Oligodendrocyte Maturation/Myelination in NG2 Null Mice

修订后的申请简介 A1申请的审查员认为，成功完成所有 该计划中的项目。预算和赠款文本之间的差异已在#年得到更正 当前版本。该公司副总裁兼首席运营官Karin Etheram女士的来信 伯纳姆研究所，载于第二节末尾和附录。这封信表明， 作为对该计划承诺的一部分，伯纳姆研究所将提供两台电生理钻机 从切片和细胞培养中记录。现在还明确指出，两个研究伙伴， Hadieh Badie-Mahdavi博士和Barbara Frederte博士将为核心A的工作贡献他们的专业知识 从第一年开始。他们的努力已经减少到评审员建议的50%。这也是 反映了由于取消前一个项目1而减少的需求。另一方面，兰施特博士的努力 手，已经增加到15%，因为她有能力投入更多的精力来准备混合 神经胶质细胞培养，也是因为她需要会诊来评估硫酸肝素的效果 蛋白多糖和NG2，根据评价者的建议，在Ranvier的节点上。 申请书的修订部分在左边空白处用一条线表示。整体而言 未标记为统一应用程序样式而引入的编辑修改。 目标 这个程序的目标是分析神经元和神经胶质细胞之间交换的分子信号 突触和有髓轴突。该计划已经确定了细胞表面成分与 现在打算研究这些分子在体内和组织中的功能 贴近体内相互作用的培养模型。本计划项目的核心A将提供 基础设施和实现这一目标的专业知识。核心将为以下两个方面提供支持 工作，通过电生理学分析神经元功能，并在适当的情况下模拟神经元-神经胶质细胞的相互作用 文化体系。 电生理学部分增加了一个新的研究维度，将为该计划提供 功能和活动依赖研究的工具和诀窍，旨在获得关于 神经元对神经胶质细胞或神经胶质衍生配体的反应发生的电生理变化。现在是时候了 显然，神经元-神经胶质细胞的相互作用不仅有助于髓鞘的功能特性，而且还有助于 突触。将电生理学部分添加到该计划将克服以前的限制 并使程序能够从功能上分析由于基因中断而导致的功能缺陷 神经细胞表面蛋白及其相关的信号转导通路。电生理学 该方法旨在分析神经冲动水平上的功能性神经元-神经胶质细胞相互作用 轴突的传导，以及突触功能和可塑性。电生理记录将 评估因表达或改变而导致的神经细胞膜特性的改变 胶质蛋白多糖的功能。该计划将使用转基因和基因敲除小鼠，这些小鼠已经在 并且有足够长的存活时间来进行电生理分析。 电生理记录将在脑片、单个细胞、 和孤立的神经。切片记录将集中于确定神经胶质蛋白eaffin-A3在 LTP和LTD过程中突触效能的变化。单细胞记录将评估ePhin的贡献- A3通过其神经元受体EphA4与神经元的兴奋性和可塑性有关。整个神经记录将 检测髓鞘功能障碍引起的复合动作电位传导速度变化 护套。这些不同层次的录音对于获得对分子的洞察是必要的 在个别项目中概述的神经元-神经胶质细胞串扰的相互作用。 拟议工作的第二个关键方面是探索细胞内神经元与神经胶质细胞的相互作用。 使用来自基因操纵的小鼠的材料进行检测。这是通过使用适当的 模仿体内特定相互作用的原代神经培养系统。具体地说，核心A将提供 神经元与髓鞘形成雪旺细胞或少突胶质细胞共培养研究 髓鞘生成。利用海马神经细胞培养研究神经胶质细胞肾上腺素-A3在调节中的作用 突触功能和可塑性将是目前核心的延续。

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.