Quantitative Mapping of Molecules and Release Properties at Nerve Terminals

描述(申请人提供)：大脑中的信息流是通过将电信息转化为化学信息并在化学突触处再回来而调节的。突触由关键的细胞器组成，这些细胞器协调进出质膜的膜交通平衡。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了解。我们最近开发了一种灵敏的方法，使我们能够表征同一细胞中许多神经末梢突触前功能的异质性。这些新方法首次允许我们在单个突触水平上确定释放概率和易释放池的测量。这个项目的目标是测试关于这些属性异质性的起源的假设。我们将测试这种异质性源于神经递质释放的两个不同分子控制点的突触到突触的可变性。在这样做的同时，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。第一个目标将检查MUNC-13-1的丰度是否解释了异质性，MUNC-13-1是胞吐作用的关键调节因子。为了做到这一点，我们将检测来自MUNC-13-1-ECFP敲打小鼠的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末梢上这些调节分子的绝对数量，然后将其与同一神经末梢的功能读数进行比较。此外，我们还将使用基于shRNA的对该蛋白质的操纵，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属性的映射方法，并确定功能如何与每个突触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的神经递质释放的关键参数。最后，我们将研究基于G蛋白的突触调制在突触群体中是如何变化的，以及它是如何影响功能的。 与公共健康相关：大脑中的信息流是通过将电子信息转化为化学信息并在化学突触上再传递回来的。人脑的功能依赖于精心编排，将神经递质满载的囊泡输送到神经末梢的位置，在那里它们可以根据需要传递这种化学信息。许多已知的帕金森氏病、偏头痛和精神分裂症等疾病的基因突变与控制突触功能的蛋白质有关。我们的工作旨在从分子水平上了解这一机制，以更好地确保未来对这些类型的神经元疾病的治疗取得成功。

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. PUBLIC HEALTH RELEVANCE: Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. The functioning of the human brain relies on the careful orchestration of delivering neurotransmitter-laden vesicles to sites at nerve terminals where they can be used to deliver this chemical message on demand. Many known genetic mutations in diseases such as Parkinson's disease, migraine headache and schizophrenia are linked to proteins that control synapse function. Our work is aimed at understanding the machinery at a molecular level to better ensure the success of future therapies for these types of neuronal diseases.