基于光遗传学技术的帕金森病运动障碍的丘脑底核神经环路解析研究

Parkinson’s disease (PD) is a common neurodegenerative disease, featured with death of dopaminergic neurons in the substantia nigra pars compacta, dopamine depletion in the basal ganglia, and motor deficits. L-dopa compensation initially relieves motor symptoms, however, its efficacy degrades with the progress of PD. In this situation, deep brain stimulation (DBS) is chosen to improve PD treatment. The subthalamic nucleus (STN) is one of the most effective targets of DBS. Whereas, the therapeutic outcomes of STN DBS keenly depend on precise electrode placement. Several elegant studies demonstrated that glutamatergic innervation from motor cortex mediates STN-DBS effects, but the underlying neural circuit pathways remain mysterious. Accumulating evidence suggests that the STN projects to the internal segment of globus pallidus (GPi), the substantia nigra pars reticulata/compacta (SNr/SNc), and the ventral pallidum (VP). We hypothesize that motor cortex selectively innervates a particular STN subdivision, which in turn projects to distinct downstream nuclei and modulates movement, and modulation of this pathway relieves parkinsonian motor deficits.. We will apply the following strategies to test our hypothesis. We will use retrograde tracing to selectively label mouse STN neurons projecting to the GPi, the SNr, and the VP, and will employ recently invented brain clearing protocol (passive CLARITY technique) to visualize the localization of these neurons. We will combine optogenetics and brain slice patch-clamp recordings to characterize glutamatergic synaptic inputs from motor cortex to STN neurons and from STN to the downstream nuclei (GPi, SNr/SNc, and VP). In in vivo situation, we will perform optogenetic manipulation and calcium imaging (fiber photometry) to analyze the roles of each STN projections in motor function, including locomotion and balancing. Finally, we will examine the damages occurred in motor cortex-STN- GPi/ SNr/ VP in PD mouse models to determine which STN projections can mitigate parkinsonian motor deficits. . The overall goal of this study is to provide topological and projection information of motor-related STN neurons, and to define the STN pathways that 1) effectively regulate motor functions, 2) are damaged in PD, and 3) possess therapeutic potential for parkinsonian motor dysfunctions. These results will provide invaluable information for precise treatment of movement disorders in PD.

深部脑刺激（DBS）丘脑底核(STN)可有效地控制帕金森氏病(PD)患者的运动障碍症状。电极的精确定位是获得理想疗效的前提，但其机制尚待阐明。运动皮层向STN的投射有可能是介导STN-DBS疗效的关键因素，但该投射在STN中的靶位神经元及其神经通路还不清楚。本研究假设运动皮层支配特定区域的STN神经元，后者又通过选择性地调节某些下游核团而影响运动功能，该通路可用于治疗PD运动障碍。我们将整合逆向示踪、透明脑、脑片电生理学、光遗传学和动物行为学等多种技术，研究①STN中不同投射神经元的空间分布及其受运动皮层的选择性支配；② STN中不同投射神经元对运动的调节作用；③ 在PD模型中，运动皮层→STN→下游核团的两级突触联系的损伤，并探讨修复这些损伤对运动障碍(运动速度和平衡能力)的缓解作用。为STN-DBS电极的精确定位和刺激模式的优化提供理论依据。

本项目对丘脑底核(STN)神经元及其纤维投射进行了细致的形态学、电生理学、药理学和光遗传学研究，揭示了不同投射在运动调控中的作用，在帕金森病小鼠模型中的可塑性变化、以及对局部短时和长时电刺激的反应。验证了本研究的假设，STN神经元通过选择性地调节某些下游核团而影响运动功能，也丰富了深部脑刺激（DBS）治疗帕金森病(PD)运动障碍的神经环路机制。. 本项目主要结果有：. ① STN神经元投射至黑质致密区（SNc）、黑质网状区（SNr）、内侧苍白球（GPi）、外侧苍白球和腹侧苍白球，且某些STN神经元既投射至SNr也投射至GPi；. ② STN向SNc多巴胺能神经元的投射促进运动，而向SNr和GPi的投射抑制运动，投射至腹侧苍白球的投射不改变运动；. ③ 在PD模型中，STN-SNc投射因多巴胺神经元的死亡而中断，STN-SNr投射功能受抑制，而STN-GPi投射被增强，抑制后两者均能缓解运动障碍，STN-SNr和STN-GPi投射强度的变化可能参与运动障碍的代偿和病理生理机制；. ④ 在PD模型中，STN-SNr神经元的突触输入不变，STN-GPi神经元的抑制性突触输入受损，因此，短时电刺激引起多数STN-GPi神经元兴奋，长时电刺激则抑制这些神经元，提示STN-GPi神经元可能是DBS缓解PD运动障碍的重要细胞类型；. ⑤ 在PD模型中，STN-GPi投射更易被多巴胺受体激动剂阿朴吗啡所抑制。. 因此，本项目通过多方面的证据提示STN-GPi神经元及其投射可能是DBS和药物治疗PD运动障碍的重要靶点。