Study Of Discharge Changes And Neural Pathway Between M1 And STN In Hemiparkinsonian Rats

Parkinson’s disease(PD)is a neurodegenerative disease.Its pathogenesis is mainly the degeneration and loss of dopaminergic(dopamine,DA)neurons in substantia nigra of the midbrain,which in turn triggers the dysfunction of related nuclei of basal ganglia(BG),and then causes a series of symptoms of PD.The symptoms are characterized by resting tremor,rigidity and bradykinesia.Clinical treatment of PD is mainly drug treatment with levodopa,but long-term use will cause many side effects.At present,deep brain stimulation(DBS)targeting the subthalamic nucleus(STN)is widely used to improve the symptoms of PD.At the same time,STN is an important relay station in the cortical-basal ganglia-thalmocortical circuit,and it modulate the information fow in both direct and indirect pathways.And it has extensive connections with the BG and the primary motor cortex(M1).M1 is also the main motor output nucleus in the cortico-basal ganglia network,which plays a vital role in motor function.There is a hyper-direct pathway between M1 and STN,which makes it easier to complete the overall motion adjustment control with high efficiency.From the perspective of neurophysiology,the essence of brain function is realized by electrical signal conduction.In PD state,DA neurons in substantia nigra of midbrain are degenerated and lost,which leads to abnormal neuroelectrical activity in the brain,which disrupts normal physiological electrical activity and thus produces a series of related clinical symptoms.Among them,the abnormal electrical activity of STN,as the target of DBS stimulation,plays an important role.The spike frequency,discharge pattern,local field potential(LFP)oscillation activity of STN and the change of internal network structure are closely related to the occurrence of PD symptoms.It has been proved that the clinical symptoms of PD can be relieved by stimulating STN and its pathway.And the decrease and increase of STN burst discharge will lead to the decrease and increase of movement disorder respectively.However,the abnormal discharge of STN is not autonomous,which may be triggered by the input from M1,because the increase of M1 activity and the enhancement of M1-STN input in PD will lead to excessive abnormal discharge in STN.In addition,M1 can not only drive STN to generate abnormal discharge,but also be driven by STN discharge with specific frequency LFP,thus generating abnormal motion.Although many previous studies have explored how STN and M1 control the start and maintenance of motion in PD,the changes of electrical signal activity under specific motion conditions are still unclear,and the anatomical connectivity of the hyper-direct pathway remains to be clarified.People have done a lot of pathophysiological research on STN and M1 in PD,but the mechanism of abnormal discharge and LFP frequency change between STN,M1 and M1-STN and the relationship between them in regulating information transmission in cortical circuits is still unclear.For this purpose,in this study,firstly,we recorded in the STN and M1 of the PD model brain through the microelectrode array implanted in the brain by in vivo multi-channel recording technology,and analyzed the spike and LFP of the PD model brain neurons and their corresponding behavioral changes in the awake state by offline sorting neuroexplorer,MATLAB and other software.Further,from the anatomical perspective,analyzing the anatomical basis of electrophysiological and conduction pathway changes of STN and M1 in PD model brain may better understand the mechanism of brain functioning in physiological or pathological state,and thus propose solutions for the treatment of PD.In the Subsequent experiments,anterograde adeno-associated virus and retrograde AAV2/retro-GFP neural tracers were injected into M1 and other related brain nuclei through brain stereotaxic location,and the transfected neurons in each nucleus and the neuronal projection pathways among them were observed by immunofluorescence technique.Explore and reveal the characteristics of abnormal electrical activity of STN and M1 in PD-related basal ganglia-thalamus-cortex circuits from different levels of neurophysiology,anatomy and function,and clarify the key roles of STN and M1 in PD-related circuits.It provides a theoretical basis for the occurrence mechanism of PD.Methods:(1)The physiological signals of extracellular discharge collected by microelectrodes implanted into target nuclei in vivo are used to analyze and compare the characteristics of Electroencephalogram activity in PD rats under specific behaviors in real time,which has a higher accuracy advantage in time and space to reflect the correlation between brain dysfunction and corresponding motor abnormalities.Firstly,a stable PD rat model was established by injecting 6-hydroxydopamine into unilateral medial forebrain bundle.Two arrays of self-made16-channel metal electrodes were implanted into the M1 and STN of the same rat.And we simultaneously recorded the spikes and LFPs of STN and M1,during the rest and the treadmill movement.Finally,we used offline Sorter,neuroexplorer and matlab to analyze the electrophysiological signals to study the effects of dopamine lesion on extracellular discharge frequency,discharge mode,local field potential oscillation activity of STN and M1 and electrophysiological activity of internal structure of M1-STN pathway.(2)Virus-injection experiment: The significance of neural tracers(which can be anterograde,from the cell body to the axon;or retrograde,from the axon to the cell body)is to explain the anatomical connections of the nervous system through tracing.Thus providing a tissue anatomical basis for the architecture of neural pathways.Adeno-associated virus has the advantages of high security,nonsynaptic,strong diffusion ability and so on.it is a common viral tool for neural circuit tracing.Firstly,the glass microtube injection system was established,and then the virus tracer was injected into different normal rats by using AAV vector stereotactic injection technology: the anterograde virus tracer AAV2/9-GFP was injected into unilateral M1region;Retrovirus tracer AAV2/Retro-GFP was injected into unilateral STN area;Retrovirus tracer AAV2/retro-GFP was injected into unilateral M1 region.Subsequently,virus tracers were injected into different PD rats: AAV2/9-GFP was injected into M1 area of the injured side;AAV2/retro-GFP was injected into the STN area of the injured side.Finally,qualitative and quantitative methods were used to analyze and observe the expression of the tracer virus in the two nuclei and its projection sites,so as to explore the relationship between the anatomical connection pathways of the normal M1 and STN,as well as the changes of the connection pathways of M1 and STN in PD.This combination method of spatial and temporal specific electrophysiological techniques and anatomical techniques was implemented in the same animal brain,It helps to better understand how structural changes affect function and analyze possible roles in the pathogenesis of PD.Results:(1)Our results showed the firing rate and firing pattern of STN were all altered by dopamine lesion during rest and treadmill movement.However,only the firing rate and firing pattern of putative pyramidal neurons(BS neurons)in M1 were altered by dopamine lesion during the two states,whereas interneurons(NS neurons)were not.(2)Dopamine lesion induced significant effects on the LFP power of STN and M1 during rest and treadmill movement.The power of lesioned rats was decreased in the low-frequency(0.7-12 Hz),but the power was increased in the high-frequency(12-35 Hz),as compared with the control rats during the two states.(3)Dopamine lesion altered the LFP-LFP and spike-LFP relationships between STN and M1 during rest and treadmill movement.(4)There is a bidirectional projection relationship between M1 and STN.AAV-injection can inhibit the exercise behavior of rats.(5)Our study showed that dopamine lesion impairs anatomical connectivity of the M1-STN hyper-direct pathway.Conclusion and significance:Dopamine lesion induced changes in M1-STN hyper-direct pathway,including electrophysiological activities and neural pathway connectivity.The firing rate and firing pattern of neurons in STN and M1 changed in 6-OHDA lesioned rats.Dopamine lesion caused the change of electrophysiological correlation between STN and M1.In terms of neural pathway connectivity,there was a bidirectional projection relationship between STN and M1.And the AAV-injection can inhibit the exercise behavior of rats.The projection from M1 to STN was significantly reduced in PD state,indicating that 6-OHDA lesion impaired the anatomical connectivity of M1-STN hyper-direct pathway.These results expand our understanding that the interaction between STN and M1 plays an important role in influencing STN activities.It shows that in order to get the best treatment of DBS,we should consider the fine electrophysiological factors at the level of M1 and STN,and the M1-STN hyper-direct pathway is a potential therapeutic target for PD,which deserves further exploration.