Super-Resolution Fluorescence Microscopy of Synaptic Plasticity on Unmodified Brain Slices in Health and Tauopathy

神经元通信发生在称为突触的细胞间连接处，这种突触可以动态地加强 并削弱了突触的可塑性。而突触可塑性是学习和记忆的基础，异常 可塑性与突触丧失和记忆力下降有关。突触可塑性在一定程度上是通过 谷氨酸门控AMPA受体(AMPAR)水平的变化。距离尺度是~10-25 nm，因此， 需要~10 nm的分辨率来确定像纳米结构域和纳米柱这样的结构。测量这样的 自然脑片中纳米级的变化是困难的，因为细胞和 蛋白质，特别是在海马体中。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中以成像AMPAR，很少有 将高分辨率应用于脑切片：那些确实有分辨率的切片，只看边缘附近(几微米深)或使用 敲入表位标记亚单位的小鼠。此外，蛋白质降解的方法，如膨胀法和 清除，涉及广泛的组织处理。因此，未经修改的脑片上的SRFM被认为是 “金本位制”。该技术提案(PAR-22-127)的目标是开发能够 在突触可塑性过程中分离突触和非突触区域的天然表面AMPAR 由于健康和紧张症而导致记忆丧失的脑片。辨别突触与非突触 细胞膜上的突触AMPAR需要多色SRFM技术来确定空间分辨率。 围绕表面AMPAR的突触蛋白的连接。在我们未发表的作品中，我们选择性地将其标记为原生的 200微米厚脑片细胞表面Ampar亚单位GluA2-4的化学标记 探员打了CAM2-Alexa647。固定后切片至~30微米，在突触后标记 蛋白质，Hmer 1，第二个SRFM染料(CF568)。然后使用双色3D对Ampar和Hmer 1进行成像 DSTORM具有像差校正显微镜和可变形反射镜，可获得20x20x90 nm的3D分辨率。 在本地的脑片上涂上药水。这是以前从未实现过的。在具体目标1中，我们将扩大这一范围 努力实现分辨率约为11 x 11 x 40 nm的三色SRFM，以确定AMPAR与基风的距离 或RIM1在突触前终末，从而识别突触AMPAR与非突触AMPAR。过度磷酸化 Tau将被贴上第四种SRFM颜色的标签。一种新的自干涉SRFM技术，称为SELFI，以及 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(&lt；10 nm)，通过将本地大脑切片连续切片成“薄”的切片(0.7~4微米)，并以数字方式重现- 将他们的图像压缩到原来的厚度。配备了新的相机，激光，荧光染料和软件， 我们预计速度会提高30-100倍，可能会将3D dSTORM和SELFI扩展到 大脑。我们将在成熟的表面AMPAR变化系统中验证这些技术，包括HIP- 海马区突触可塑性、恐惧诱导的学习和牵张症。

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.