Multiparametric Biosensor Imaging in Brain Slices

破译神经编码将需要解构复杂和相互交织的信号机制， 在大脑中驱动细胞兴奋性、突触可塑性和电路动力学。这一根本目标有 极具挑战性，因为解开多个信号的时间和空间相互作用 通路需要对单个细胞和多个神经元内的多个网络进行协调观察 在完整的电路内。知识上的巨大差距仍然存在，因为我们目前用来跟踪 分子活性不适合于一次调查不止一个记者。在此，我们建议 通过开发一种新的方法来同时对多个 单个神经元内的定量FRET生物传感器，使用荧光各向异性报告器(FLARE)。 许多针对典型信号通路的耀斑，包括钙、cAMP和MAPK级联， 以多种颜色构建，允许在单个传感器中同时成像多达三个传感器 准备，无论是在相同的道路上，还是在免费的道路上。我们提出了三个目标来验证和进一步 开发这项技术，使其适用于研究小鼠急性切片和培养切片中的细胞和电路 大脑在神经编码过程中。我们将首先采用一种光学切片显微镜方法， 有利于荧光偏振成像，称为双反相选择性平面照明 显微镜(DiSPIM)，用于耀斑成像。我们还将扩展耀斑调色板，以包括 突触功能(RAC、CaMKII)和膜兴奋性(电压)。FLARE-SPIM的建造 该仪器将使对两个高价值神经元电路的原理研究成为可能。第一，挑战极限 亚细胞空间分辨率、Flare-SPIM成像将在单个 长时程增强诱导过程中急性海马脑片中的树突棘。第二，推动 在细胞时间分辨率的限制下，我们将跟踪电压、钙、PKA和 器官培养中神经元活动昼夜振荡期间的ERK活性 视交叉上核脑片。总之，这些研究将为系统探索奠定基础。 对细胞内的神经调节和神经元电路的研究，为 信号通路之间的空间和时间相互作用。通过与其他Brain计划的合作 解决类似问题的小组，这一基础工作将可扩展到添加传感器套件，这些传感器套件 可视化协调细胞活动的节点，揭示和测量神经编码的复杂性 完整的大脑回路。

Deciphering neural coding will require deconstructing the complex and intertwined signaling mechanisms that drive cellular excitability, synaptic plasticity, and circuit dynamics in the brain. This fundamental objective has been extremely challenging because unraveling the temporal and spatial interactions of multiple signaling pathways requires coordinated observation of multiple networks within individual cells and multiple neurons within intact circuits. Large gaps in knowledge remain because our current tools for tracking the dynamics of molecular activity are poorly suited for investigating more than one reporter at a time. Here, we propose to tackle this constraint through development of a novel methodology for simultaneous optical imaging of multiple quantitative FRET biosensors within single neurons, using FLuorescence Anisotropy Reporters (FLAREs). Numerous FLAREs targeting canonical signaling pathways, including calcium, cAMP, and the MAPK cascade, have been constructed in several colors allowing simultaneous imaging of up to three sensors in a single preparation, either in the same or complimentary pathways. We propose three aims to validate and further develop this technology to tailor it for studying cells and circuitry in acute and cultured slices from the mouse brain during neural coding. We will first adapt an optical sectioning microscopy method that is highly advantageous for fluorescence polarization imaging, known as dual-inverted Selective Plane Illumination Microscopy (diSPIM), for FLARE imaging. We will also expand the FLARE palette to include key regulators of synaptic function (Rac, CaMKII) and membrane excitability (voltage). Construction of the FLARE-SPIM instrument will enable proof of principle studies on two high-value neuronal circuits. First, pushing the limits of subcellular spatial resolution, FLARE-SPIM imaging will be performed on key signaling molecules in single dendritic spines in acute hippocampal brain slices during induction of long-term potentiation. Second, pushing the limits of cellular temporal resolution, we will track the rhythmic fluctuations of voltage, calcium, PKA and ERK activities during circadian oscillations of neuronal activity exhibited in organotypically-cultured suprachiasmatic nucleus brain slices. Together, these studies will lay the foundation for systematic exploration of neuromodulation within cells and neuronal circuitry, providing critical and unprecedented new insights for the spatial and temporal interactions between signaling pathways. Through collaboration with other Brain Initiative groups working on similar problems, this foundational work will be scalable to add suites of sensors that visualize nodes of coordinated cellular activity and reveal and measure the complexity of neural coding within intact brain circuits.