Interrogating Central Circuits with Laser Ablation: Studies in the mammalian res

描述(申请人提供)：这项R21发展研究助学金利用多光子激光技术在体外对工作的神经元网络进行成像，然后测试组成神经元如何通过特定细胞的激光消融对网络功能做出贡献。具体地说，已经开发出一种计算机控制的系统，可以使用多光子荧光显微镜检测神经元，将这些细胞的位置存储在内存中，然后依次对目标神经元进行激光消融，同时监测神经回路的电生理功能。长波长脉冲激光的使用提供了前所未有的三维组织病变的特异性和可控性。这一重大的技术发展为询问网络行为的细胞基础以及网络功能的病理生理崩溃提供了强大的新工具。这个项目的重点是呼吸行为的神经生成和控制。特别是，这些研究探索了位于人类和所有哺乳动物脑干下部的专门的吸气节律产生部位的电路特性，该部位被称为preBotzinger复合体(PreBotC)。特异性AIM 1将评估前BotC的细胞组成。转基因小鼠模型将被用来将荧光标记应用于前BotC中遗传上不同的神经元亚群，然后选择性和连续地损伤它们，以测试它们在心律失常发生中各自的作用。来自同源结构域转录因子Dbx1的神经元组成前BotC的基本节律生成核的假设将得到专门的测试。特定的AIM 2将评估当节律性活跃的前BotC神经元被顺序删除时，呼吸功能是否以及如何恶化。在这里，目标神经元的选择将基于吸气节律活动，而不是遗传来源。这组实验将作为一个一般的疾病模型，以检查具有中心病因的呼吸道病理背后的细胞机制。总而言之，这个R21项目将提供有关神经生成和呼吸控制的重要新信息。这一新知识对人类的健康和健康非常重要，因为呼吸是一种坚持不懈的、不可或缺的人类行为，它维持着动态平衡和生命本身。在接下来的项目中，这项新技术--用于在体外检测并消融累积序列中的神经元--将被应用于询问运动和咀嚼节奏产生网络，这些网络也可以在体外的脊髓和后脑标本中进行研究。事实上，这种损伤系统将广泛适用于研究任何大脑区域的体外网络。 与公共健康相关：呼吸是一种至关重要的人类行为，对维持动态平衡和生命本身至关重要。这个项目将促进我们对产生和控制呼吸节奏的脑干神经回路的细胞组成的理解，并研究当产生呼吸节奏的神经元逐渐死亡时，呼吸功能是如何崩溃的。所获得的新知识将作为治疗和预防具有中枢神经病因学的呼吸疾病的基础，并阐明奠定节律性运动行为基础的电路水平的特性。

DESCRIPTION (provided by applicant): This R21 Developmental Research Grant harnesses multi-photon laser technology to image a working neuronal network in vitro, and then test how constituent neurons contribute to network function via cell-specific laser ablation. Specifically, a computer-controlled system has been developed to detect neurons using multi-photon fluorescence microscopy, store the locations of these cells in memory, and then laser-ablate the target neurons one at a time, in sequence, while monitoring the function of the neural circuit electrophysiologically. Use of a long- wavelength pulsed laser provides unprecedented specificity and control of the lesion in three- dimensional tissue. This significant technological development provides a powerful new tool for interrogating the cellular bases for network behaviors, as well as pathophysiological breakdown in network function. This project focuses on the neural generation and control of breathing behavior. In particular, the investigations probe the circuit properties of the specialized inspiratory rhythm-generating site called the preBotzinger Complex (preBotC) located in the lower brainstem of humans and all mammals. SPECIFIC AIM 1 will evaluate the cellular composition of the preBotC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct subpopulations of neurons in the preBotC, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. The hypothesis that neurons derived from the homeodomain transcription factor Dbx1 comprise the essential rhythm- generating kernel of the preBotC will be specifically tested. SPECIFIC AIM 2 will evaluate whether and how respiratory function deteriorates when rhythmically active preBotC neurons are sequentially deleted. Here the target neurons will be selected on the basis of inspiratory rhythmic activity, rather than genetic origin. This set of experiments will serve as a general disease model to examine the cellular mechanisms underlying respiratory pathologies that have a central etiology. In summary, this R21 project will provide significant new information regarding the neural generation and control of breathing. This new knowledge is important for human health and wellness given that breathing is a relentless and indispensable human behavior that maintains homeostasis and life itself. In subsequent projects this new technique - for detecting and then ablating neurons in a cumulative sequence in vitro - will be applied to interrogate locomotor and masticatory rhythm-generating networks that can be also be studied in spinal cord and hindbrain preparations in vitro. Indeed, this lesioning system will be broadly applicable to studying networks in vitro from any brain region. PUBLIC HEALTH RELEVANCE: Breathing is a vital human behavior that is essential to maintain homeostasis and life itself. This project will advance our understanding of the cellular composition of brainstem neural circuits that generate and control breathing rhythms, and examine how respiratory function breaks down as respiratory rhythm-generating neurons progressively die. The new knowledge obtained will serve as a foundation for the treatment and prevention of respiratory disorders with a central neural etiology, and elucidate circuit-level properties that underlie rhythmic motor behaviors in general.