Multiparametric Biosensor Imaging in Brain Slices

脑切片多参数生物传感器成像

• 批准号: 9214054
• 负责人: Thomas A Blanpied
• 金额: $ 63.58万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 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/9214054
• 关键词: AMPA Receptors; Actins; Action Potentials; Acute; Address; Biochemical Process; Biosensor; Brain; Calcium; Cells; Circadian Rhythms; Code; Collaborations; Color; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Data Collection; Dendritic Spines; Detection; Development; Dimensions; Event; Exhibits; Fluorescence Anisotropy; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Foundations; Gene Expression; Goals; Hippocampus (Brain); Hour; Image; In Situ; Individual; Knowledge; Laboratories; Life; 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; 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; image reconstruction; insight; instrument; microscopic imaging; molecular dynamics; neuronal circuitry; neuroregulation; novel; optical imaging; polymerization; quantitative imaging; receptor; relating to nervous system; research study; response; rho; sensor; suprachiasmatic nucleus; synaptic function; synaptogenesis; temporal measurement; tool; trafficking; voltage; working group

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.

解密的神经编码将需要解构复合且相互交织的信号传导机制 驱动大脑中的细胞兴奋性，突触可塑性和电路动力学。这个基本目标有 非常具有挑战性，因为揭开了多个信号的时间和空间相互作用 途径需要对单个细胞和多个神经元内多个网络进行协调观察 在完整的电路中。知识的较大差距仍然存在，因为我们当前用于跟踪动态的工具 分子活性不适合一次研究一个以上的记者。在这里，我们建议 通过开发新的方法来解决这种限制 使用荧光各向异性报道（耀斑），单神经元内定量的FRET生物传感器。 针对规范信号通路的许多耀斑，包括钙，cAMP和MAPK级联 已经用几种颜色构建，可以同时在单个中同时成像三个传感器 准备，无论是相同还是免费的途径。我们提出了三个目标，以进一步验证 开发该技术来调整它，以研究小鼠急性和培养的切片中的细胞和电路 神经编码期间的大脑。我们将首先适应高度的光学切片显微镜方法 对荧光偏振成像的有利优势，称为双向内选择平面照明 显微镜（Dispim），用于耀斑成像。我们还将扩大耀斑调色板，包括 突触功能（RAC，CAMKII）和膜兴奋性（电压）。耀斑刺激的构造 仪器将对两个高价值神经元电路进行原则研究证明。首先，推动极限 亚细胞空间分辨率，将在单个关键信号分子上执行耀斑SPIM成像 长期增强期间，急性海马脑切片中的树突状刺。第二，推 细胞时间分辨率的极限，我们将跟踪电压，钙，PKA和 在细胞型培养中表现出的神经元活性的昼夜节律振荡期间的ERK活动 脑部核脑切片。这些研究将共同​​为系统探索奠定基础 细胞和神经元回路内的神经调节的作用，为临界和前所未有的新见解提供 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 研究类似问题的小组，这项基础工作将是可扩展的，以添加套件的传感器套件 可视化协调的细胞活性的节点，并揭示和测量神经编码的复杂性 完整的脑电路。