Super-Resolution Microscopy of Neuronal Synapses with Advanced Imaging Tools

使用先进成像工具对神经元突触进行超分辨率显微镜检查

• 批准号: 10299205
• 负责人: Hee Jung Chung
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10299205
• 关键词: 3-Dimensional; Alzheimer' s Disease; Avidin; Binding; Biological Sciences; Caliber; Cell membrane; Cells; Chemicals; Clinical; Color; Communication; Cross-Linking Reagents; DNA; DNA Probes; Diffusion; Disadvantaged; Dyes; Event; Evolution; Fluorescence; Fluorescence Microscopy; Future; Glutamate Receptor; Grant; Hippocampus (Brain); Homer 1; Image; Imaging Device; Individual; Label; Lateral; Learning; Length; Ligands; Long-Term Potentiation; Maintenance; Measurement; Measures; Memory; Methods; Microscopy; Molecular; N-Methylaspartate; NMDA receptor A1; Neurodegenerative Disorders; Neurons; Neurosciences; Neurotransmitter Receptor; Parkinson Disease; Phase; Physiological; Positioning Attribute; Process; Proteins; Quantum Dots; Resolution; Site; Speed; Structure; Sulfhydryl Compounds; Surface; Synapses; Synaptic Cleft; System; Techniques; Technology; Testing; Thinness; Time; base; crosslink; density; dimer; experimental study; facsimile; fluorophore; imager; improved; innovation; nanobodies; nanometer; presynaptic; receptor; response; single molecule; stoichiometry; temporal measurement; tool; trafficking

Significance: The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact on basic bioscience and likely to future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (known as iGluRs) are dynamically involved in neuron- to-neuron communication across the thin (≈30 nm) synapse; when dysregulated, neurodegenerative diseases result, such as Alzheimer’s and Parkinson’s diseases. We—and others—have tracked these events with nanometric resolution using super-resolution fluorescence microscopy (SRFM). Using small probes—quantum dots (≈12 nm diameter) and other photostable fluorophores, developed in our lab in the preceding grant—we came up with some surprises. We find that a large fraction of the AMPARs reside in the synapse where their mobility is restricted; during long-term-potentiation (LTP, a molecular underpinning of memory formation), we’ve quantified their numbers and find during their maintenance phase that their lateral diffusion is rare; NMDARs have extra-synaptic nanodomains which may keep their numbers from rising during LTP. But are these, and other results, correct? To validate these preliminary results, we will measure the placement and diffusion of the iGluRs, primarily AMPARs, using three different SRFM techniques, each one having its own advantages and disadvantages. We will also determine the 3D-orientation of the synapse, the effect of probe size and type, the details of LTP activation, and quantitatively determine the number of iGluRs at each synapse. The results between the three techniques will be compared. Innovation: Each SRFM technique has new aspects, particularly with respect to neuroscience. First, we will improve the PALM/STORM technique (one type of SRFM) to test the distribution and dynamics of iGluRs more accurately. We will use new probes—nanobodies and scFv’s—against post-synaptic proteins and iGluRs, and test new sQDs and new cross-linking reagents against iGluRs. We will also determine the orientation and position of the synaptic zone by labeling neuroligin and various presynaptic proteins, such as Bassoon and RIM1/2, first under basal conditions and then with chemical LTP (cLTP). Second, we will use and develop PAINT, another form of SRFM, which has recently been shown to have a 100× increase in speed with excellent spatial resolution—≈5 nanometers in 0.2 sec. We will show that quantitative-PAINT can be applied to fixed neurons and can be used to measure cLTP on an individual synapse. And for the first time, we will apply PAINT to a living neuron under physiological conditions to measure AMPAR dynamics. With PAINT, we will be able to test how many iGluRs there are per synapse, whether they are synaptic or extra-synaptic, and how the number of iGluRs change with cLTP. Third, we will utilize a fluorogenic activating protein (FAP) with iGluRs and show that the number of receptors can be measured in living neurons with nanometric resolution, no background, and potentially fast response to cLTP. This method will therefore provide another test of iGluR structure & dynamics.

意义：测量纳米神经元通讯分子机制的能力 空间尺度将对基础生物科学产生巨大影响，并可能对未来的临床神经科学产生巨大影响。在 特别是，AMPA 型和 NMDA 型谷氨酸受体（称为 iGluR）动态地参与神经元 跨薄（约 30 nm）突触的神经元通讯；当失调时，神经退行性疾病 结果，例如阿尔茨海默病和帕金森病。我们和其他人已经跟踪这些事件 使用超分辨率荧光显微镜（SRFM）获得纳米分辨率。使用小型探针——量子 点（直径约 12 nm）和其他耐光荧光团，是我们实验室在之前的资助中开发的 - 我们 带来了一些惊喜。我们发现大部分 AMPAR 驻留在突触中 行动受限；在长时程增强（LTP，记忆形成的分子基础）期间，我们 量化了它们的数量，发现在它们的维持阶段，它们的横向扩散很少见； NMDAR 具有突触外纳米结构域，这可能会阻止其数量在 LTP 期间增加。但这些是吗？ 其他结果，对吗？为了验证这些初步结果，我们将测量 iGluR，主要是 AMPAR，使用三种不同的 SRFM 技术，每种技术都有自己的优点和 缺点。我们还将确定突触的 3D 方向、探针尺寸和类型的影响、 LTP 激活的详细信息，并定量确定每个突触处 iGluR 的数量。结果 将对这三种技术进行比较。 创新：每种 SRFM 技术都有新的方面，特别是在神经科学方面。首先，我们将 改进 PALM/STORM 技术（SRFM 的一种）以测试 iGluR 的分布和动态 准确。我们将使用新的探针——纳米抗体和 scFv——来对抗突触后蛋白和 iGluR，并且 针对 iGluR 测试新的 SQD 和新的交联试剂。我们还将确定方向和位置 首先通过标记 Neuroligin 和各种突触前蛋白（例如 Bassoon 和 RIM1/2）来对突触区进行标记 在基础条件下，然后用化学 LTP (cLTP)。其次，我们将使用和开发另一个PAINT SRFM 形式，最近被证明速度提高了 100 倍，并且具有出色的空间 分辨率—0.2 秒内约 5 纳米。我们将证明定量-PAINT可以应用于固定神经元 并可用于测量单个突触上的 cLTP。这是我们第一次将 PAINT 应用到生活中 神经元在生理条件下测量 AMPAR 动态。通过 PAINT，我们将能够测试如何 每个突触有许多 iGluR，无论它们是突触内的还是突触外的，以及 iGluR 的数量如何 随 cLTP 改变。第三，我们将利用带有 iGluR 的荧光激活蛋白 (FAP)，并证明 可以在没有背景的情况下以纳米分辨率测量活体神经元中的受体数量 对 cLTP 的潜在快速响应。因此，该方法将提供 iGluR 结构和动力学的另一个测试。