A genetic toolkit for targeted connectomics of specific neuronal types

用于特定神经元类型的靶向连接组学的遗传工具包

• 批准号: 9089114
• 负责人: David M. Berson
• 金额: $ 23.36万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089114
• 关键词: Axonal Transport; Biological Preservation; Brain; Brain region; Budgets; Cell membrane; Cells; Cellular Structures; Chimeric Proteins; Communities; Computers; Data; Dendrites; Dependovirus; Dyes; Electron Microscope; Electron Microscopy; Electrons; Enzymes; Fiber; Fingerprint; Fluorescence; Generations; Genetic; Genetically Modified Animals; Green Fluorescent Proteins; Health; Histocytochemistry; Human; Image; Injection of therapeutic agent; Knock-in Mouse; Label; Light; Link; Location; Maps; Masks; Membrane; Membrane Proteins; Methods; Microscopic; Microscopy; Molecular; Molecular Genetics; Mus; Neurons; Neurosciences; Organelles; Output; Partner in relationship; Peroxidases; Population; Presynaptic Terminals; Process; Proteins; Protocols documentation; Reagent; Recombinant adeno-associated virus (rAAV); Recombinants; Reporter; Resolution; Scientist; Serotyping; Structure; Synapses; Synaptic Vesicles; Tissue Sample; Tissues; Viral; Viral Vector; Virus; base; brain volume; cell type; design; design and construction; flexibility; fluorescence microscope; fluorophore; genetic technology; improved; mammalian genome; nanoscale; neural circuit; neuronal cell body; novel; optogenetics; postsynaptic; presynaptic; protein biomarkers; protein transport; public health relevance; recombinase-mediated cassette exchange; reconstruction; relating to nervous system; selective expression; tool; two-photon

 DESCRIPTION (provided by applicant): Serial blockface electron microscopy (SBFEM) is revolutionizing the mapping of neural microcircuits. Small volumes of brain can be fully reconstructed at nanometer-scale resolution, providing a complete description of the form and location of all synaptic inputs to a single cell. For very local inputs, the identity (and synaptic inputs and outputs) of these presynaptic cells can also be reconstructed. This technical breakthrough parallels the transformative impact of genetically modified animals and viruses for characterizing and manipulating molecularly defined neuronal cell types. Using viruses, cre-lox technology, optogenetics, and chemogenetics, distributed functional circuits can be imaged, mapped, activated, silenced or deleted. This proposal seeks to bridge the divide between these approaches. We propose to perfect a method we have devised exploiting molecular-genetic technologies to mark defined cell types with an electron-dense label for SBFEM analysis. Specifically, we have generated a recombinant adeno-associated virus (AAV) that delivers a Cre-dependent genetic construct to infected cells. Exclusively in Cre-expressing cells, the viral payload expresses a membrane-targeted marker protein comprising a fusion of a fluorescent protein (membrane-targeted green fluorescent protein - mGFP) to a recombinant peroxidase enzyme (APEX2). Pilot data show that infected Cre-expressing cells strongly express the fusion protein throughout the membrane (soma, dendrites, axons and terminals). Its bright fluorescence permits detailed confocal analysis; enzyme histochemical processing reveals the same structures by electron-dense marking visible after SBFEM sectioning and imaging. Already, the method has great promise for targeted `connectomic' analysis of Cre-expressing neurons in any brain region and of their output synapses in remote neural structures. Here, we aim to improve and extend the method. Our aims for this proposal are: 1) to optimize the histochemical protocols and design of viral constructs to mark specific cellular structures or compartments without masking synaptic vesicles and other organelles; and 2) to expand the potential applications of the method, by restricting the fluorescent and ultrastructural labeling t neurons that innervate specific targets; and by generating a knock-in mouse line that expresses the marker through mating to a Cre driver line or injection of Cre-expressing viruses.

 描述（由应用程序提供）：串行区块电子显微镜（SBFEM）正在革新神经微电路的映射。可以在纳米尺度的分辨率下完全重建少量大脑，从而完整描述了所有突触输入到单个单元的形式和位置。对于非常本地的输入，身份（和突触 这些突触前细胞的输入和输出也可以重建。这一技术突破与转基因动物和病毒在表征和操纵分子定义的神经元细胞类型方面的变革性影响相似。使用病毒，CRE-LOX技术，光遗传学和化学遗传学，可以成像，映射，激活，沉默或删除分布式功能电路。该提议旨在弥合这些方法之间的鸿沟。我们建议完善一种我们设计的方法，利用分子遗传学技术来标记具有电子致密标签以进行SBFEM分析的定义细胞类型。具体而言，我们已经生成了一种重组腺相关病毒（AAV），该病毒将依赖CRE的遗传构建体提供给感染细胞。该病毒有效载荷仅在表达CRE的细胞中，表达了含膜的标记蛋白，该标记蛋白包括荧光蛋白（拟膜靶向的绿色荧光蛋白-MGFP）融合到重组过氧化物酶酶（APEX2）。试点数据表明，感染的表达Cre的细胞在整个膜上强烈表达融合蛋白（SOMA，树突，轴突和末端）。它的明亮荧光允许详细的共聚焦分析。酶组织化学处理通过SBFEM切片和成像后可见的电子致密标记揭示了相同的结构。该方法已经对任何大脑区域中表达CRE的神经元及其远程神经元输出突触的表达神经元的靶向“连接组”分析有很大的希望。在这里，我们旨在改进和扩展方法。我们的目标是：1）优化病毒构建体的组织化学方案和设计，以标记特定的细胞结构或隔室，而无需掩盖突触蔬菜和其他细胞器； 2）通过限制支配特定靶标的荧光和超微结构标记的T神经元来扩展该方法的潜在应用；并通过生成一条敲入小鼠线，该线路通过交配到CRE驱动线或注入表达CRE的病毒来表达标记。