Dissecting functional subgroups and closed-loop circuits between the pedunculopontine nucleus and the basal ganglia

摘要 桥脑脚核(PPN)与基底节相互作用以实现平稳运动，是一种 帕金森氏病(PD)深部脑刺激(DBS)的靶点。帕金森病是最常见的 神经退行性运动障碍，以静止性震颤、僵硬、缓慢运动和姿势为特征 不稳定。尾部靶向DBS刺激改善患者和啮齿动物帕金森病模型的步态损害 而在啮齿动物帕金森病模型中，以鸟嘴为靶点的DBS使其恶化。有效的以PPN为目标的DBS可以归因于 与闭合环路引起的PPN尾侧α振荡增加和神经元增多有关 跨PPN的活动。PPN靶向DBS在患者和啮齿动物模型中的相互矛盾的结果 提示PPN内的功能异质性以及其吻侧和尾侧亚区之间的差异。至 了解DBS如何以不同的方式调节PPN内的运动，解剖功能性 PPN内的亚群，并确定它们与基底节的通信方式。在这项建议中，我 帕金森病选择性退行性变与步态相关的胆碱能PPN神经元 减损。我将使用体外全细胞膜片钳电生理学、光遗传学、钙成像和 追溯微珠标记(1)以表征固有电生理特性并鉴定分子 区分成年小鼠头端和尾端胆碱能PPN神经元的标记，(2)鉴定和 全面映射抑制性突触输入到PPN的区域连接性，以及(3)识别 抑制性基底节核和PPN之间的闭合环路。我们的初步数据显示， 两个主要的抑制性基底节核团，黑质网状部和苍白球 在轴突成像中，外侧核(GPE)不同地投射到PPN的吻侧和尾侧。将光遗传学用于 全细胞膜片钳和钙成像，我们可以确认这些解剖的功能连通性 轴突投射和表征来自SNR和GPE的PPN上的抑制电流。vbl.使用 免疫染色，我将测试在PPN中选择性表达的五种蛋白质的差异表达 与其他大脑区域进行比较，以确定可用作遗传访问工具的分子标记 吻侧和尾侧PPN。利用光遗传学和逆行珠标记法，我将确定SNR-和 GPE投射的PPN神经元也从各自的基底节核接受抑制输入，形成 闭环系统。这项提议将全面描述和绘制区域互联互通的特征和地图 SNR和GPE到胆碱能PPN并识别闭合环路，可以帮助我们了解PPN是如何 与基底节相互作用以调节运动，以及它的变性如何导致运动障碍 警察。这些发现将指导新的药理靶点和DBS靶向治疗帕金森病的PPN回路 病人。

Abstract The pedunculopontine nucleus (PPN) interacts with the basal ganglia to enable smooth movement and is a target for deep brain stimulation (DBS) in Parkinson's disease (PD). PD is the most common neurodegenerative motor disorder, characterized by resting tremor, rigidity, slow movement, and postural instability. Caudal-targeted DBS stimulation improves gait impairment in both patients and rodent PD models whereas rostral-targeted DBS worsens it in rodent PD models. Effective PPN-targeted DBS may be attributed to increasing alpha oscillations in the caudal PPN generated by closed-loop circuits and increasing neuronal activity across the PPN. Contradictory outcomes of PPN-targeted DBS in both patients and rodent models suggest functional heterogeneity within the PPN and differences between its rostral and caudal subregions. To understand how DBS differentially modulates movement within the PPN, it is critical to dissect the functional subpopulations within the PPN and determine how they communicate with the basal ganglia. In this proposal, I focus on the cholinergic PPN neurons whose selective degeneration in PD also correlates with gait impairment. I will use ex vivo whole-cell patch clamp electrophysiology, optogenetics, calcium imaging, and retro bead labeling (1) to characterize the intrinsic electrophysiological properties and identify a molecular marker that differentiates rostral and caudal cholinergic PPN neurons in adult mice, (2) to characterize and comprehensively map the regional connectivity of inhibitory synaptic inputs to the PPN, and (3) to identify closed-loop circuits between the inhibitory basal ganglia nuclei and the PPN. Our preliminary data show that the two major inhibitory basal ganglia nuclei, the substantia nigra pars reticulata (SNr) and globus pallidus externus (GPe), differentially project to the rostral and caudal PPN in axon imaging. Using optogenetics with whole cell patch clamp and calcium imaging, we can confirm the functional connectivity of these anatomical axonal projections and characterize inhibitory currents from the SNr and GPe on the PPN. Using immunostaining, I will test the differential expression of five proteins selectively expressed in the PPN compared to other brain regions to identify a molecular marker that can be used as a tool to genetically access the rostral and caudal PPN. Using optogenetics and retrobead-labeling, I will determine whether SNr- and GPe-projecting PPN neurons also receive inhibitory input from the respective basal ganglia nuclei, forming a closed-loop circuit. This proposal will comprehensively characterize and map the regional connectivity of the SNr and GPe to the cholinergic PPN and identify closed-loop circuits that can help us understand how the PPN interacts with the basal ganglia to modulate movement and how its degeneration underlies motor deficits in PD. These findings will guide new pharmacological targets and DBS-targeting of the PPN circuit in PD patients.