Multiparametric Biosensor Imaging in Brain Slices

脑切片多参数生物传感器成像

基本信息

批准号：
9449901
负责人：
Thomas A Blanpied
金额：
$ 7.52万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9449901
关键词：
AMPA Receptors Actins Action Potentials Acute Address BRAIN initiative Biochemical Process Biosensor Brain Brain Diseases Calcium Cells Circadian Rhythms Code Collaborations Color Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeleton Data Data Collection Dendritic Spines Detection Development Dimensions Disease Event Exhibits Fluorescence Anisotropy Fluorescence Polarization Fluorescence Resonance Energy Transfer Foundations Gene Expression Goals Hippocampus (Brain)Hour Image In Situ Individual Knowledge Laboratories Light Lighting Link Location Long-Term Potentiation MAP Kinase Gene MYLK gene Measurement Measures Mediating Membrane Methodology Methods Microscope Microscopy Molecular Monitor Monomeric GTP-Binding Proteins Mus Neurons Noise Optics Organism Output Pathway interactions Peptide Signal Sequences Periodicity Phase Phosphotransferases Phototoxicity Physiological Preparation Property Reporter Research Resolution Signal Pathway Signal Transduction Signaling Molecule Slice Specimen Stimulus Surface Synapses Synaptic plasticity Technology Time Validation Work calmodulin-dependent protein kinase II circadian pacemaker electrical property experience experimental study image reconstruction imaging approach insight instrument microscopic imaging molecular dynamics neuronal circuitry neuroregulation novel optical imaging polymerization quantitative imaging receptor relating to nervous system response rho sensor suprachiasmatic nucleus synaptic function synaptogenesis temporal measurement tool trafficking voltage working group

项目摘要

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.

破译神经编码需要解构复杂且相互交织的信号机制驱动大脑中的细胞兴奋性、突触可塑性和回路动力学。这一基本目标已非常具有挑战性，因为解开多种信号传导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。由于我们目前用于跟踪动态的工具，知识方面仍然存在巨大差距分子活性不太适合一次调查多个报告者。在此，我们建议通过开发一种同时光学成像的新方法来解决这一限制使用荧光各向异性报告器（FLARE）在单个神经元内进行定量 FRET 生物传感器。许多 FLARE 针对经典信号通路，包括钙、cAMP 和 MAPK 级联，具有多种颜色，允许在单个传感器中同时成像最多三个传感器以相同或互补的途径进行准备。我们提出三个目标来验证和进一步开发这项技术，使其适合研究小鼠急性切片和培养切片中的细胞和电路神经编码期间的大脑。我们将首先采用光学切片显微镜方法，该方法高度有利于荧光偏振成像，称为双倒置选择性平面照明显微镜 (diSPIM)，用于耀斑成像。我们还将扩展 FLARE 调色板，以包括以下关键监管机构突触功能（Rac、CaMKII）和膜兴奋性（电压）。 FLARE-SPIM 的构建该仪器将能够对两个高价值神经元电路进行原理验证研究。首先，突破极限 FLARE-SPIM 成像将在单次成像中对关键信号分子进行亚细胞空间分辨率长期增强诱导期间急性海马脑切片中的树突棘。二、推为了突破细胞时间分辨率的限制，我们将跟踪电压、钙、PKA 和器官典型培养中神经元活动昼夜节律振荡期间的 ERK 活性视交叉上核脑切片。这些研究将为系统探索奠定基础细胞和神经元回路内的神经调节，为信号通路之间的空间和时间相互作用。通过与其他大脑计划的合作致力于解决类似问题的小组，这项基础工作将可扩展以添加传感器套件，可视化协调细胞活动的节点并揭示和测量内部神经编码的复杂性完整的大脑回路。