Tissue Culture

组织培养

• 批准号: 7185426
• 负责人: BARBARA RANSCHT
• 金额: $ 22.64万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7185426
• 关键词: Action Potentials; Appendix; Axon; Binding; Brain; Budgets; CSPG4 gene; Cell surface; Cells; Chromosome Pairing; Coculture Techniques; Communication; Consultations; Cultured Cells; Defect; Dimensions; Disruption; Electrophysiology (science); EphA4 Receptor; Ephrin-A3; Genetic; Goals; Grant; Hand; Heparan Sulfate Proteoglycan; Hippocampus (Brain); Individual; Institutes; Knockout Mice; Knowledge; Left; Letters; Ligands; Membrane; Membrane Proteins; Modeling; Modification; Molecular; Mus; Myelin; Myelin Sheath; Nerve; Neuroglia; Neurons; Oligodendroglia; Postdoctoral Fellow; Preparation; Property; Proteins; Proteoglycan; Ranvier' s Nodes; Research; Research Design; Research Infrastructure; Role; Schwann Cells; Signal Transduction; Signal Transduction Pathway; Slice; Synapses; System; Text; Time; Transgenic Organisms; Work; editorial; in vivo; insight; neuronal excitability; programs; relating to nervous system; response; synaptic function; tissue culture; tool

INTRODUCTION TO REVISED APPLICATION The reviewers of the A1 application considered the Core essential for the successful accomplishment of all the projects in the Program. Discrepancies between the budget and the grant text have been corrected in the current revision. The letter from Ms. Karin Eastham, Vice President and Chief Operating Officer of the Burnham Institute, is enclosed at the end of section II and also in the Appendix. This letter states that the Burnham Institute will provide, as part of its commitment to this Program, two rigs for electrophysiological recording from slices and cell cultures. It is also now clearly stated that both Research Associates, Dr. Hadieh Badie-Mahdavi and Dr. Barbara Fredette, will contribute their expertise to the work in Core A starting in year 1. Their effort has been reduced to 50%, as recommended by the reviewers. This also reflects decreased needs due to elimination of the previous Project 1. Dr. Ranscht's effort, on the other hand, has been increased to 15% due to her ability to dedicate increased effort to the preparation of mixed neuron-glia cultures and also due to the need for her consultation to evaluate the effects of heparan sulfate proteoglycans and NG2 at the nodes of Ranvier, as recommended by the reviewers. The revised portions of the application are indicated with a line on the left margin. The overall editorial modifications introduced to unify the style of the application are not marked. Objectives The goal of this Program is to analyze the molecular signals exchanged between neurons and glia at synapses and in myelinated axons. The program has identified cell surface components implicated in neuron-glia communication and now intends to study the function of these molecules in vivo and in tissue culture models that closely mimic the in vivo interactions. Core A of this Program Project will provide the infrastructure and the expertise to accomplish this goal. The Core will offer support for two aspects of the work, analysis of neuronal function by electrophysiology and modeling neuron-glia interactions in suitable culture systems. The Electrophysiology component adds a new research dimension that will provide the Program with the tools and know-how for functional and activity-dependent studies designed to acquire knowledge of the electrophysiological changes occurring in neurons in response to glial cells or glial-derived ligands. It is now apparent that neuron-glia interactions contribute to the functional properties not only of myelin but also of synapses. Addition of the electrophysiology component to the Program will overcome previous limitations and enable the Program to functionally analyze functional defects resulting from the genetic disruption of neural cell surface proteins and their associated signal transduction pathways. The electrophysiological approach is geared towards the analysis of functional neuron-glia interactions at the level of nerve impulse conduction along axons, as well as synaptic function and plasticity. Electrophysiological recordings will assess the modifications in neuronal membrane properties resulting from changes in the expression or function of glial proteoglycans. The Program will employ transgenic and knockout mice that are already in hand and have a sufficiently long survival time for conducting the electrophysiological analyses. Electrophysiological recordings will be conducted on brain slices, single cells, and isolated nerves. Slice recordings will focus on determining the role of the glial protein ephrin-A3 in synaptic efficacy changes during LTP and LTD. Single-cell recording will assess the contribution of ephrin- A3 to neuronal excitability and plasticity through its neuronal receptor, EphA4. Whole nerve recording will detect conduction velocity changes in the compound action potential caused by malfunction of the myelin sheath. Recordings at these different levels are necessary for gaining insights into the molecular interactions that underlie neuron-glia crosstalk as outlined in the individual projects. The second critical aspect of the proposed work is to probe neuron-glia interactions at the cellular level using material from genetically manipulated mice. This is effectively accomplished using suitable systems of primary neural cultures that mimic specific in vivo interactions. Specifically, Core A will provide the co-cultures of neurons and myelin-forming Schwann cells or oligodendrocytes for studies of myelinogenesis. The use of hippocampal cultures for studies on the influence of glial ephrin-A3 in regulating synaptic function and plasticity will be a continuation of the current Core.

修订申请简介 A1应用程序的审阅者认为成功完成所有人的核心是必不可少的 计划中的项目。预算和赠款文本之间的差异已得到纠正 当前的修订。 Karin Eastham女士的信，副总裁兼首席运营官 伯纳姆学院（Burnham Institute）在第二节末尾和附录中被封闭。这封信指出 伯纳姆研究所将作为对该计划的承诺的一部分提供两个用于电生理的钻机 从切片和细胞培养物记录。现在也清楚地指出，两个研究伙伴， Hadieh Badie-Mahdavi博士和Barbara Fredette博士将为核心的工作贡献他们的专业知识 从第一年开始。根据审稿人的建议，他们的努力已减少到50％。这也是如此 反映由于消除了上一个项目1而导致的需求减少。Ranscht博士的努力，另一个 手，由于她有能力将努力提高到混合准备的能力，因此已增加到15％ 神经元培养物，也是由于她需要咨询来评估硫酸乙酰肝素的影响 根据审稿人的建议，Ranvier节点的蛋白聚糖和NG2。 应用程序的修订部分用左边边缘的一条线表示。总体 引入的编辑修改旨在统一应用程序的样式。 目标 该程序的目的是分析神经元和神经胶质之间交换的分子信号 突触和髓鞘轴突。该程序已经确定了与 神经元 - 胶体通信，现在打算研究这些分子在体内和组织中的功能 培养模型，这些模型紧密模仿体内相互作用。该计划项目的核心A将提供 基础架构和实现这一目标的专业知识。核心将为两个方面的两个方面提供支持 通过电生理学和在合适的神经元间相互作用进行神经元功能的工作，分析神经元功能 培养系统。 电生理组件添加了一个新的研究维度，该维度将为程序提供 旨在获取知识的功能和活动依赖性研究的工具和专业知识 对神经胶质细胞或神经胶质衍生的配体的神经元中发生的电生理变化。现在 显然，神经元-GLIA相互作用不仅有助于髓磷脂的功能特性 突触。在程序中添加电生理组件将克服以前的限制 并使该程序能够通过遗传破坏引起的功能缺陷来分析 神经细胞表面蛋白及其相关的信号转导途径。电生理学 方法旨在分析神经冲动水平的功能性神经元 - 胶质相互作用 沿轴突以及突触功能和可塑性传导。电生理记录将 评估由于表达式变化或 神经胶质蛋白聚糖的功能。该程序将采用已经存在的转基因和淘汰小鼠 手，有足够长的生存时间进行电生理分析。 电生理记录将在大脑切片，单细胞上进行 和孤立的神经。切片记录将集中于确定神经胶质蛋白Ephrin-A3在 LTP和LTD期间的突触功效变化。单细胞记录将评估ephrin-的贡献 A3通过其神经元受体Epha4到神经元兴奋性和可塑性。整个神经记录将 检测由髓磷脂故障引起的复合动作电位的传导速度变化 鞘。在这些不同级别上的记录对于获得分子的见解是必要的 如各个项目中概述的神经元格利亚串扰的互动。 拟议的工作的第二个关键方面是探测细胞上的神经元 - 胶质相互作用 使用遗传操纵小鼠的材料的水平。使用合适的 模仿体内相互作用的特异性特异性神经培养的系统。具体来说，核心A将提供 用于研究 脊髓衰变。使用海马培养物来研究神经胶质Ephrin-A3在调节中的影响 突触功能和可塑性将是当前核心的延续。