Multiparametric Biosensor Imaging in Brain Slices

脑切片多参数生物传感器成像

• 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
• 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

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.

破译神经编码将需要解构复杂和交织的信号机制， 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 这是非常具有挑战性的，因为解开多重信号的时间和空间相互作用 通路需要协调观察单个细胞和多个神经元内的多个网络 在完整的电路中。知识上的巨大差距仍然存在，因为我们目前用于跟踪 分子活性不太适合于一次研究一个以上的报道分子。在此，我们建议 通过开发一种新的方法来解决这一限制， 定量FRET生物传感器内的单个神经元，使用荧光各向异性报告（FLAREs）。 许多FLARE靶向经典信号通路，包括钙、cAMP和MAPK级联， 已经被构造成几种颜色，允许在单个传感器中同时成像多达三个传感器。 准备，无论是在相同的或互补的途径。我们提出了三个目标，以验证和进一步 开发这项技术，使其适用于研究小鼠急性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， 这对于荧光偏振成像是有利的，称为双反转选择性平面照明 显微镜检查（diSPIM），用于FLARE成像。我们还将扩展FLARE选项板，以包括以下主要监管机构 突触功能（Rac，CaMKII）和膜兴奋性（电压）。FLARE-SPIM的构建 仪器将能够证明两个高价值的神经元回路的原则研究。第一，挑战 亚细胞空间分辨率，FLARE-SPIM成像将在单个细胞中对关键信号分子进行 长时程增强诱导过程中急性海马脑片中的树突棘。第二，推 细胞时间分辨率的限制，我们将跟踪电压，钙，PKA和 ERK活性在神经元活动的昼夜节律振荡中表现出在器官型培养的 视交叉上核脑切片。这些研究将共同为系统的探索奠定基础 细胞内的神经调节和神经元回路，提供关键和前所未有的新见解， 信号通路之间的空间和时间相互作用。通过与其他脑倡议组织的合作， 工作在类似问题上的小组，这项基础工作将是可扩展的，以添加传感器套件， 可视化协调细胞活动的节点，并揭示和测量神经编码的复杂性， 完整的脑回路