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

用激光烧蚀探究中央回路：哺乳动物资源研究

• 批准号: 7876465
• 负责人: Christopher A. Del Negro
• 金额: $ 17.34万
• 依托单位: COLLEGE OF WILLIAM AND MARY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7876465
• 关键词: Ablation; Affect; Behavior; Brain Stem; Brain region; Breathing; Cells; Characteristics; Computers; Development; Disease model; Dorsal; Etiology; Figs - dietary; Fluorescence Microscopy; Foundations; Frequencies; Generations; Genetic; Health; Homeostasis; Human; Image; In Vitro; Investigation; Knowledge; Lasers; Lesion; Life; Location; Mammals; Measurable; Medial; Memory; Microscopy; Monitor; Motor; Motor Neurons; Motor output; Neonatal; Network-based; Neurons; Neurosciences; Output; Pathology; Physiologic pulse; Physiological; Play; Population; Preparation; Prevention; Property; Research Project Grants; Respiration Disorders; Respiratory Failure; Respiratory physiology; Rodent; Role; Site; Slice; Specificity; Spinal Cord; System; Techniques; Technology; Testing; Time; Tissues; Transgenic Mice; Work; base; central pattern generator; hindbrain; homeodomain; insight; mouse model; multi-photon; neural circuit; neurodevelopment; preBotzinger complex; public health relevance; relating to nervous system; research study; respiratory; response; tool; transcription factor

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.

描述（由申请人提供）：这项R21发展研究资助利用多光子激光技术在体外对工作神经元网络进行成像，然后通过细胞特异性激光消融测试组成神经元如何对网络功能做出贡献。具体地说，已经开发了一种计算机控制的系统，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，然后依次一次一个地激光消融目标神经元，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特异性和三维组织中病变的控制。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学故障提供了一个强大的新工具。这个项目的重点是神经生成和呼吸行为的控制。特别是，调查探测专门的吸气节律产生网站称为preBotzinger复杂（preBotC）位于人类和所有哺乳动物的下脑干的电路特性。SPECIFIC AIM 1将评估preBotC的细胞组成。转基因小鼠模型将被用来应用荧光标签的遗传不同的亚群的神经元在preBotC，然后选择性地和连续的病变，以测试它们各自的作用，在节奏。将具体测试来源于同源域转录因子Dbx 1的神经元包含preBotC的基本节律产生核心的假设。SPECIFIC AIM 2将评估当有节奏活性的preBotC神经元被顺序删除时，呼吸功能是否以及如何恶化。在这里，靶神经元将基于吸气节律活动而不是遗传起源来选择。这组实验将作为一个通用的疾病模型，以检查具有中心病因的呼吸道病理学的细胞机制。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。这一新知识对人类健康和健康很重要，因为呼吸是一种无情的、不可或缺的人类行为，可以维持体内平衡和生命本身。在随后的项目中，这种新技术-用于检测然后在体外累积序列中消融神经元-将被应用于询问运动和咀嚼节律生成网络，这些网络也可以在体外脊髓和后脑制剂中进行研究。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。 公共卫生相关性：呼吸是一种至关重要的人类行为，对维持体内平衡和生命本身至关重要。这个项目将推进我们对产生和控制呼吸节律的脑干神经回路的细胞组成的理解，并研究呼吸功能如何随着呼吸节律产生神经元的逐渐死亡而分解。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系统疾病的基础，并阐明通常作为节律性运动行为基础的回路水平特性。