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: 8019489
• 负责人: Christopher A. Del Negro
• 金额: $ 19.57万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019489
• 关键词: Ablation; Affect; Behavior; Brain Stem; Brain region; Breathing; Cells; Characteristics; Complex; Computers; Development; Disease model; Dorsal; Etiology; 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 发展研究补助金利用多光子激光技术对工作神经元网络进行体外成像，然后测试组成神经元如何通过细胞特异性激光烧蚀对网络功能做出贡献。具体来说，我们开发了一种计算机控制系统，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为三维组织中的病变提供了前所未有的特异性和控制。这一重大技术发展为研究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强大的新工具。该项目的重点是呼吸行为的神经生成和控制。特别是，这些研究探讨了位于人类和所有哺乳动物下脑干的称为前博辛格复合体（preBotC）的专门吸气节律生成部位的回路特性。具体目标 1 将评估 preBotC 的细胞组成。转基因小鼠模型将用于将荧光标签应用于 preBotC 中遗传上不同的神经元亚群，然后选择性地连续损伤它们以测试它们各自在节律发生中的作用。来自同源域转录因子 Dbx1 的神经元构成 preBotC 的重要节律生成内核的假设将得到专门测试。 SPECIFIC AIM 2 将评估当有节奏的活跃 preBotC 神经元被顺序删除时，呼吸功能是否以及如何恶化。这里，目标神经元将根据吸气节律活动而不是遗传起源来选择。这组实验将作为一般疾病模型来检查具有中心病因的呼吸系统病理学的细胞机制。总之，这个 R21 项目将提供有关呼吸的神经生成和控制的重要新信息。鉴于呼吸是维持体内平衡和生命本身的一种持续且不可或缺的人类行为，这一新知识对人类健康和保健非常重要。在后续项目中，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：呼吸是一种重要的人类行为，对于维持体内平衡和生命本身至关重要。该项目将增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，并研究随着产生呼吸节律的神经元逐渐死亡，呼吸功能如何崩溃。获得的新知识将作为治疗和预防具有中枢神经病因的呼吸系统疾病的基础，并阐明一般节律性运动行为背后的回路水平特性。

项目成果

Automated cell-specific laser detection and ablation of neural circuits in neonatal brain tissue.

自动细胞特异性激光检测和新生儿脑组织神经回路消融。

• DOI: 10.1113/jphysiol.2012.247338
• 发表时间: 2013
• 期刊: The Journal of physiology
• 作者: Wang,Xueying;Hayes,JohnA;Picardo,MariaCristinaD;DelNegro,ChristopherA
• 通讯作者: DelNegro,ChristopherA

Interactions of persistent sodium and calcium-activated nonspecific cationic currents yield dynamically distinct bursting regimes in a model of respiratory neurons.

• DOI: 10.1007/s10827-010-0311-y
• 发表时间: 2011-10
• 期刊: JOURNAL OF COMPUTATIONAL NEUROSCIENCE
• 影响因子: 1.2
• 作者: Dunmyre, Justin R.;Del Negro, Christopher A.;Rubin, Jonathan E.
• 通讯作者: Rubin, Jonathan E.

Flufenamic acid as an ion channel modulator.

• DOI: 10.1016/j.pharmthera.2013.01.012
• 发表时间: 2013-05
• 期刊: PHARMACOLOGY & THERAPEUTICS
• 影响因子: 13.5
• 作者: Guinamard, Romain;Simard, Christophe;Del Negro, Christopher
• 通讯作者: Del Negro, Christopher