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

开发体内四色双光子成像策略

• 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
• 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

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疾病表现为突触完整性、稳定性、 以及依赖经验的选择，导致连接缺陷和功能紊乱。然而，我们有能力 研究这些障碍如何影响突触结构和功能受到以下困难的严重限制 在活着的大脑中可视化突触，并跟踪它们的不同蛋白质成分。我们建议发展 并测试新的标签和显微镜配置，可以同时实时跟踪多达 在完整的小鼠大脑中的四种细胞蛋白质。下面的目标提出了两个四色星座 旨在解决不同类别的实验问题。对双光子的调整 能够同时满足这两个目的的显微镜设计主要是在探测路径上。目标1：实现 开发和实施光谱分辨双光子显微镜，用于同时跟踪 四种蛋白质位于突触裂隙的两侧。这一目标中的四色星座旨在 除了两个突触后蛋白和一个 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，在丘脑传入基因标签的背景下。在这个场景中，tdTomato将丘脑 传入，EYFP作为细胞填充物，PSD95-TEAL标记成熟的兴奋性突触，PSD93与FAR融合 红色荧光团(IRFP682)标记未成熟的兴奋性突触。有了这个标签，人们可以问这样的问题 AS，丘脑神经支配与成熟的PSD95阳性棘和未成熟的PSD93的比率和动力学是什么 正脊椎？这种4色组合也可以用来监测脑内任何两个突触后标记 与任何细胞类型的特定传入标签组合。目标2：开发和实现频谱解析 双光子显微镜用于同时跟踪最多四个突触后荧光团，其中之一 是绿色的。在绿色范围内有几种类别的荧光团将特别有用 与结构标记相结合。最值得注意的是，对pH敏感的绿色荧光蛋白突变体，超级黄道PHluorin(SEP) 用于标记和跟踪突触受体，以及GCaMP钙传感器家族，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。在这里锥体神经元将共表达：一个红细胞填充(MScarlet)标记 树突形态、PSD95-iRFP682和PSD93-BFP(短波长蓝)标记成熟和未成熟 兴奋性突触和SEP-GluR1。然后我们可以问这样的问题，比如，动力是什么 AMPA受体插入成熟PSD95阳性与未成熟PSD93阳性脊柱的比较？这是四色 SEP结合也可用于标记和追踪NMDA或GABA受体亚基。互换 用于GCaMP钙传感器的SEP标记受体将进一步扩大这种4色的可能性 通过启用活动报告器与突触标记的集成来实现星座。