Quantitative Mapping of Molecules and Release Properties at Nerve Terminals

神经末梢分子和释放特性的定量图谱

• 批准号: 8389677
• 负责人: Timothy Aidan Ryan
• 金额: $ 40.15万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-02 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389677
• 关键词: Accounting; Action Potentials; Agonist; Axon; Back; Baclofen; Brain; Calcium Channel; Calibration; Cell membrane; Cells; Characteristics; Chemical Synapse; Chemicals; Complement; Disease; Dose; Ensure; Equilibrium; Exocytosis; Fluorescence; Future; GTP-Binding Proteins; Gene Mutation; Goals; Heterogeneity; Human; Individual; Knock-in Mouse; Link; Maps; Measures; Mediating; Membrane Protein Traffic; Methods; Migraine; Molecular; Molecular Probes; Mus; N-Type Calcium Channels; Nerve; Neurons; Neurotransmitters; Optical Methods; Optics; PHluorin; Parkinson Disease; Pharmacology; Physiological; Population; Presynaptic Terminals; Probability; Procedures; Property; Proteins; Q-Type Calcium Channels; Schizophrenia; Site; Specificity; Stimulus; Synapses; Testing; Time; Variant; Vesicle; Work; base; controlled release; neurotransmitter release; overexpression; presynaptic; public health relevance; research study; response; small hairpin RNA; success; synaptic function

DESCRIPTION (provided by applicant): Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. Synapses are made up of crucial cellular machineries that orchestrate a balance of membrane traffic to and from the plasma membrane. Our goal is to develop a detailed quantitative understanding of the synapse both in terms of physiological responses to action potential stimuli as well as the molecular underpinnings of its function. We recently developed sensitive approaches that allow us to characterize the heterogeneity of presynaptic function across many nerve terminals from the same cell. These new methods allow us for the first time to determine release probabilities and measures of readily-releasable pools at the single synapse level. The goal of this project is to test hypotheses about the origin of the heterogeneity of these properties. We will test the idea that this heterogeneity arises from synapse to synapse variability in two different molecular control points of neurotransmitter release. In doing so we will also obtain new and rich information about these control points at the single synapse level. The first aim will examine if the abundance of a Munc-13-1, a critical regulator of exocytosis, accounts for the heterogeneity. To do this we will examine the biophysical parameters of release at synapses in neurons derived from Munc-13-1-ECFP knockin mice. Calibration procedures will allow us to determine the absolute number of these regulatory molecules at each nerve terminal which will then be compared to functional readouts at the same terminal. We will additionally use shRNA-based manipulation of this protein will allow us to determine the molecular dose-response relationship for function at a very detailed level. Our second Aim will use this same mapping approach of presynaptic properties and determine how function is correlated with the specific types of calcium channels present at each synapse and how the abundance of functional channels influences key parameters of neurotransmitter release at the single synapse level. Finally we will examine how G-protein based modulation of synapses varies from across a population of synapses and how it impacts function.

描述（由申请人提供）：大脑中的信息流是通过将电气信息转移到化学信息中并在化学突触时再次返回的。突触是由关键的细胞机械组成的，它们协调了往返质膜的膜流量的平衡。我们的目标是根据对动作电位刺激的生理反应以及其功能的分子基础，对突触进行详细的定量理解。我们最近开发了敏感的方法，使我们能够表征来自同一细胞的许多神经末端的突触前功能的异质性。这些新方法使我们首次确定在单个突触水平上易于释放的池的释放概率和度量。该项目的目的是检验有关这些特性异质性的起源的假设。我们将测试这种异质性是由神经递质释放的两个不同分子控制点突触变异引起的。这样一来，我们还将在单个突触级别获得有关这些控制点的新信息。第一个目的将检查是否有胞吞作用的关键调节剂Munc-13-1是异质性的。为此，我们将检查源自MUNC-13-1-ECFP敲击蛋白小鼠的神经元中突触中释放的生物物理参数。校准程序将使我们能够确定每个神经末端的这些调节分子的绝对数量，然后将其与同一末端的功能读数进行比较。我们还将使用基于SHRNA的该蛋白质的操作，使我们能够在非常详细的水平下确定功能的分子剂量反应关系。我们的第二个目标将使用相同的突触前特性的映射方法，并确定功能与每个突触处的特定类型的钙通道的相关性以及功能通道的丰度如何影响单个突触水平上神经递质释放的关键参数。最后，我们将研究基于G蛋白的突触调制如何与突触群体之间的变化以及它如何影响功能。