A genetic toolkit for targeted connectomics of specific neuronal types

 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。小体积的大脑可以在纳米级分辨率下完全重建，提供对单个细胞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 输入和输出）也可以被重建。这一技术突破与转基因动物和病毒对表征和操纵分子定义的神经元细胞类型的变革性影响平行。利用病毒、cre-lox技术、光遗传学和化学遗传学，分布式功能电路可以被成像、映射、激活、沉默或删除。这项建议旨在弥合这些办法之间的分歧。我们建议完善我们设计的方法，利用分子遗传学技术标记定义的细胞类型与电子致密标签SBFEM分析。具体来说，我们已经产生了一种重组腺相关病毒（腺相关病毒），它将依赖于Cre的遗传构建体传递到受感染的细胞。仅在表达Cre的细胞中，病毒有效载荷表达膜靶向标记蛋白，其包含荧光蛋白（膜靶向绿色荧光蛋白-mGFP）与重组过氧化物酶（APEX 2）的融合物。初步数据表明，感染的Cre表达细胞强烈表达融合蛋白的整个膜（索马，树突，轴突和终端）。其明亮的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。已经，该方法有很大的希望有针对性的'连接组学'分析的Cre表达神经元在任何大脑区域和他们的输出突触在远程神经结构。在这里，我们的目标是改进和扩展的方法。我们的目标是：1）优化组织化学方案和病毒构建体的设计，以标记特定的细胞结构或隔室，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支配特定靶点的t神经元，扩大该方法的潜在应用;以及通过与Cre驱动系交配或注射表达Cre的病毒来产生表达标记物的敲入小鼠系。

 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.