ANALYSIS OF MOTOR PATTERN SWITCHING BY DOPAMINE

描述（由申请人提供）：帕金森病中最令人衰弱的问题是阻断在不同运动模式之间切换的能力，包括运动所需的那些。这是由于黑质中多巴胺神经元的变性导致多巴胺能信号传导的丧失而引起的。这项研究的长期目标是研究多巴胺能功能的变化如何促进运动模式转换。我们最近已经证明，强大的遗传模型秀丽隐杆线虫类似于人类，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。具体来说，C。秀丽线虫在干燥的环境中爬行，但悬浮在水中时游泳。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对于从游泳到爬行的转变是必要的，也是足够的。我们还发现，多巴胺神经元的缺失导致了C区运动模式转换时的不动。elegans-与帕金森病患者惊人的相似。在人类和C. elegans建立了这种模式生物作为一个有吸引力的系统，在其中确定这些转换困难的神经分子基础。此外，还存在一个基本完整的C。线虫的神经系统，加上它正好包含八个多巴胺能神经元的事实，意味着我们可以以前所未有的细节来研究多巴胺信号。这项研究提出了两个核心问题：首先，多巴胺信号如何促进转换到适当的运动程序，其次，当多巴胺信号被破坏时，运动程序的转换如何变得功能失调？这两个问题在三个具体目标中得到解决，这些目标利用了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业知识。体内秀丽隐杆线虫神经元：（1）我们将通过细胞消融和通过光激活离子通道激活和抑制神经元来确定哪些神经元在爬行和游泳之间的转换中起重要作用。(2)我们将确定这些神经元在完整动物中的作用，因为它们在具有功能性钙成像的微流体室中在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如何影响这些神经元的膜电流和活动。从这些研究中发现的原理有可能提高对多巴胺如何用于人类运动模式之间切换以及运动模式启动和切换如何在帕金森病中变得功能障碍的理解。 公共卫生相关性：启动运动（例如坐起来和吞咽）和在运动模式之间切换（例如行走和伸手）的能力对于日常生活至关重要。这些行为在帕金森病中严重受损。在美国，帕金森病的估计费用每年超过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，在一个C。elegans帕金森病模型。我们的建议旨在利用C的分子操纵的能力和容易性。elegans来解决保守的运动模式转换的多巴胺能机制，以及消除多巴胺神经元后如何干扰转换。

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.