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

基本信息

批准号：
10729062
负责人：
Hee Jung Chung
金额：
$ 187.08万
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：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10729062
关键词：
3-Dimensional AMPA Receptors Acute Age Age Months Alzheimer&apos s Disease Area Automation Brain Brain region Buffers Cell Density Cell membrane Cell surface Cellular Structures Chemicals Color Communication Computer software DLG4 gene Dementia Dendritic Spines Disease Dissociation Dyes Electrophysiology (science)Environment Epitopes Excitatory Synapse Fluorescence Fluorescence Microscopy Fluorescent Dyes Fright Frontotemporal Dementia Glutamate Receptor Glutamates Goals Health Hippocampus Homer 1 Human Image Imaging Techniques Individual Intercellular Junctions Knock-in Mouse Label Lasers Learning Length Long-Term Potentiation Masks Measures Mediating Memory Memory Loss Methods Microscope Microtomy Mus Mutation Nerve Neurons Optics Phase Physiology Postsynaptic Membrane Presynaptic Terminals Proteins Resolution Sampling Silicone Oils Site Slice Speed Structure Surface Synapses Synaptic plasticity System Tauopathies Techniques Technology Testing Thick Thinness Tissues Visualization Work brain tissue conditioned fear density detector digital fluorophore hippocampal pyramidal neuron hyperphosphorylated tau imaging capabilities improved light scattering meter mutant nano nanobodies nanocolumn nanometer nanometer resolution nanoscale neuron loss postsynaptic preservation presynaptic reconstruction sample fixation tau Proteins ultra high resolution unpublished works

项目摘要

Neuronal communication occurs at intercellular junctions called synapses, which can dynamically strengthen and weaken — termed synaptic plasticity. While synaptic plasticity underlies learning and memory, abnormal plasticity is associated with synapse loss and memory decline. Synaptic plasticity is expressed, in part, by changes in the level of glutamate-gated AMPA receptors (AMPARs). The distance scale is ~10-25 nm, and thus, ~10 nm resolution is needed to ascertain structures like nanodomains and nanocolumns. Measuring such changes at the nanometer level in native brain slices is difficult due to the extraordinary high density of cells and proteins, particularly in the hippocampus. While many super-resolution fluorescence microscopy (SRFM) tech- niques (most commonly dSTORM) exist to image AMPARs in dissociated neuronal culture, very few have been applied with high resolution to brain slices: the ones that do, look only near the edge (a few µm deep) or use knock-in mice of epitope-tagged subunits. In addition, methods to decrowd proteins, such as expansion and clearing, involve extensive tissue manipulation. As a result, SRFM on unmodified brain slices is considered the ‘gold standard’. The goal of this technology proposal (PAR-22-127) is to develop new forms of SRFM that can isolate native surface AMPARs in both synaptic and non-synaptic domains during synaptic plasticity on unmod- ified brain slices as a function of health and tauopathy that cause memory loss. To identify synaptic vs non- synaptic AMPARs on the cell membrane necessitates multi-color SRFM techniques to define the spatial resolu- tion of synaptic proteins surrounding surface AMPARs. In our unpublished work, we selectively labeled native AMPAR subunits GluA2-4 on the cell surface of live 200 µm thick brain slices using a small chemical labeling agent called CAM2-Alexa647. After fixation and sectioning to ~30 µm, the slices were labeled on the postsynaptic protein, Homer1, with a second SRFM dye (CF568). AMPAR and Homer1 were then imaged using two-color 3D dSTORM with an aberration corrected microscope and deformable mirrors, resulting in 20x20x90 nm 3D reso- lution on a native brain slice. This has not previously been accomplished. In Specific Aim 1, we will extend this work to achieve 3-color SRFM with ~ 11 x 11 x 40 nm resolution to determine AMPAR distances from Bassoon or RIM1 in the presynaptic terminal, thereby identifying synaptic vs non-synaptic AMPARs. Hyperphosphorylated tau will be labeled with a 4th SRFM color. A new self-interferometric SRFM technique, called SELFI, along with 3D-dSTORM, will also be used with the goal of simplifying the optics. In Specific Aim 2, we aim for an improved resolution (< 10 nm) by serially slicing the native brain slices into “thin” sections (0.7~4 µm) and digitally recom- bining their images to the original thickness. Equipped with new cameras, lasers, fluorescent dyes, and software, we expect a 30-100-fold speed improvement, possibly extending 3D dSTORM and SELFI to other parts of the brain. We will validate these techniques in well-established systems for surface AMPAR change including hip- pocampal synaptic plasticity, fear-induced learning, and tauopathy.

神经元通信发生在称为突触的细胞间连接处，可以动态增强和弱 - 称为合成可塑性。虽然合成可塑性是学习和记忆的基础，但异常可塑性与突触丧失和记忆力下降有关。突触可塑性部分通过谷氨酸门控AMPA受体（AMPARS）的水平变化。距离尺度为〜10-25 nm，因此需要〜10 nm的分辨率来确定纳米构和纳米序列等结构。测量这样的由于细胞的高密度和蛋白质，特别是在海马中。而许多超分辨率荧光显微镜（SRFM）技术在分离的神经元培养中存在图像AMPAR的Niques（最常见的DSTORM），很少有用高分辨率涂在大脑切片中：那些这样做的，只看在边缘附近（深几µm）或使用表位标签亚基的敲门鼠。另外，删除蛋白质的方法，例如扩展和清除，涉及广泛的组织操作。结果，未修饰的大脑切片上的SRFM被认为 “黄金标准”。该技术建议的目标（PAR-22-127）是开发新形式的SRFM形式突触可塑性期间突触型和非突触结构域的分离天然表面AMPAR在非模型上 IFIDIED大脑切片是导致记忆力丧失的健康和tauopathy的函数。确定突触与非 - 细胞膜上的突触AMPAR需要多色SRFM技术来定义空间分辨率 - 表面AMPAR周围的突触蛋白的影响。在未发表的作品中，我们有选择地标记为本地 AMPAR亚基GLUA2-4在活体200 µm厚脑切片的细胞表面上使用小型化学标记特工称为CAM2-ALEXA647。固定并截至约30 µm之后，将切片标记在突触后。蛋白质，HOMER1，具有第二个SRFM染料（CF568）。然后使用两色3D成像AMPAR和HOMER1 DSTORM具有纠正的显微镜和可变形的镜子，导致20x20x90 nm 3D reso- 在本地大脑切片上流式传输。以前尚未实现这一目标。在特定目标1中，我们将扩展用〜11 x 11 x 40 nm分辨率实现3色SRFM的工作，以确定与巴松的AMPAR距离或突触前末端中的RIM1，从而识别突触与非突触的AMPAR。高磷酸化 Tau将用第4个SRFM颜色标记。一种新的自我开采计量学SRFM技术，称为selfi，以及 3D-DSTORM也将用于简化光学的目标。在特定目标2中，我们的目标是改进分辨率（<10 nm）通过串行将天然脑切片切成“薄”部分（0.7〜4 µm），并以数字方式推荐将他们的图像凸出到原始厚度。配备新的相机，激光器，荧光染料和软件，我们希望提高30-100倍的速度，可能将3D DSTORM和SEFFI扩展到脑。我们将验证这些技术在公认的系统中，以进行表面AMPAR变化，包括髋关节 Pocampal突触可塑性，恐惧引起的学习和tauopathy。