Quantitative Mapping of Molecules and Release Properties at Nerve Terminals

描述（由申请人提供）：大脑中的信息流是通过电信息转化为化学信息并在化学突触中再次传递的传导来调节的。突触是由至关重要的细胞机器组成的，它协调了进出质膜的膜交通的平衡。我们的目标是在对动作电位刺激的生理反应以及其功能的分子基础方面对突触进行详细的定量了解。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梢的突触前功能的异质性。这些新方法使我们第一次能够在单个突触级别确定释放概率和可释放池的度量。这个项目的目标是测试关于这些属性异质性起源的假设。我们将测试这种异质性是由突触到突触的两个不同的神经递质释放分子控制点的变异性引起的。在这样做的过程中，我们还将在单个突触水平上获得关于这些控制点的新的和丰富的信息。第一个目标是检查是否大量的Munc-13-1，一个关键的胞吐调节因子，解释了这种异质性。为此，我们将检查Munc-13-1-ECFP敲入小鼠神经元突触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终端上这些调节分子的绝对数量，然后将其与同一终端的功能读数进行比较。此外，我们将使用基于shrna的蛋白质操作，这将使我们能够在非常详细的水平上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映射方法，并确定功能如何与每个突触中存在的特定类型的钙通道相关，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蛋白的突触调节如何在突触种群中变化以及它如何影响功能。

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.