A method for diffraction-limited spot measurements of membrane potential in situ

一种原位衍射极限点测量膜电位的方法

• 批准号: 8500479
• 负责人: Thomas S Otis
• 金额: $ 36.67万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500479
• 关键词: Action Potentials; Area; Axon; Ballistics; Brain; Brain Diseases; Cells; Communication; Coupled; Cultured Cells; Custom; Data; Dendritic Spines; Detection; Development; Dyes; Effectiveness; Energy Transfer; Fluorescence; Frequencies; Goals; Health; Hour; In Situ; Individual; Injection of therapeutic agent; Label; Lasers; Light; Lighting; Measurement; Measures; Membrane; Membrane Potentials; Methods; Monitor; Neuronal Plasticity; Neurons; Neurosciences; Optical Methods; Optical reporter; Optics; Pattern; Photons; Phototoxicity; Preparation; Reaction; Reliance; Reporter; Reporting; Scanning; Signal Transduction; Site; Slice; Source; Speed; Spottings; Stimulus; Synapses; System; Technology; Tracer; base; charge coupled device camera; design; dipicrylamine; flexibility; liquid crystal; nervous system disorder; neural circuit; non-genetic; non-invasive monitor; novel; novel strategies; optical sensor; prevent; relating to nervous system; research study; response; sensor; tool; two-photon; voltage

DESCRIPTION (provided by applicant): Two major impediments in the study of neural plasticity are the lack of tools for monitoring electrical signals in very small compartments such as dendritic spines, and the limited methods for studying electrical activity in groups of interacting neurons. For these reasons many neuroscientists agree on the need for optical sensors of membrane potential that are easy to use, have limited photoxicity, and the speed and sensitivity required for detection of individual action potentials (APs) in single neurons. In this application we propose to develop and apply a novel two-component optical approach for sensing membrane potential, based on Fvrster resonance energy transfer (FRET). A critical feature of our method is that it relies on the widely used neuronal tracer dye, DiO, as a donor in the FRET reaction while dipicrylamine (DPA), a molecule whose membrane partitioning is voltage sensitive serves as the acceptor. Preliminary data show that large and rapid fractional fluorescence changes (56 % per 100 mV, D ~ 0.1 ms) are observed in response to membrane depolarization of cultured cells. In cultured neurons and in neurons in brain slices, AP-induced optical signals are nearly 3-fold larger than with any other reporter, making it possible to detect subthreshold activity and APs in single trials from membrane areas less than a square micron. The application is organized into three aims. Aim 1 proposes to further characterize the system, establishing the effectiveness of two-photon sources, and carefully measuring the effects of DPA on electrical excitability. Using a stationary laser spot approach, Aim 2 proposes to measure membrane potential in single dendritic spines, a subcellular compartment regarded as a key site of neural plasticity. Aim 3 utilizes "diolistics" to label groups of functionally similar neurons to demonstrate that the DiO/DPA system can be used to monitor activity in small neural circuits. The proposal seeks to establish a robust and flexible new method for non-invasive monitoring of electrical signal flow within single neurons and anatomically-defined neural circuits. We expect that this new experimental approach will enable rapid progress in the study of neural plasticity both in preparations that are genetically-amenable and those that are not.

描述（由申请人提供）：神经可塑性研究的两个主要障碍是缺乏用于监测树突棘等非常小的隔室中的电信号的工具，以及研究相互作用的神经元组中的电活动的方法有限。由于这些原因，许多神经科学家一致认为需要易于使用、光毒性有限的膜电位光学传感器，以及检测单个神经元中的单个动作电位 (AP) 所需的速度和灵敏度。在此应用中，我们建议开发并应用一种基于 Fvrster 共振能量转移 (FRET) 的新型双组分光学方法来传感膜电位。我们的方法的一个关键特征是，它依赖于广泛使用的神经元示踪染料 DiO 作为 FRET 反应中的供体，而二辛胺 (DPA)（一种膜分配对电压敏感的分子）作为受体。初步数据表明，响应培养细胞的膜去极化，观察到大而快速的荧光分数变化（每 100 mV 56%，D ~ 0.1 ms）。在培养的神经元和脑切片的神经元中，AP 诱导的光信号比任何其他报告基因大近 3 倍，使得在单次试验中从小于一平方微米的膜区域检测阈下活动和 AP 成为可能。该应用程序分为三个目标。目标 1 建议进一步表征该系统，确定双光子源的有效性，并仔细测量 DPA 对电兴奋性的影响。 Aim 2 建议使用固定激光点方法测量单个树突棘的膜电位，树突棘是一种被视为神经可塑性关键部位的亚细胞区室。目标 3 利用“diolistics”来标记功能相似的神经元组，以证明 DiO/DPA 系统可用于监测小神经回路的活动。该提案旨在建立一种稳健且灵活的新方法，用于非侵入性监测单个神经元和解剖学定义的神经回路内的电信号流。我们期望这种新的实验方法将使神经可塑性研究取得快速进展，无论是在适合遗传的制剂还是不适合遗传的制剂中。