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

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，以及研究相互作用神经元群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的膜电位光学传感器，具有有限的光毒性，以及检测单个神经元中单个动作电位（APs）所需的速度和灵敏度。在这个应用中，我们提出了一种基于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点是，它依赖于广泛使用的神经元示踪染料，DiO，作为FRET反应的供体，而二丙胺（DPA），其膜分配是电压敏感的分子作为受体。初步数据表明，在培养细胞的去极化过程中，观察到大而快速的荧光分数变化（56% / 100 mV, 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因子大近3倍，这使得在小于1平方微米的膜区域单次试验中检测阈下活性和ap成为可能。该应用程序被组织为三个目标。目标1提出进一步表征该系统，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提出测量单个树突棘的膜电位，树突棘是一种亚细胞区室，被认为是神经可塑性的关键部位。目的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强大而灵活的新方法，用于对单个神经元和解剖定义的神经回路内的电信号流进行无创监测。我们期望这种新的实验方法将使神经可塑性的研究取得快速进展，无论是在基因可接受的制备中还是在基因不可接受的制备中。公共卫生相关性：目前在回路内单个神经元水平上测量神经活动的技术受到严重限制，这阻碍了对许多神经信号功能失调的脑部疾病的理解。提出了一种测量神经回路活动的无创光学方法；这种新颖的策略应该能够促进我们对许多神经系统疾病的潜在机制的理解。

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. PUBLIC HEALTH RELEVANCE: Current technologies for measurement of neural activity at the level of single neurons within circuits are severely limited and this prevents progress in understanding many brain diseases in which neural signaling is dysfunctional. This proposal presents a noninvasive optical method for measuring neural circuit activity; this novel strategy should enable advancements in our understanding of many of the underlying mechanisms of neurological diseases.