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。最重要的功能元件之一是电压门控钙通道，它将电信号转换为钙离子流量，以高度非线性的方式驱动神经递质的释放。我们最近开发了灵敏的方法，使我们能够表征这些通道的关键属性，以及在它们的自然环境-神经末梢-控制它们的电信号。本项目的目标是确定决定VGCC功能的关键机制的分子基础。第一个目标将研究一种新形式的适应性可塑性的机制，即神经末梢VGCC数量的变化进而改变电信号(动作电位)的形状。这些实验利用了一种新兴的基因编码荧光电压指示器技术，使人们能够定量测量神经末梢的动作电位波形以及它是如何控制的。我们的第二个目标将使用另一项新技术，使我们能够确定VGCC通常在活动区停留多长时间，它们是否可以通过细胞内突触前间隙循环，以及这些动态是如何受活动、钙内流和一些突触前蛋白控制的。第三个目标将研究不同的活动区蛋白，特别是Munc13和RIM1，如何在神经末梢控制VGCC功能的不同功能和细胞生物学方面。

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 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. One of the most important functional elements is the voltage-gated calcium channel as it converts the electrical signal into a flux of calcium that drives neurotransmitter release in a highly non-linear fashion. We recently developed sensitive approaches that allow us to characterize key properties of these channels and the electrical signal that controls them in their native environment, the nerve terminal. The goal of this project is determine the molecular basis of key mechanisms that determine VGCC function. The first aim will examine the mechanisms of a novel form of adaptive plasticity whereby changes in VGCC number at nerve terminals in turn changes the shape of the electrical signal (the action potential). These experiments make use of an emerging technology of genetically-encoded fluorescent voltage indicators that allow one to quantitatively measure the action potential waveform in the nerve terminal and how it is controlled. Our second Aim will use another new technology allowing us to determine how long VGCCs typically stay resident in the active zone, whether they can recycle through an intracellular presynaptic compartment and how these dynamics are controlled by activity, calcium influx and a number of presynaptic proteins. The third Aim will examine how different active zone proteins, in particular Munc13 and Rim1, control different functional and cell biological aspects of VGCC function at nerve terminals.