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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neuron loss postsynaptic preservation presynaptic reconstruction sample fixation tau Proteins ultra high resolution unpublished works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.

神经元通讯发生在称为突触的细胞间连接处，并减弱突触可塑性。虽然突触可塑性是学习和记忆的基础，可塑性与突触丧失和记忆衰退有关。突触可塑性部分表现为谷氨酸门控AMPA受体（AMPAR）水平的变化。距离尺度为~10-25 nm，因此，需要~10 nm的分辨率来确定纳米畴和纳米柱等结构。测量这种由于细胞密度非常高，蛋白质，尤其是海马体。虽然许多超分辨率荧光显微镜（SRFM）技术， niques（最常见的dSTORM）存在于分离的神经元培养物中对AMPAR进行成像，但很少有高分辨率应用于大脑切片：那些只看边缘附近（几微米深）或使用表位标记亚基的敲入小鼠。此外，减少蛋白质聚集的方法，如扩增和清理，涉及广泛的组织操作。因此，在未经修饰的脑切片上的SRFM被认为是 “黄金标准”。这项技术提案（PAR-22-127）的目标是开发新形式的SRFM，在未修饰突触可塑性过程中，在突触和非突触结构域中分离天然表面AMPAR，将脑切片作为健康和导致记忆丧失的tau蛋白病的功能。为了区分突触和非突触- 细胞膜上的突触AMPAR需要多色SRFM技术来定义空间分辨率。 AMPAR周围的突触蛋白的作用。在我们未发表的工作中，我们选择性地标记了native AMPAR亚基GluA 2 -4在活的200 µm厚脑切片的细胞表面上使用小的化学标记名为CAM 2-Alexa 647的代理。固定并切片至约30 µm后，在突触后膜上标记切片。蛋白Homer 1与第二SRFM染料（CF 568）的杂交。然后使用双色3D成像技术对AMPAR和Homer 1进行成像， dSTORM与像差校正显微镜和变形镜，导致20 x20 x90 nm的3D分辨率，在大脑切片上的免疫反应。这在以前是没有做到的。在具体目标1中，我们将扩展这一点努力实现分辨率约为11 x 11 x 40 nm的3色SRFM，以确定与巴松管的AMPAR距离或RIM 1，从而识别突触与非突触AMPAR。过度磷酸 tau将用第四SRFM颜色标记。提出了一种新的自干涉SRFM技术，称为SELFI，沿着 3D-dSTORM也将用于简化光学系统。在具体目标2中，我们的目标是改进分辨率（< 10 nm），通过连续将天然脑切片切成“薄”切片（0.7~4 µm），并进行数字记录，将它们的图像与原始厚度合并。配备了新的相机，激光，荧光染料和软件，我们预计30-100倍的速度提高，可能会将3D dSTORM和SELFI扩展到其他部分，个脑袋我们将在完善的系统中验证这些技术，用于表面AMPAR变化，包括髋关节- 突触可塑性、恐惧诱导的学习和tau蛋白病。