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（应用程序提供）：神经元通过其细胞机制的电势的波动来传达信息。全细胞贴片夹具是测量这些波动的金标准技术，是一种艺术形式，需要每天只能在几个单元格上执行的精湛技巧。这主要仅限于体外实验，一些体内实验，并且在清醒大脑中的应用非常有限。 Forest博士（MIT的Boyden博士和合作者）开发了一个机器人，该机器人通过分析临时电极阻抗变化的临时序列来自动执行小鼠活体大脑中的斑块夹紧。使用它，他们在小鼠皮质和海马中表现出良好的产量，吞吐量和记录质量。借助这种“自动捕获”机器人，可以常规地访问神经元的电气和分子特性，体内实验的系统和可扩展性，以及从根本上进行新型的单细胞分析。在过去的12个月中，该团队在学术研究实验室中安装了15名自动捕捞者，获得了全球媒体报道，并导致了Forest博士和博伊登博士的邀请，向巴拉克·奥巴马（Barack Obama）总统宣布了《大脑倡议》。目前尚无公开的实验证明了两种或多个突触连接的神经元的体内细胞内记录。我们建议利用自动捕获器靶向解剖学上良好的子电路，以显着增加识别突触连接对的几率。具体而言，我们将利用小鼠颤音/晶须途径中的丘脑皮质回路作为模型实验系统，在该系统中，从丘脑到体感（触觉）皮质的输入层的项目大量收敛。斯坦利实验室在该电路中具有丰富的刺激和电生理记录的经验，并且是唯一的 使用细胞外技术从合成连接的神经元对成功记录的一些实验室。我们旨在证明和表征在麻醉和清醒的小鼠大脑中功能电路的第一个简单的细胞内记录，以揭示其神经元网络动力学。在这个36个月的计划中，斯坦利和森林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，在从事相关项目的十个研究生的帮助下，将开发单个（AIM 1）和Dual（AIM 2,3）（AIM 2,3）为麻醉和Awake设置的机器人进行自动匹配机器人。成功将首次允许活大脑中的突触效率数量，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的革命中受益匪浅一样，我们相信神经科学将从我们发起的体内自动化革命中受益匪浅，并在这里提出扩展。