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

基本信息

批准号：
8327806
负责人：
LINNAEA E OSTROFF
金额：
$ 15.4万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-06 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8327806
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

项目摘要

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.

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，大脑的所有功能都通过它来表达。绘制和表征这些连接是神经科学的基础，因为如果大脑系统的结构未知，就无法理解它。几十年来，神经解剖学示踪剂一直被用来揭示大脑大部分区域的连接性，但它们提供的信息是有限的。示踪剂是沿着轴突运输的化合物，可以使它们的起源、目的地或两者都可视化。现有示踪剂的特异性难以控制，导致结果不一致且不可靠。为了准确地绘制连接图，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，理想情况下，突触形态的电镜研究应该在标记的连接上进行。由于超微结构严重受损，现有的少数与 EM 兼容的示踪剂与形态学研究不兼容。我们对我们感兴趣的系统——成年大鼠外侧杏仁核在电镜水平上进行了广泛的形态学和神经解剖学示踪研究。我们建议开发新型示踪剂，为光学显微镜和电镜标记特定细胞，同时保留高质量的超微结构用于形态学研究。使用病毒载体，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 可以在不使组织接触破坏 EM 超微结构的去污剂的情况下进行可视化。这样可以很好地保存组织形态，同时将 HRP 限制在膜上，防止标签遮盖任何细胞器。我们将膜结合 HRP 基因置于两个不同启动子之一的控制之下。第一个是钙/钙调蛋白依赖性蛋白激酶 II 启动子，它将限制标记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（传统示踪剂的摄取则不然），这将是可用的最具空间和功能特异性的示踪剂。第二个启动子来自活性调节的细胞骨架相关蛋白 Arc，它的表达是为了响应强烈的突触激活和行为体验。这将允许识别在学习和可塑性实验期间激活的细胞的轴突和树突，以便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具有广泛的应用。