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出突触完整性，稳定性，稳定性的受损 以及与经验有关的选择，导致接线缺陷和扰动功能。但是我们的能力 研究这种疾病如何影响突触结构和功能受到严重限制 可视化活大脑中的突触并跟踪其多样的蛋白质成分。我们建议发展 并测试新的标签和显微镜配置，这些配置可以同时实时跟踪 在完整的小鼠大脑的背景下，四个细胞蛋白。下面的目的提出了两个4色星座 旨在解决不同类别的实验问题。对两光子的调整 可以容纳这两个目标的显微镜设计主要是在检测路径中。目标1：到 开发和实施光谱解决的两光子显微镜，以同时跟踪到 四个蛋白质位于突触裂缝的两侧。此目标中的4色星座旨在 除了两种突触后蛋白和A外，还可以在体内监测突触前的传入标签 突触后细胞填充。我们将标记两个兴奋性后突触后标记，它们被认为是相互排斥的， 在丘脑传入遗传标签的背景下，PSD95和PSD93。在这种情况下，tdtomato标记丘脑 传入，EYFP用作细胞填充，PSD95-TEAL标签成熟的兴奋性突触，PSD93融合到远处 红色荧光团（IRFP682）标记不成熟的兴奋性突触。有了这个标签，一个人可能会问这样的问题 AS，丘脑神经与成熟的PSD95阳性刺与不成熟psd93的比率和动力学是什么 阳性刺？这种4色的组合也可用于监视任何两个突触后标签 与任何细胞类型的特定传入标签结合使用。目的2：开发和实施光谱解决 两光子显微镜用于同时跟踪多达四个突触后荧光团，其中之一 是绿色的。绿色范围内有几种类别的荧光团，这将特别有用 与结构标记结合。最值得注意的是，对pH敏感的GFP突变体，超黄道素（SEP） 用于标记和跟踪突触受体以及钙传感器的GCAMP家族，广泛用于监测 神经元活性。不幸的是，由于 它们的排放光谱重叠。在这里，金字塔神经元将共表达：红细胞填充（mscarlet）标记 树突形态，PSD95-IRFP682和PSD93-BFP（短波长蓝色），标记成熟和不成熟 兴奋性突触和sep-glur1。然后，我们可以提出诸如动态的问题 AMPA受体插入到成熟的PSD95阳性与未成熟pSD93阳性棘中？这个四色 组合也可以用于标记和跟踪具有SEP的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大该4色的可能性 通过启用与突触标记的活动报告的整合来通过星座。