ANALYSIS OF MOTOR PATTERN SWITCHING BY DOPAMINE

描述(申请人提供)：帕金森氏症中最令人衰弱的问题阻碍了在不同的运动模式之间切换的能力，包括那些运动所需的模式。这是由于黑质中的多巴胺神经元退化而导致的多巴胺能信号的丧失。这项拟议研究的长期目标是调查多巴胺能功能的变化如何有助于运动模式的转换。我们最近证明了线虫强大的遗传模型与人类相似，因为多巴胺信号是在不同形式的运动行为之间切换的绝对要求。具体地说，线虫在干燥的环境中爬行，但悬浮在水中时会游泳。通过结合行为分析、光遗传学和神经元消融，我们发现多巴胺的释放是从游泳过渡到爬行的必要条件和充分条件。我们还发现，在线虫的运动模式之间切换的时刻，多巴胺神经元的丢失恰恰会导致不动--这与帕金森氏症患者有着惊人的相似之处。人类和线虫中多巴胺信号中断的影响之间的对应关系建立了这种模式生物作为一个有吸引力的系统，在其中识别这些转换困难的神经分子基础。此外，线虫神经系统基本完整的接线图的存在，以及它恰好包含八个多巴胺能神经元的事实，意味着我们可以前所未有的详细地研究多巴胺信号。这项拟议的研究解决了两个核心问题：第一，多巴胺信号如何促进切换到适当的运动程序；第二，当多巴胺信号中断时，运动程序的切换如何变得功能障碍？利用我们在定量行为分析和光遗传学以及电生理学和钙成像方面的独特专长，我们将通过三个具体目标解决这两个问题：(1)通过细胞消融，以及通过激活和抑制具有光活化离子通道的神经元，我们将确定哪些神经元在爬行和游泳之间的切换中起着至关重要的作用。(2)我们将通过功能钙成像来确定这些神经元在完整动物身上的作用，因为它们在微流体室中的爬行和游泳之间切换。(3)我们将通过膜片钳电生理学研究多巴胺对这些神经元的膜电流和活动的影响。从这些研究中发现的原理有可能提高对多巴胺如何被用来在人类的运动模式之间切换，以及帕金森病中运动模式的启动和切换如何变得功能障碍的理解。 与公共健康相关：启动运动(例如，坐起和吞咽)以及在运动模式之间切换(例如，行走和伸手)的能力对日常生活至关重要。这些行为在帕金森氏症中严重受损。在美国，帕金森氏症每年的成本估计超过250亿美元。在这里，我们提出了第一个类似的运动功能障碍的例子，在线虫帕金森氏病模型。我们的建议旨在利用线虫分子操作的能力和简便性来解决运动模式转换的保守多巴胺能机制，以及在消除多巴胺神经元后转换是如何受到干扰的。

DESCRIPTION (provided by applicant): The most debilitating problems in Parkinson's disease block the ability to switch between distinct motor patterns including those required for locomotion. This is caused by loss of dopaminergic signaling due to the degeneration of dopamine neurons in the substantia nigra. The long-term objective of the proposed research is to investigate how changes in dopaminergic function contribute to motor pattern switching. We have recently demonstrated that the powerful genetic model Caenorhabditis elegans resembles humans in that dopamine signaling is an absolute requirement for switching between distinct forms of locomotory behavior. Specifically, C. elegans crawls in a dry environment but swims when suspended in water. By combining behavioral analysis, optogenetics, and neuronal ablation, we have found that dopamine release is both necessary and sufficient to transition from swimming to crawling. We have also found that loss of dopamine neurons results in immobility precisely at the moment of switching between motor patterns in C. elegans - a striking parallel with Parkinson's disease patients. The correspondence between the effects of disruption of dopamine signaling in humans and C. elegans establishes this model organism as an attractive system in which to identify the neuromolecular basis for these switching difficulties. Moreover, the existence of an essentially complete wiring diagram of the C. elegans nervous system together with the fact that it contains exactly eight dopaminergic neurons means that we can study dopamine signaling in unprecedented detail. The proposed research addresses two central questions: First, how does dopamine signaling facilitate a switch to an appropriate motor program, and second, how does switching of motor programs become dysfunctional when dopamine signaling is disrupted? These two questions are addressed in three specific aims that capitalize on our unique expertise in quantitative behavioral analysis and optogenetics as well as electrophysiology and calcium imaging from identified C. elegans neurons in vivo: (1) We will determine which neurons have essential roles in the switch between crawling and swimming with cell ablation and through activation and inhibition of neurons with light-activated ion channels. (2) We will identify the roles of these neurons in intact animals as they switch between crawling and swimming in a microfluidic chamber with functional calcium imaging. (3) We will investigate how dopamine influences the membrane currents and activity of these neurons by performing patch-clamp electrophysiology. The principles uncovered from these studies have the potential to improve understanding of how dopamine is used to switch between motor patterns in humans and how motor pattern initiation and switching becomes dysfunctional in Parkinson's disease. PUBLIC HEALTH RELEVANCE: The ability to initiate movement (e.g. sitting up and swallowing) and switch between motor patterns (e.g. walking and reaching) is essential for everyday life. These behaviors are severely compromised in Parkinson's disease. In the United States, the estimated costs for Parkinson's disease amount to well over $25 billion each year. Here we present the first example of analogous motor dysfunction in a C. elegans model of Parkinson's disease. Our proposal seeks to leverage the power and ease of molecular manipulation of C. elegans to resolve conserved dopaminergic mechanisms for motor pattern switching as well as how switching is perturbed after elimination of dopamine neurons.