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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：能够在纳米水平上测量神经元通讯的分子机制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。在特别地，AMPA-和NMDA-型谷氨酸受体（称为iGluRs）动态地参与神经元- 通过薄（约30 nm）突触的神经元通信;当失调时，神经退行性疾病结果，如阿尔茨海默氏症和帕金森氏症。我们和其他人一直在跟踪这些事件，纳米分辨率使用超分辨率荧光显微镜（SRFM）。使用小探针-量子点（直径约12纳米）和其他光稳定的荧光团，在我们的实验室开发的前赠款，我们带来了一些惊喜我们发现大部分AMPAR位于突触中，在长时程增强（LTP，记忆形成的分子基础）过程中，量化了它们的数量，并发现在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，这可能会阻止它们的数量在LTP期间上升。但这些，其他结果，对吗？为了验证这些初步的结果，我们将测量的位置和扩散的 iGluR，主要是AMPAR，使用三种不同的SRFM技术，每一种都有自己的优点，缺点我们还将确定突触的3D方向，探针大小和类型的影响， LTP激活的细节，并定量确定每个突触处iGluR的数量。结果这三种技术将进行比较。创新：每种SRFM技术都有新的方面，特别是在神经科学方面。一是改进PALM/STORM技术（SRFM的一种），以进一步检测iGluRs的分布和动态准确地我们将使用针对突触后蛋白和iGluRs的新探针-纳米抗体和scFv，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，通过标记神经连接素和各种突触前蛋白，如巴松管和RIM 1/2，在基础条件下，然后用化学LTP（cLTP）。其次，我们将使用和开发PAINT，另一个 SRFM的一种形式，最近已被证明具有100倍的速度增加，具有良好的空间分辨率-在0.2秒内达到105纳米。我们将表明，定量PAINT可以应用于固定的神经元并可用于测量单个突触上的cLTP。我们将第一次将油漆应用于神经元在生理条件下测量AMPAR动力学。通过PAINT，我们将能够测试如何每个突触都有许多iGluR，无论它们是突触还是突触外的，以及iGluR的数量如何改变cLTP第三，我们将利用荧光激活蛋白（FAP）与iGluRs，并表明，受体的数量可以在活体神经元中以纳米分辨率测量，没有背景，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种测试。

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.

具有不同尺寸荧光探针的突触和突触外AMPA受体的超分辨率成像。

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。