Development of genetically encoded neural tracers for electron microscopy

用于电子显微镜的基因编码神经示踪剂的开发

• 批准号: 8176619
• 负责人: LINNAEA E OSTROFF
• 金额: $ 22.9万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-06 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8176619
• 关键词: Adult; Amygdaloid structure; Architecture; Area; Axon; Behavioral; Binding; Brain; Calcium/calmodulin-dependent protein kinase; Cell Density; Cell membrane; Cells; Characteristics; Cytoskeleton; Data; Dendrites; Dependovirus; Destinations; Detergents; Development; Electron Microscopy; Functional disorder; Gene Expression; Genes; Goals; Horseradish Peroxidase; Immediate-Early Genes; Infection; Injection of therapeutic agent; Label; Lateral; Learning; Light; Literature; Maps; Membrane; Methods; Microscopic; Morphology; Neuroanatomy; Neurobiology; Neurons; Neurosciences; Organelles; Output; Phaseolus vulgaris leucoagglutinin; Proteins; Rattus; Reporter Genes; Research; Site; Specificity; Structure; Synapses; System; Temporal Lobe; Testing; Tissue Preservation; Tissues; Tracer; Training; Transfection; Travel; Viral; Viral Vector; Virus; Work; brain cell; calmodulin-dependent protein kinase II; conditioned fear; excitatory neuron; experience; in vivo; interest; light microscopy; nervous system disorder; neural circuit; neuronal cell body; novel; prevent; promoter; relating to nervous system; research study; response; retrograde transport; success; tool; uptake

DESCRIPTION (provided by applicant): The connections between neurons compose the basic structure of the brain, through which all of its functions are expressed. Mapping and characterizing these connections is fundamental to neuroscience, as no brain system can be understood if its architecture is unknown. Neuroanatomical tracers have been used for decades to reveal connectivity in much of the brain, but the information they provide is limited. Tracers are compounds which are transported along axons and allow their origin, destination, or both to be visualized. It is difficult to control the specificity of existing tracers, which leads to inconsistent and unreliable results. To accurately map connectivity, the existence of synaptic connections between identified neurons must be verified at the electron microscopy (EM) level. Furthermore, EM studies of synapse morphology should ideally be carried out on labeled connections. The few existing tracers that are compatible with EM are not compatible with morphological studies due to severely compromised ultrastructure. We have conducted extensive morphological and neuroanatomical tracer studies at the EM level on our system of interest, the adult rat lateral amygdala. We propose to develop novel tracers which will label specific cells for light microscopy and EM while preserving high quality ultrastructure for morphological studies. Using a viral vector, we will express a membrane-targeted form of the EM label horseradish peroxidase (HRP) in adult rat neurons. Unlike current tracers, HRP can be visualized without subjecting tissue to detergents which damage EM ultrastructure. This allows good preservation of tissue morphology, while restricting the HRP to the membrane prevents the label from obscuring any cellular organelles. We will place the membrane-bound HRP gene under the control of one of two different promoters. The first, the calcium/calmodulin-dependent protein kinase II promoter, will restrict expression of the label to excitatory neurons. Combined with the fact that viral transfection is restricted to cell bodies (which conventional tracer uptake is not), this will be the most spatially and functionally specific tracer available. The second promoter will be from the activity-regulated cytoskeleton-associated protein Arc, which is expressed in response to strong synaptic activation and behavioral experience. This will allow identification of the axons and dendrites of cells activated during learning and plasticity experiments such that their synapses can be specifically examined. The tools we propose to create will have a broad range of applications in neuroanatomy, neurobiology, and plasticity studies throughout the brain. PUBLIC HEALTH RELEVANCE: Mapping and characterizing the connectivity between brain cells is fundamental to understanding how the brain works. Many neurological diseases involve dysfunction of particular brain circuits or connections, and it is essential to elucidate and examine these connections in both normal and diseased brains. The goal of this project is to develop novel tools to map neural connections and visualize them clearly at the microscopic level.

描述（申请人提供）：神经元之间的连接构成了大脑的基本结构，所有的功能都是通过它来表达的。绘制和描述这些联系是神经科学的基础，因为如果大脑系统的结构未知，就无法理解它。几十年来，神经解剖示踪剂一直被用于揭示大脑大部分区域的连通性，但它们提供的信息有限。示踪剂是沿着轴突运输的化合物，可以使它们的起点、终点或两者都可视化。现有示踪剂的特异性难以控制，导致结果不一致、不可靠。为了准确地映射连接，必须在电子显微镜（EM）水平上验证已识别神经元之间突触连接的存在。此外，突触形态学的EM研究最好是在标记的连接上进行。现有的几种与EM相容的示踪剂由于超微结构严重受损而与形态学研究不相容。我们对我们感兴趣的成年大鼠外侧杏仁核系统进行了广泛的形态学和神经解剖学示踪研究。我们建议开发新的示踪剂，在光镜和电镜下标记特定的细胞，同时为形态学研究保留高质量的超微结构。利用病毒载体，我们将在成年大鼠神经元中表达EM标记辣根过氧化物酶（HRP）的膜靶向形式。与目前的示踪剂不同，HRP可以可视化，而不需要将组织置于破坏EM超微结构的洗涤剂中。这样可以很好地保存组织形态，同时将HRP限制在膜上可以防止标签掩盖任何细胞器。我们将把膜结合的HRP基因置于两种不同启动子之一的控制之下。首先，钙/钙调素依赖性蛋白激酶II启动子，将限制标签的表达到兴奋性神经元。结合病毒转染仅限于细胞体的事实（传统示踪剂摄取不受限制），这将是可用的最具空间和功能特异性的示踪剂。第二个启动子将来自活性调控的细胞骨架相关蛋白Arc，该蛋白在强烈的突触激活和行为体验下表达。这将允许识别在学习和可塑性实验中激活的细胞的轴突和树突，从而可以专门检查它们的突触。我们建议创建的工具将在整个大脑的神经解剖学，神经生物学和可塑性研究中有广泛的应用。