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

基本信息

批准号：
9975253
负责人：
Hee Jung Chung
金额：
$ 31.57万
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 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9975253
关键词：
3-Dimensional AMPA Receptors Alzheimer&apos s Disease Basic Science Behavior Biological Biological Sciences Biology Caliber Caring Cells Chemicals Clinical Color Communication DLG1 gene DLG4 gene Detection Diffusion Dyes Ensure Event Fluorescence Fluorescent Probes Future Generations Glutamate Receptor Goals Grant Grant Review Health Hour Image Imaging Device Integral Membrane Protein Label Learning Ligands Long-Term Depression Long-Term Potentiation Measurement Measures Memory Methods Microscope Microscopy Minority Misinformation Molecular Monitor N-Methylaspartate Neurodegenerative Disorders Neurons Neurosciences Neurotransmitter Receptor Optics Output Paper Parkinson Disease Photons Problem Solving Process Proteins Publishing Quantum Dots Regulation Resolution S-nitro-N-acetylpenicillamine Sampling Site Stroke Surface Synapses Synaptic Cleft Synaptic plasticity Technology Temperature Testing Time base density design experimental study fluorophore microscopic imaging nanometer nanoscale prevent receptor single molecule tool trafficking

项目摘要

Abstract The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact in both basic bioscience and in future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (AMPARs/NMDARs, known as iGluRs) are involved in neuron-to-neuron communication across synapses, where these receptors contribute to learning and memory, and when dysregulated, to neurodegenerative diseases including Alzheimer's, Parkinson's and complications from strokes. A critical mechanistic event is the transport of iGluRs into and out of synapses (or parts of synapses) in a dynamic process called synaptic plasticity. A revolution is underway because of the recent ability to resolve these events at the nanometer-scale using fluorescence super-resolution microscopy (FSRM). However significant inherent problems with this technology have led to confounding results and misinformation. The biggest problem has been with the fluorescent probes used to image receptors: conventional organic fluorescent probes last only a few seconds; commercial (and big) quantum dots (bQDs), despite their exceptional brightness and photostability, are over 20 nm in diameter and are too large to fit inside the synaptic cleft where iGluRs are active. We recently overcame this problem through an R21, which enabled us to develop small quantum dots (sQDs) that are <10 nm in diameter. They specifically label iGluRs in the synaptic cleft, which is just ~20-30 nm wide. The sQDs do this with tremendous brightness and stability, resulting in FSRM images in 3-dimensions with 100 ms time-resolution for greater than 2 minutes of continuous excitation. In contrast, bQD-labeled AMPARs are predominantly stuck in the extra-synaptic space because steric hindrance prevents them from going inside. We have recently extended these findings with a newer sQD that is completely stable, and with small organic fluorophores that we now show are stable enough, on live neurons (which previously had been too photolabile for such measurements.) Our findings, some of which have been published in 3 papers resulting from our R21 grant, may have tremendous implications for basic science and health: the surface mobility and trafficking of iGluRs, which depend on the ease of diffusion inside and outside of synapses, regulates synaptic efficacy. Here we wish to understand the distribution and dynamics of iGluRs, both within the synapses and between synapses, using our new sQDs and other new photoactivatable fluorescent proteins and some organic fluorophores. For this, a number of new advances in optics, probe design, and care with receptor monovalency are necessary. After these technical problems are solved (which will be useful to answer many different biological questions), we will validate the biology that we have observed, and to apply these to proof-of-principle experiments involved in two key biological questions: 1) In what way do receptors move into and around the synapse during homeostatic and synaptic plasticity? 2) Do endocytosed receptors communicate with each other between synapses on the same neuron?

抽象的在纳米空间尺度上测量神经元通讯的分子机制的能力将对基础生物科学和未来的临床神经科学产生巨大影响。特别是，AMPA- 和 NMDA 型谷氨酸受体（AMPAR/NMDAR，称为 iGluR）参与神经元间的传递突触间的交流，这些受体在其中有助于学习和记忆，以及何时失调，导致神经退行性疾病，包括阿尔茨海默氏症、帕金森氏症和并发症笔画。一个关键的机制事件是 iGluR 进出突触（或部分突触）的运输在称为突触可塑性的动态过程中。一场革命正在进行，因为最近有能力使用荧光超分辨率显微镜（FSRM）在纳米尺度上解决这些事件。然而，这项技术的重大固有问题导致了令人困惑的结果和错误信息。最大的问题是用于成像受体的荧光探针：传统的有机探针荧光探针只能持续几秒钟；商业（和大）量子点（bQD），尽管它们卓越的亮度和光稳定性，直径超过 20 nm，太大而无法放入突触内 iGluRs 活跃处的裂缝。我们最近通过 R21 克服了这个问题，这使我们能够开发直径 <10 nm 的小型量子点 (sQD)。他们专门标记突触中的 iGluR 裂缝，宽度仅为 20-30 nm。 sQD 具有极高的亮度和稳定性，从而实现了这一点 3 维 FSRM 图像，时间分辨率为 100 毫秒，连续激励时间超过 2 分钟。相比之下，bQD 标记的 AMPAR 主要停留在突触外空间，因为空间位阻障碍物阻止他们进入。我们最近用更新的 sQD 扩展了这些发现是完全稳定的，并且我们现在证明在活神经元上具有足够稳定的小有机荧光团（以前对于此类测量来说太不耐光了。）我们的发现，其中一些已经被由我们的 R21 资助产生的 3 篇论文发表，可能对基础科学和健康：iGluR 的表面流动性和贩运，这取决于内部和外部扩散的难易程度突触，调节突触功效。在这里我们希望了解 iGluR 的分布和动态，在突触内和突触之间，使用我们的新 SQD 和其他新的光激活荧光蛋白和一些有机荧光团。为此，光学、探针等领域的多项新进展设计和注意受体单价是必要的。这些技术问题解决后（即将有助于回答许多不同的生物学问题），我们将验证我们所拥有的生物学观察到的，并将这些应用于涉及两个关键生物学问题的原理验证实验：1）在稳态和突触可塑性过程中，受体以什么方式进入突触及其周围？ 2) 做内吞受体在同一神经元的突触之间相互通信？