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

描述(申请人提供)：神经可塑性研究中的两个主要障碍是缺乏监测树突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有限方法。出于这些原因，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单个动作电位(AP)所需的速度和灵敏度。在这一应用中，我们建议开发和应用一种新的基于Fvrster共振能量转移(FRET)的双分量光学方法来感测膜电位。我们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，而二苦胺(DPA)作为受体，DPA是一种膜分配是电压敏感的分子。初步数据表明，培养细胞的膜去极化过程中观察到大而快速的分数荧光变化(56%/100 mV，D~0.1ms)。在培养的神经元和脑片中的神经元中，AP诱导的光信号几乎是任何其他报告的3倍，这使得在不到一平方微米的膜区域的单次试验中检测到阈值下的活动和AP成为可能。该申请被组织成三个目标。目的1建议进一步描述该系统，建立双光子源的有效性，并仔细测量DPA对电兴奋性的影响。使用固定激光光斑方法，Aim 2建议测量单个树突棘的膜电位，树突棘是被认为是神经可塑性的关键部位的一个亚细胞室。目的3利用“神经生物学”来标记功能相似的神经元组，以证明Dio/DPA系统可以用来监测小神经回路的活动。该提案寻求建立一种健壮和灵活的新方法，用于对单个神经元和解剖定义的神经回路内的电信号流动进行非侵入性监测。我们预计，这种新的实验方法将使神经可塑性的研究取得快速进展，无论是在基因上可服从的制剂中，还是在那些不能遗传的制剂中。

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.