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

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:能够在纳米尺度上测量神经元通讯的分子机制

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:
  • 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.
Synapses without tension fail to fire in an in vitro network of hippocampal neurons
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:
  • 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。
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Hee Jung Chung其他文献

Hee Jung Chung的其他文献

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{{ truncateString('Hee Jung Chung', 18)}}的其他基金

Super-Resolution Fluorescence Microscopy of Synaptic Plasticity on Unmodified Brain Slices in Health and Tauopathy
健康和 Tau 病未修饰脑切片突触可塑性的超分辨率荧光显微镜
  • 批准号:
    10729062
  • 财政年份:
    2023
  • 资助金额:
    $ 37.68万
  • 项目类别:
Dynamic changes in PIP2 binding sites and their impact on axonal targeting and function of epilepsy-associated KCNQ/Kv7 channels
PIP2 结合位点的动态变化及其对癫痫相关 KCNQ/Kv7 通道的轴突靶向和功能的影响
  • 批准号:
    10744934
  • 财政年份:
    2023
  • 资助金额:
    $ 37.68万
  • 项目类别:
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:
    10467027
  • 财政年份:
    2017
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Small Quantum Dots and Advanced Imaging Tools
使用小量子点和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:
    9975253
  • 财政年份:
    2017
  • 资助金额:
    $ 37.68万
  • 项目类别:
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
  • 财政年份:
    2017
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Small Quantum Dots and Advanced Imaging Tools
使用小量子点和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:
    9384063
  • 财政年份:
    2017
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
  • 批准号:
    9160604
  • 财政年份:
    2016
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
  • 批准号:
    9918990
  • 财政年份:
    2016
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
  • 批准号:
    9478382
  • 财政年份:
    2016
  • 资助金额:
    $ 37.68万
  • 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
  • 批准号:
    9274105
  • 财政年份:
    2016
  • 资助金额:
    $ 37.68万
  • 项目类别:
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