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: 9322330
• 负责人: David M. Berson
• 金额: $ 19.25万
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9322330
• 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; Fluorescence; Generations; Genetic; Genetic Fingerprintings; 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; 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（由申请人提供）：串行块面电子显微镜（SBFEM）正在彻底改变神经微电路的映射，可以在纳米级分辨率下完全重建小体积的大脑，从而提供所有突触输入的形式和位置的完整描述。对于非常局部的输入，身份（和突触）。 这些突触前细胞的输入和输出）也可以通过使用病毒、cre-lox 技术、光遗传学和化学遗传学、分布式技术来重建。功能电路可以被成像、映射、激活、沉默或删除。我们建议完善我们利用分子遗传技术设计的方法。具体来说，我们生成了一种重组腺相关病毒 (AAV)，可将 Cre 依赖性基因构建体专门传递到表达 Cre 的细胞中。有效负载表达膜靶向标记蛋白，其中包含荧光蛋白（膜靶向绿色荧光蛋白 - mGFP）与重组过氧化物酶（APEX2）的融合物。 Cre 表达细胞在整个膜（体、树突、轴突和末端）中强烈表达融合蛋白，其明亮的荧光允许进行详细的共聚焦分析；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。 ，该方法对于任何大脑区域中表达 Cre 的神经元及其在远程神经结构中的输出突触的定向“连接组”分析具有很大的前景。在这里，我们的目标是改进和扩展该方法。该提案的目的是：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区室，而不掩盖突触小泡和其他细胞器；2）通过限制荧光和超微结构标记神经元，支配特定目标；并通过与 Cre 驱动系交配或注射 Cre 表达病毒来产生表达标记的敲入小鼠系。

项目成果

Local axonal morphology guides the topography of interneuron myelination in mouse and human neocortex.

局部轴突形态指导小鼠和人类新皮质中神经元髓鞘形成的地形。

• DOI: 10.7554/elife.48615
• 发表时间: 2019
• 期刊: eLife
• 影响因子: 7.7
• 作者: Stedehouder,Jeffrey;Brizee,Demi;Slotman,JohanA;Pascual-Garcia,Maria;Leyrer,MeganL;Bouwen,BibiLj;Dirven,ClemensMf;Gao,Zhenyu;Berson,DavidM;Houtsmuller,AdriaanB;Kushner,StevenA
• 通讯作者: Kushner,StevenA