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: 9036880
• 负责人: Ehud Isacoff
• 金额: $ 70.57万
• 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/9036880
• 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 cell body; neuronal circuitry; next generation; novel; novel strategies; operation; public health relevance; 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（由申请人提供）：理解大脑功能的基础是将特定神经元的时空放电模式与其功能连接联系起来，并确定这些连接的强度和经验依赖性调节的能力，基因编码的光学指示器已经发生了革命性的变化。这两项努力都使得动作电位和突触传递能够被检测到，但这些方法面临两个主要障碍：1）过于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，2）目前还没有方法可以测量体内突触传递的概率（Pr）和突触的定量大小，导致突触强度连接和经验依赖性变化的机制尚未解决。第一个问题来自于缺乏针对活动指标的能力。到数量足够少的特定细胞，以便可以在密集的环境中确定它们的形态和物理连接性，然后允许物理图像与活动和连接性相关。为了应对这一挑战，我们提出了一种可推广的策略。这创建“开启”基因编码的活动指示器这些指示器是对一些现有的最好的神经活动和突触传递指示器进行合理修改的，使指示器能够被光激活以提供类似高尔基体的功能。查看细胞形态并报告动作电位和突触输入，因为指示器是通过光打开的（与高尔基染色等随机标记方法不同）， 人们可以选择特定的目标细胞，并对那些过程严重重叠的密集细胞进行功能成像，同时知道哪个过程属于哪个细胞，从而允许一种简单形式的优雅连接映射。第二个问题来自于缺乏突触特异性量子方法。尽管我们对突触功能有丰富的了解，但突触反应的大规模定量分辨率成像将是对实验神经科学工具包的有力补充，有助于解决突触强度的动态变化如何影响感觉、行动、学习和记忆。由于缺乏有效的工具，减少了脑切片等离体准备工作，我们对学习和行为过程中体内突触功能的了解极其有限，克服这一技术差距的新方法将弥合突触和电路水平之间的鸿沟。对清醒、行为动物的高信噪比脊柱水平钙成像可以解决这一差距。为了应对这一挑战，我们建议开发突触靶向钙指示剂，使兴奋性突触传递能够成像。由于这是一种成像方法，因此它可以提供突触特定的信息，而电生理记录无法轻松地将分布在神经元树突树上的大量输入的测量结果汇总在一起。对行为动物的研究将允许直接评估可能构成学习基础的突触功效的动态波动，它还将开辟全新的研究途径，探索突触功效的变化如何影响基本方面。 Isacoff、Scott 和 Adesnik 之间的合作使这些新工具能够在三种模型生物（斑马鱼、果蝇和小鼠）的脑回路分析和行为中得到验证。