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

描述（由申请人提供）：神经可塑性研究中的两个主要障碍是缺乏用于监测非常小的隔室（如树突棘）中的电信号的工具，以及用于研究相互作用的神经元组中的电活动的方法有限。出于这些原因，许多神经科学家一致认为需要一种易于使用、光敏性有限、检测单个神经元中的个体动作电位（AP）所需的速度和灵敏度的膜电位光学传感器。在本申请中，我们提出开发和应用一种新的双组分光学方法用于传感膜电位，基于Fvrster共振能量转移（FRET）。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，二O，作为FRET反应中的供体，而dipicrylamine（DPA），其膜分区是电压敏感的分子作为受体。初步数据显示，响应于培养细胞的膜去极化，观察到大而快速的分数荧光变化（56%/100 mV，D ~ 0.1 ms）。在培养的神经元和脑切片中的神经元中，AP诱导的光学信号比任何其他报告者大近3倍，使得有可能在小于1平方微米的膜面积的单次试验中检测阈下活动和AP。该应用程序分为三个目标。目的1提出进一步表征系统，建立双光子源的有效性，并仔细测量DPA对电兴奋性的影响。使用固定的激光点的方法，目的2提出测量膜电位在单个树突棘，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”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中的活动。该提案旨在建立一种强大而灵活的新方法，用于无创监测单个神经元和解剖学定义的神经回路内的电信号流。我们希望这种新的实验方法将使神经可塑性研究的快速进展，无论是在遗传上适合和那些不适合的准备工作。公共卫生相关性：目前用于在电路内的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障碍的脑部疾病的理解。该提案提出了一种用于测量神经回路活动的非侵入性光学方法;这种新策略应该能够促进我们对神经系统疾病的许多潜在机制的理解。

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.