN.To further examine this previously unidentified STN-ANT circuit,we next used the Cre recombinase-dependent viral expression method.EYFP-positive cells were observed in ANT with Cre recombinase expressed.Furthermore,retrograde viral tracing was used to further verify the STN-ANT connection.On the other hand,we examined synaptic connection in STN-ANT circuit by Ch R2-assisted in vitro whole cell recordings.We observed EPSCs in the presence of TTX and 4-AP,which were Na~+blocker and K~+blocker,respectively.2.Increased activity of ipsilateral ANT in PD model rodentWe established hemi-parkinsonian rodent models by injecting 6-OHDA,and then examined expression level of c-fos 1.5 h after apomorphine intraperitoneal injection.A clearly enhanced expression of c-fos in ipsilateral,but not in contralateral,ANT in hemi-parkinsonian model mice was found.In contrast,a similar expression of c-fos in bilateral ANT in normal mice was observed.We then tested whether this increased activity was caused by the hyperactive STN in PD model mice.Ablating ipsilateral STN neurons by ibotenic acid(IBO)markedly decreased c-fos expression in ipsilateral ANT neurons.In contrast,the enhanced c-fos expression in ipsilateral ANT neurons was not affected when vehicle was used in the ipsilateral STN.Furthermore,to characterize this increased activity in vivo,we did multi-channel electrophysiological recordings in freely behaving rats.The firing rate in the ipsilateral ANT neurons was greatly increased than that in the contralateral ANT of PD model rats(Con:7.96(8.14)Hz,Ips:22.94(47.2)Hz).What’s more,we found a distinct band of LFP activity in the beta range(15-35 Hz)in ipsilateral ANT of PD model rats The total power of LFP between 15-35Hz was markedly increased in PD model rats.3.Suppressing STN-ANT activity corrected motor deficits in PD model miceAfter ablation of ipsilateral ANT neurons by IBO,the time of passing the beam(AA:4.13±0.23 s;IBO:3.36±0.16 s)and the number of apomorphine induced rotations(AA:6.43±0.76;IBO:0.52±0.52)were greatly shorted or decreased in PD model mice.Furthermore,optical fiber and virus expressing e Np HR were implanted and injected into ipsilateral ANT.We inhibited ANT activity by yellow light stimulation and found that motor deficits was alleviated in PD model mice.Then we silenced STN-ANT circuit by optogenetics method and found that the time of passing the beam(laser off:4.36±0.49 s;laser on:3.72±0.39 s)and the number of apomorphine induced rotations(laser off:10.67±2.09;laser on:5.88±1.11)were also markedly reduced compared with no optical stimulation.4.STN-ANT synaptic plasticity is crucial for PD motor deficitsWe recorded the evoked AMPAR-EPSCs and NMDAR-EPSCs in ANT neurons and found that AMPAR/NMDAR currents ratio was greatly increased in ipsilateral STN-ANT circuit of PD model mice compared with that in normal mice.Furthermore,the rectification index of AMPARs,reflecting the Glu R2-lacking AMPARs on the membrane,in the ipsilateral STN-ANT circuit of PD model mice was markedly increased compared with that in normal mice.The results supported that augmentation of postsynaptic transmission in ANT neurons was mediated by the increased membrane expression of Glu R1.We used phosphorylation site-specific antibodies and found that Glu R1-S845 was greatly increased in ipsilateral ANT of PD model mice.Application of H-89 in the ipsilateral ANT slice also recovered ratio of AMPAR/NMDAR current(a CSF:3.85±0.90;H-89:1.57±0.17)and corrected PD motor deficits(Balance beam test:a CSF:4.31±0.26 s;H-89:3.67±0.18 s.Apomorphine induced rotation test:a CSF:10.86±1.04;H-89:7.93±0.93).To specifically prevent the phosphorylation of Glu R1-S845,a TAT-S845 was designed to generate a cell-permeable peptide that contained a sequence spanning the PKA phosphorylation site in Glu R1.The peptide recovered the ratio of AMPAR/NMDAR current in PD model mice,as well as correcting PD motor deficits.ConclusionsOur study was the first to identify the crucial role of STN-ANT plasticity in motor deficits of PD model rodents.We found that(1)ANT was an newly identified downstream nucleus of STN;(2)suppressing STN-ANT pathway significantly improved motor deficits of PD model rodents,which was related to increased synaptic strength in STN-ANT circuit mediated by aberrant postsynaptic AMPA glutamate receptor Glu R-S845 phosphorylation.This work expanded the current view of the motor control loop in the basal ganglia and provided a novel perspective to understand mechanisms of PD motor deficits after progressive loss of dopamine.At the same time,it provided a new target for strengthening military combat capabilities in the face of harsh environment and heavy work stress.