Novel tools for cell-specific imaging of functional connectivity and circuit operations

用于功能连接和电路操作的细胞特异性成像的新工具

• 批准号: 9343283
• 负责人: Ehud Isacoff
• 金额: $ 17.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-23 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9343283
• 关键词: Action Potentials; Address; Animal Model; Animals; Axon; Behavior; Body Weight Changes; Brain; Calcium; Cells; Cellular Morphology; Cognition; Collaborations; Complex; Dendrites; Disease; Drosophila genus; Electrophysiology (science); Engineering; Environment; Esthesia; Face; Fishes; Functional Imaging; Glutamates; Golgi Apparatus; Health; Image; Individual; Knowledge; Label; Learning; Light; Mammals; Maps; Measurement; Measures; Mediating; Memory; Methods; Morphology; Mus; Neocortex; Neurons; Neurosciences; Neurotransmitters; Noise; Nutritional; Optics; Pattern; Physical activity; Physiological; Population; Preparation; Probability; Process; Regulation; Reporter; Reporting; Research; Resolution; Signal Transduction; Slice; Staining method; Stains; Synapses; Synaptic Transmission; System; Taste Perception; Time; Trees; Validation; Vertebral column; Vertebrates; Weight; Zebrafish; awake; base; brain cell; calcium indicator; cell type; cognitive function; experience; fly; genetic approach; imaging modality; in vivo; learned behavior; meetings; neural circuit; neuronal circuitry; next generation; novel; novel strategies; operation; receptive field; relating to nervous system; response; synaptic function; tool; voltage

 DESCRIPTION (provided by applicant): Fundamental to understanding brain function is the ability to relate the spatio-temporal firing patterns of specific neurons to their functional connectivity and determine the strength and experience-dependent regulation of those connections. Genetically-encoded optical indicators have revolutionized both endeavors, enabling action potentials and synaptic transmission to be detected, but these approaches face two major hurdles: 1) overly dense expression often makes it impossible to trace the morphology and hence connectivity of specific neurons, and 2) there has been no method to measure the probability of synaptic transmission (Pr) and quantal size of synapses in vivo, leading synaptic strength connections and the mechanism of experience- dependent change unresolved. The first problem emerges from a lack of ability to target activity indicators to specific cells that are few enough in number so that their morphology and physical connectivity could be determined in a densely packed environment, to then permit the physical picture to be related to activity and connectivity. To meet this challenge, we propose a generalizable strategy for the creation of "turn-on" genetically-encoded activity indicators. These indicators are rationally modified from some of the best existing indicators of neural activity and synaptic transmission. The re-engineering enables the indicators to be activated by light to provide a Golgi-like view of cell morphology and report on action potentials and synaptic input. Because the indicators are turned on by light (unlike in the random labeling methods like the Golgi stain), one can select specific target cells and functionally image densely packed cells whose processes heavily overlap while knowing which process belongs to which cell, thereby permitting a simple form of elegant connectivity mapping. The second problem emerges from the lack of a method for synapse-specific quantal analysis. Large-scale quantal resolution imaging of synaptic responses would represent a powerful addition to the experimental neuroscience toolkit to help address how dynamic changes in synaptic strength contribute to sensation, action, learning and memory. Despite a wealth of knowledge on synaptic function in reduced ex vivo preparations, such as brain slices, due to the lack of effective tools, our knowledge of synaptic function in vivo during learning and behavior is extremely limited. A new approach that overcomes this technical gap would bridge the divide between synaptic and circuit level analyses in awake, behaving animals. High signal-to-noise spine level calcium imaging in behaving animals could address this gap. To meet this challenge we propose to develop synaptically-targeted calcium indicators that enable excitatory synaptic transmission to be imaged with quantal resolution simultaneously at hundreds to thousands of connections. Because this is an imaging method, it provides synapse-specific information that one cannot readily obtain from electrophysiological recordings that lump together measurements from a large number of inputs distributed over a neuron's dendritic tree. Optical quantal analysis in behaving animals would permit direct assessment of the dynamic fluctuations in synaptic efficacy that may underlie learning. It will also open whole new avenues of research that could explore how changes in synaptic efficacy contribute to fundamental aspects of sensation, action, and higher cognitive function. The collaboration between Isacoff, Scott and Adesnik enables these new tools to be validated for in vivo applications in brain circuit analysis and behavior in three model organisms: zebrafish, fruitfly and mouse.

 描述（由适用提供）：理解大脑功能的基础是将特定神经元的空间发射模式与其功能连通性联系起来，并确定这些连接的强度和经验依赖性调节。遗传编织的光学指标已彻底改变了这两个努力，使动作潜力和突触传播能够被发现，但是这些方法面临两个主要障碍：1）过度密集的表达通常使得无法追踪特定神经元的形态和连接性，并且不可能衡量连接的突触（PR）的可能性（PR）的可能性（PR），并且是突触的可能性（PR）的范围（PR）的可能性（PR）。经验依赖变化的机制尚未解决。第一个问题来自缺乏将活动指标靶向数量少的特定细胞的能力，因此可以在荒芜的包装环境中确定它们的形态和身体连通性，从而允许物理图片与活动和连通性有关。为了应对这一挑战，我们提出了一种可普遍的策略，以创建“转旋”一般编码的活动指标。这些指标是根据神经元活动和突触传播的一些现有最佳现有指标进行合理修改的。重新设计使指标能够被光激活，以提供类似高尔基的细胞形态视图，并报告动作电位和突触输入。由于指示器是通过光打开的（与Golgi染色（例如Golgi染色）的随机标记方法不同），所以 人们可以选择特定的目标单元格，并在功能上图像无填充的单元，其过程大量重叠，同时知道哪个过程属于哪个单元，从而允许一种简单的优雅连接映射形式。第二个问题来自缺乏突触特异性定量分析的方法。突触反应的大规模量化分辨率成像将代表实验性神经科学工具包的强大补充，以帮助解决突触强度的动态变化如何有助于感觉，动作，学习和记忆。尽管由于缺乏有效的工具，在减少的离体制剂（例如脑切片）中有丰富的知识，但在学习和行为过程中我们对体内突触功能的了解非常有限。一种克服这一技术差距的新方法将弥合醒着的综合和电路水平分析，表现为动物。在行为动物中，高信号到噪声脊柱水平的钙成像可以解决这一差距。为了应对这一挑战，我们建议开发出突触靶向的钙指标，以同时使用数百至数千个连接的数量分辨率对兴奋性突触传播进行成像。因为这是一种成像方法，所以它提供了突触特异性的信息，这些信息无法轻易从电生理记录中获得，这些记录将分布在神经元的树突树上的大量输入中汇总在一起。行为动物中的光学定量分析将允许直接评估可能是学习基础的突触效率的动态波动。它还将开放全新的研究途径，该途径可以探索突触效率的变化如何促进感觉，动作和更高认知功能的基本方面。 Isacoff，Scott和Adesnik之间的合作使这些新工具能够在三种模型生物的脑电路分析和行为中的体内应用进行验证：斑马鱼，果蝇和鼠标。