Presynaptic Mechanisms at Ribbon-class Synapses

描述(申请人提供)：这项工作的长期目标是了解耳朵、眼睛和其他感觉系统中感觉受体细胞的突触传递机制，这些细胞在带状突触上使用分级传递。这种特殊形式的化学信号似乎是一种适应，允许传递有关感觉输入的微小变化的信息，这些信息在使用动作电位的传统突触传递过程中会丢失。拟议的实验将研究这些突触的生理学、解剖学和生物化学。这项研究将以青蛙和斑马鱼为模型物种，研究耳朵中的感觉受体(毛细胞)。斑马鱼的工作还将研究视网膜中的光感受器和双极细胞，以及侧线器官中的毛细胞。所有这些叫声都有带状突触。之所以选择这些物种进行研究，是因为有细胞和分子工具可以用来回答突触生理学的基本问题。就青蛙而言，也有丰富的信息可供建立。在过去的10年里，几个实验室开发了一些技术，可以使用紧密密封的电压钳来测量膜电容的微小变化，从而在毫秒级的时间尺度上观察突触传输，从而对突触传输进行详细的电生理分析。这种生理学方法将结合电子断层扫描和新近发展的膜示踪染料来研究突触小泡周期带状突触的超微结构。目的是测试有关突触“带”功能的几个关键假说，这些突触是以突出的解剖学特征命名的。该项目还将专注于两种主要蛋白质(钙视黄素和小白蛋白3)，这两种蛋白质被认为通过捕获和运输钙离子离开突触，在这些细胞的突触传递中发挥核心作用。该项目将调查重要的生化特性，这些特性决定了这些蛋白质与钙结合的速度，它们可以隔离多少钙，以及它们在细胞内扩散的速度。这些特性是理解毛细胞突触传递的核心，并与这些蛋白和相关钙结合蛋白在保护中风和其他脑损伤期间钙超载方面的拟议功能具有更广泛的相关性。斑马鱼研究人员在过去十年中开发的遗传和分子工具将允许直接测试一种蛋白质(Ribeye)的功能，这种蛋白质最近被确定为带状结构的主要组成部分。

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the mechanism of synaptic transmission in sensory receptor cells in the ear, eye and other sensory systems that use graded transmission at ribbon-class synapses. This specialized form of chemical signaling appears to be an adaptation to allow the transmission of information about small changes in sensory input that would be lost during conventional synaptic transmission that uses action potentials. The proposed experiments will investigate the physiology, anatomy and biochemistry of these synapses. The work on sensory receptors (hair cells) in the ear will use frogs and zebrafish as model species. The zebrafish work will also study photoreceptors and bipolar cells in the retina, and hair cells in the lateral line organs. All of these calls have ribbon-class synapses. These species were chosen for study because of the cellular and molecular tools that are available to answer fundamental questions in synaptic physiology. In the case of frogs, there is also a wealth of information already available upon which to build. During the past 10 years, several laboratories have developed techniques that allow detailed electrophysiological analysis of synaptic transmission using tight-seal voltage clamp to measure small changes in membrane capacitance to observe synaptic transmission on a millisecond time scale. This physiological method will be used in conjunction with electron tomography and recently developed membrane tracer dyes to study the ultra structure of the synaptic vesicle cycle ribbon synapses. The goal is to test several key hypotheses concerning the function of the synaptic "ribbon", the prominent anatomical feature for which these synapses are named. The project will also focus on two major proteins (calretinin and parvalbumin 3) that are believed to serve central roles in synaptic transmission in these cells by capturing and transporting calcium ions away from the synapses. The project will investigate the important biochemical properties that determine how fast these proteins bind calcium, how much calcium they can sequester, and how fast they can diffuse within the cell. These properties are central to understanding synaptic transmission in hair cells, and have a wider relevance to the proposed function of these and related calcium-binding proteins in protection from calcium overload during strokes and other brain injuries. The genetic and molecular tools developed by zebrafish researchers during the past decade will allow a direct test the function of a protein (Ribeye) that has recently been identified as a major component of the ribbon.