Developing a Strategy for 4-Color in Vivo Two-Photon Imaging

开发体内四色双光子成像策略

基本信息

批准号：
10577846
负责人：
Elly Nedivi
金额：
$ 19.37万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2024-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10577846
关键词：
AMPA Receptors Address Affect Bite Brain Brain Diseases Calcium Categories Cell Surface Receptors Cell surface Cells Color Communication Complex Cre driver DLG4 gene Detection Development Disease Disease model Environment Erythrocytes Excitatory Synapse Family GABA Receptor Genetic Glutamate Receptor Goals Image Impairment Individual Knockout Mice Label Mediating Microscope Microscopic Microscopy Molecular Monitor Morphology Mus N-Methyl-D-Aspartate Receptors Nerve Degeneration Neurons PHluorin Proteins Pyramidal Cells Reporter Side Site Source Structure Synapses Synaptic Cleft Synaptic Receptors Synaptophysin Testing Thalamic structure Time Transgenic Organisms Vertebral column Visualization cell type design experience fluorophore hippocampal pyramidal neuron in vivo in vivo monitoring in vivo two-photon imaging mutant nerve supply optical spectra postsynaptic presynaptic pup receptor scaffold sensor synaptogenesis tool two photon microscopy two-photon

项目摘要

Many neurodevelopmental and neurodegenerative brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Yet our ability to investigate how such disorders affect synaptic structure and function is severely limited by the difficulty of visualizing synapses in the living brain and tracking their varied protein components. We propose developing and testing new labeling and microscope configurations that would enable simultaneous live tracking of up to four cellular proteins in the context of the intact mouse brain. The aims below put forth two 4-color constellations that are designed to address distinct classes of experimental questions. Adjustments to the two-photon microscope design that could accommodate both aims would primarily be in the detection path. Aim 1: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to four proteins situated on both sides of the synaptic cleft. The 4-color constellation in this aim is designed to enable in vivo monitoring of a presynaptic afferent label in addition to two postsynaptic proteins and a postsynaptic cell fill. We will label two excitatory post-synaptic markers that are considered mutually exclusive, PSD95 and PSD93, in the context of a thalamic afferent genetic label. In this scenario, tdTomato labels thalamic afferents, eYFP serves as a cell fill, PSD95-teal labels mature excitatory synapses, and PSD93 fused to a far red fluorophore (iRFP682) labels immature excitatory synapses. With this labeling one could ask questions such as, what is the ratio and dynamics of thalamic innervation to mature PSD95 positive spines vs immature PSD93 positive spines? This 4-color combination could also be used to monitor any two postsynaptic labels in combination with any cell-type specific afferent label. Aim 2: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to four postsynaptic fluorophores, one of these being green. There are several categories of fluorophores in the green range that would be particularly useful to combine with structural markers. Most notably, the pH-sensitive GFP mutant, Super ecliptic pHluorin (SEP) used to tag and track synaptic receptors, and the GCaMP family of calcium sensors, broadly used to monitor neuronal activity. Unfortunately, a green fluorophore is incompatible with most blue and yellow labels due to overlap of their emission spectra. Here pyramidal neurons would co-express: a red cell fill (mScarlet) to label dendritic morphology, PSD95-iRFP682 and PSD93-BFP (a short wavelength blue) to label mature and immature excitatory synapses, respectively, and SEP-GluR1. We could then ask questions such as, what are the dynamics of AMPA receptor insertion into mature PSD95 positive vs immature PSD93 positive spines? This 4-color combination could also be used to tag and track NMDA or GABA receptor subunits with SEP. Swapping the SEP-tagged receptor for a GCaMP calcium sensor would further expand the possibilities of this 4-color constellation by enabling the integration of an activity reporter with synaptic labeling.

许多神经发育和神经退行性脑部疾病表现为突触完整性、稳定性受损、和依赖经验的选择，导致接线缺陷和功能扰动。然而我们的能力研究此类疾病如何影响突触结构和功能受到困难的严重限制可视化活体大脑中的突触并追踪其不同的蛋白质成分。我们建议开发并测试新的标签和显微镜配置，以实现同时实时跟踪多达完整小鼠大脑中的四种细胞蛋白。下面的目标提出了两个 4 色星座旨在解决不同类别的实验问题。双光子的调整能够适应这两个目标的显微镜设计主要是在检测路径中。目标 1：开发并实施光谱分辨双光子显微镜，可同时跟踪多达四种蛋白质位于突触间隙两侧。此目标中的 4 色星座旨在除了两个突触后蛋白和一个突触前传入标签外，还可以进行体内监测突触后细胞填充。我们将标记两个被认为是相互排斥的兴奋性突触后标记， PSD95 和 PSD93，在丘脑传入遗传标签的背景下。在这种情况下，tdTomato 标记丘脑传入神经，eYFP 作为细胞填充，PSD95-青色标记成熟的兴奋性突触，PSD93 融合到远红色荧光团 (iRFP682) 标记未成熟的兴奋性突触。有了这个标签，人们就可以提出这样的问题：例如，成熟 PSD95 阳性棘与不成熟 PSD93 的丘脑神经支配的比率和动态是多少正脊椎？这种 4 色组合也可用于监测任意两个突触后标签与任何细胞类型特异性传入标签组合。目标 2：开发和实施光谱解析双光子显微镜可同时跟踪多达四个突触后荧光团，其中之一是绿色的。绿色范围内有几类荧光团特别有用与结构标记结合。最值得注意的是，pH 敏感的 GFP 突变体，超黄道 pHluorin (SEP) 用于标记和跟踪突触受体，GCaMP 系列钙传感器广泛用于监测神经元活动。不幸的是，绿色荧光团与大多数蓝色和黄色标签不兼容，因为它们的发射光谱重叠。这里锥体神经元将共同表达：红细胞填充（mScarlet）来标记树突形态、PSD95-iRFP682 和 PSD93-BFP（短波长蓝色）来标记成熟和未成熟分别是兴奋性突触和 SEP-GluR1。然后我们可以提出诸如动态是什么之类的问题 AMPA 受体插入成熟 PSD95 阳性与未成熟 PSD93 阳性棘的区别？这个4色组合还可用于与 SEP 一起标记和追踪 NMDA 或 GABA 受体亚基。交换 GCaMP 钙传感器的 SEP 标记受体将进一步扩展这种 4 色的可能性通过将活动报告器与突触标记相集成来构建星座。