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

基本信息

批准号：
10684709
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
项目类别：
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/10684709
关键词：
3-Dimensional Alzheimer&apos s Disease Avidin Binding Biological Sciences Cell membrane Cells Chemicals Clinical Color Communication Cross-Linking Reagents DNA DNA Probes Diameter Diffusion Disadvantaged Dissociation Dyes Event Evolution Fluorescence Fluorescence Microscopy Future Glutamate Receptor Grant Hippocampus 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 Neurodevelopmental Disorder 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 Visualization crosslink density dimer experimental study facsimile fluorophore imager improved innovation monomer nano nanobodies nanometer postsynaptic postsynaptic density protein presynaptic presynaptic density protein 95 receptor response single molecule stoichiometry superresolution microscopy temporal measurement tool trafficking ultra high resolution

项目摘要

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：测量纳米分子通信的分子机制的能力空间量表将对基本的生物科学和未来的临床神经科学产生巨大影响。在特别是，AMPA和NMDA型谷氨酸受体（称为iGlurs）动态参与神经元横跨薄（≈30nm）突触的通信；失调时神经退行性疾病结果，例如阿尔茨海默氏症和帕金森氏病。我们以及其他人 - 与使用超分辨率荧光显微镜（SRFM）的纳米分辨率。使用小问题 - 量子点（≈12nm的直径）和其他光稳定荧光团，在我们的实验室中开发的先前赠款 - 我们提出了一些惊喜。我们发现，很大一部分AMPAR位于他们的突触中流动性受到限制；在长期训练期间（LTP，记忆形成的分子基础），我们已经量化其数量并在维护阶段发现其横向扩散很少见； NMDARS 具有突触外纳米域，可以防止其数量在LTP期间增加。但是这些是其他结果，对吗？为了验证这些初步结果，我们将衡量的位置和扩散 iglurs，主要AMPAR，使用三种不同的SRFM技术，每种技术都有其自己的优势缺点。我们还将确定突触的3D方向方法，探针大小和类型的效果， LTP激活的细节，并定量确定每个突触处的iGlurs数量。结果将比较这三种技术之间。创新：每种SRFM技术都有新的方面，尤其是在神经科学方面。首先，我们会的改进棕榈/风暴技术（一种类型的SRFM），以更多地测试iGlurs的分布和动力学准确。我们将使用新的问题（纳米动物和scfv），使后突触后蛋白质和iGlurs，以及测试针对iGlurs的新SQD和新的交联试剂。我们还将确定方向和位置通过标记神经素和各种突触前蛋白（例如低音和RIM1/2）的标记，首先是合成区域在基本条件下，然后使用化学LTP（CLTP）。其次，我们将使用和开发油漆，另一个 SRFM的形式，最近已证明其速度有100倍，具有出色的空间分辨率 - ≈5纳米在0.2秒内。我们将证明可以将定量涂片应用于固定的神经元并且可用于测量单个突触上的CLTP。这是我们第一次将油漆涂在谋生在生理条件下的神经元测量AMPAR动力学。使用油漆，我们将能够测试许多iglurs都有每次突触，无论是突触还是突触外的，以及iglurs的数量用CLTP更改。第三，我们将使用iGlurs利用荧光激活蛋白（FAP），并表明可以在具有纳米分辨率的活神经元中测量接收器的数量，没有背景，并且可能对CLTP的快速响应。因此，该方法将提供iGlur结构和动力学的另一项测试。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses

DOI：
10.3389/fnmol.2020.00010
发表时间：
2020-03-13
期刊：
FRONTIERS IN MOLECULAR NEUROSCIENCE
影响因子：
4.8
作者：
Delgado, Jary Y.;Nall, Duncan;Selvin, Paul R.
通讯作者：
Selvin, Paul R.

A Revised View on the Role of Surface AMPAR Mobility in Tuning Synaptic Transmission: Limitations, Tools, and Alternative Views.

DOI：
10.3389/fnsyn.2018.00021
发表时间：
2018
期刊：
Frontiers in synaptic neuroscience
影响因子：
3.7
作者：
Delgado JY;Selvin PR
通讯作者：
Selvin PR

Synapses without tension fail to fire in an in vitro network of hippocampal neurons

DOI：
10.1073/pnas.2311995120
发表时间：
2023-12-26
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Joy，Md Saddam Hossain;Nall，Duncan L.;Saif，M. Taher A.
通讯作者：
Saif，M. Taher A.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

DOI：
10.7554/elife.27744
发表时间：
2017-07-27
期刊：
eLife
影响因子：
7.7
作者：
Lee SH;Jin C;Cai E;Ge P;Ishitsuka Y;Teng KW;de Thomaz AA;Nall D;Baday M;Jeyifous O;Demonte D;Dundas CM;Park S;Delgado JY;Green WN;Selvin PR
通讯作者：
Selvin PR

Structural Contributions to Hydrodynamic Diameter for Quantum Dots Optimized for Live-Cell Single-Molecule Tracking.

针对活细胞单分子跟踪优化的量子点的流体动力学直径的结构贡献。