In-vivo circuit activity measurement at single cell, sub-threshold resolution

单细胞体内电路活动测量，亚阈值分辨率

• 批准号: 8935946
• 负责人: Craig Forest
• 金额: $ 50.25万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8935946
• 关键词: Affect; Algorithms; Area; Arts; Automation; BRAIN initiative; Behavioral; Biological Models; Biological Neural Networks; Brain; Cells; Cellular Membrane; Code; Complex; Computer software; Development; Disease; Electrodes; Engineering; Etiology; Experimental Models; Glutamates; Gold; Head; Health; Hippocampus (Brain); Histology; Image; In Vitro; Individual; Laboratories; Laboratory Research; Life; Location; Manuals; Measurement; Measures; Medial; Membrane; Membrane Potentials; Methodology; Molecular; Molecular Biology; Morphology; Mus; Neurons; Neurosciences; Pathway interactions; Pharmacologic Substance; Property; Publishing; Research; Research Personnel; Research Priority; Resolution; Robot; Robotics; Sensory; Site; Specificity; Structure; Synapses; System; Tactile; Techniques; Technology; Testing; Thalamic structure; Time; Vibrissae; Work; awake; blind; cell type; electric impedance; electrical potential; experience; extracellular; forest; graduate student; in vivo; innovation; neuron development; novel; patch clamp; programs; research study; response; sample fixation; skills; somatosensory; success; theories; tool

 DESCRIPTION (provided by applicant): Neurons communicate information through fluctuations in the electrical potentials across their cellular membranes. Whole-cell patch clamping, the gold standard technique for measuring these fluctuations, is something of an art form, requiring great skill to perform on only a few cells per day. Thus, it has been primarily limited to in vitro experiments, a few in vivo experiments, and very limited applications in the awake brain. Dr. Forest (and collaborator Dr. Boyden at MIT) developed a robot that automatically performs patch clamping in the living brains of mice by algorithmically detecting cells through analysis of a temporal sequence of electrode impedance changes. Using it, they have demonstrated good yield, throughput, and quality of recording in mouse cortex and hippocampus. With this 'autopatching' robot enabling routine access to electrical and molecular properties of neurons, systematic and scalable in vivo experiments as well as fundamentally new kinds of single-cell analyses have become possible. In the past 12 months, the team has installed 15 autopatchers in academic research laboratories, garnered worldwide media coverage, and led to Dr. Forest's and Dr. Boyden's invitations to President Barack Obama's announcement of the BRAIN Initiative. There are currently no published experiments demonstrating in vivo intracellular recordings of two or more neurons that are synaptically connected. We propose to utilize the autopatcher to target anatomically well-studied sub-circuits to significantly increase the odds of identifying synaptically connected pairs. Specifically, we wil utilize the thalamocortical circuit in the mouse vibrissa/whisker pathway as a model experimental system, where there is a substantial convergence of projections from the thalamus to the input layer in the somatosensory (tactile) cortex. The Stanley Laboratory has extensive experience with stimulation and electrophysiological recordings in this circuit, and is one of only a few laboratories that has successfully recorded from synaptically connected pairs of neurons using extracellular techniques. Thus we aim to demonstrate and characterize the first simultaneous intracellular recording of a functional circuit in the anesthetized and awake living mouse brain to reveal its neural network dynamics. In this 36 month program, the labs of Prof. Stanley and Forest, supported by two postdoctoral researchers, two graduate research assistants, a research engineer and five undergraduates, with assistance from ten graduate students working on related projects, will develop single (Aim 1) and dual (Aim 2,3) autopatching robots for the anesthetized and awake brain. Success will allow, for the first time, quantification of synaptic efficacy in the living brain, crucial for understanding normal and pathological function. Just as molecular biology has greatly benefited from the revolution in in vitro automation, we believe that neuroscience will greatly benefit from the revolution in in vivo automation that we have launched, and here propose to extend.

 描述（由申请人提供）：神经元通过细胞膜电位的波动传递信息。全细胞膜片钳是测量这些波动的黄金标准技术，它是一种艺术形式，每天只需要几个细胞就可以完成。因此，它主要局限于体外实验，一些体内实验，以及在清醒大脑中的非常有限的应用。Forest博士（和麻省理工学院的合作者Boyden博士）开发了一种机器人，该机器人通过分析电极阻抗变化的时间序列来算法检测细胞，从而自动在小鼠的活体大脑中执行膜片钳。使用它，他们已经证明了良好的产量，吞吐量和质量的记录在小鼠皮层和海马。有了这种“自动匹配”机器人，就可以对神经元的电学和分子特性进行常规访问，系统的、可扩展的体内实验以及全新的单细胞分析成为可能。在过去的12个月里，该团队已经在学术研究实验室安装了15台自动匹配器，获得了全球媒体的报道，并导致Forest博士和Boyden博士被邀请参加美国总统巴拉克奥巴马宣布的BRAIN计划。目前还没有发表的实验证明在体内细胞内记录两个或更多个神经元的突触连接。我们建议利用自动匹配器来定位解剖学上研究得很好的子电路，以显着增加识别突触连接对的几率。具体而言，我们将利用小鼠触须/胡须通路中的丘脑皮层回路作为模型实验系统，其中存在从丘脑到体感（触觉）皮层中的输入层的大量投射的会聚。斯坦利实验室在该电路的刺激和电生理记录方面拥有丰富的经验，并且是唯一一个 一些实验室已经成功地用细胞外技术记录了突触连接的神经元对。因此，我们的目标是证明和表征的第一个同时在细胞内记录的功能电路在麻醉和清醒的活小鼠大脑，以揭示其神经网络动力学。在这个为期36个月的项目中，Stanley教授和Forest教授的实验室在两名博士后研究人员、两名研究生研究助理、一名研究工程师和五名本科生的支持下，在十名从事相关项目的研究生的帮助下，将为麻醉和清醒的大脑开发单（目标1）和双（目标2，3）自动匹配机器人。成功将首次允许量化活体大脑中的突触功效，这对于理解正常和病理功能至关重要。正如分子生物学从体外自动化革命中受益匪浅一样，我们相信神经科学也将从我们发起的体内自动化革命中受益匪浅，并在此提出扩展。