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.

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.