Regulation of photoreceptor vesicle resupply and synaptic transfer kinetics

描述(申请人提供)：基础视网膜研究的基本任务之一是了解视网膜--位于TE眼球后部的一张薄薄的感光组织--如何负责处理和传输吸收光子产生的信号。这一过程始于视网膜中的第一个突触，该突触将杆状和锥状感光细胞的信号传输到二级神经元。光感受器的信号传递能力依赖于突触带，突触带是一种特殊的结构，存在于各种神经感觉细胞中，负责提供预置的囊泡供应，通过紧张性囊泡的释放来支持信号传递。这项提议的目的是通过研究突触带如何被囊泡补充以及这种补充机制如何对视网膜视觉处理的早期阶段做出贡献来探索锥体带突触的基本信号功能。以前的研究已经描述了视网膜带状突触能够以一种依赖于突触小泡补充动力学的方式来传递对比度和亮度的方式。Thoreson实验室最近的工作表明，锥体带的补充动力学也被钙(Ca~(2+))加速，这表明调控光感受器信号编码的机制是Ca~(2+)依赖的。在听觉中继性突触Hold的花冠，钙的补充依赖于信号分子钙调蛋白。钙调蛋白可以类似地调节光感受器突触对钙的依赖，但这种可能性还有待测试。AIM 1将测试 这一假说认为钙调蛋白是锥体带小泡快速、依赖于钙离子补充的机制。此外，补充已经被认为是光感受器突触动态编码反应的主要决定因素，然而这一点也仍未得到检验。目标2将检验这一假设，即快速钙调蛋白依赖的补充模式负责编码和传输视觉反应的时间。这些目标将使用各种电生理技术，如视网膜电信号和单细胞和成对的全细胞记录，以及实时成像技术，如共聚焦钙成像，TIRF显微镜，和带有量子点的单粒子跟踪，以评估视锥细胞的突触信号。特定的药理化合物，直接传递到光感受器或施加到整个视网膜，将被用来通过钙调蛋白和钙依赖过程来操纵信号。光感受器对信号的正确编码和传输对视觉至关重要。了解视网膜在健康和疾病中的功能 在实施与现有视网膜网络集成的治疗方法时，如干细胞或视网膜植入物，这一点非常重要。此外，了解调节囊泡再供应和光感受器运输的机制，对于理解几种视网膜退行性疾病的病理生理学非常重要，因为突触蛋白在神经传递中的作用与各种视网膜疾病有关。

DESCRIPTION (provided by applicant): One of the fundamental tasks of basic retina research is to understand how the retina - a thin sheet of light- sensitive tissue at the back of te eye - is responsible for processing and transmitting signals arising from the absorption of photons. This process begins at the very first synapse in the retina, which transmits signals from rod and cone photoreceptors to second-order neurons. The signaling capabilities of photoreceptors depend the synaptic ribbon, a specialized structure found in a variety of neurosensory cells responsible for providing a supply of primed vesicles to support signaling by tonic vesicle release. The objective of this proposal is to explore the fundamental signaling capabilities of the cone ribbon synapse by examining how synaptic ribbons are replenished with vesicles and how that replenishment mechanism contributes to early stages of visual processing in the retina. Previous studies have described the ways in which retinal ribbon synapses are capable of signaling contrast and luminance in a manner dependent on the kinetics of synaptic vesicle replenishment. Recent work in the Thoreson lab has shown that the kinetics of replenishment at the cone ribbon is also accelerated by calcium (Ca2+), pointing to a Ca2+-dependent mechanism for regulating the encoding of photoreceptor signals. At the Calyx of Held, an auditory relay synapse, acceleration of replenishment by calcium depends on the signaling molecule calmodulin. Calmodulin may similarly regulate the Ca2+- dependence of replenishment at photoreceptor synapses, but this possibility has yet to be tested. Aim 1 will test the hypothesis that calmodulin is responsible for a fast, Ca2+-dependent mechanism of vesicle replenishment at the cone ribbon. Additionally, replenishment has been suggested as a major determinant of kinetic-encoding responses by the photoreceptor synapse, yet this also remains untested. Aim 2 will test the hypothesis that a fast calmodulin-dependent mode of replenishment is responsible for encoding and transmitting the timing of visual responses. These goals will be accomplished using a variety of electrophysiological techniques such as electroretinogram and single and paired whole-cell recordings as well as with live imaging techniques such as confocal calcium imaging, TIRF microscopy, and single-particle tracking with quantum dots to assess synaptic signaling by cones. Specific pharmacological compounds, delivered directly to the photoreceptor or applied to the entire retina, will be used to manipulate signaling by calmodulin and Ca2+-dependent processes. Proper encoding and transmission of signals by photoreceptors is crucial to vision. Understanding retinal function in health as well as disease is important when implementing therapeutic approaches that integrate with the existing retinal network such as stem cells or retinal implants. Moreover, an understanding of the mechanisms regulating vesicle resupply and trafficking in photoreceptors, as this proposal seeks to provide, is important in understanding the pathophysiology of several retinal degenerative diseases, as synaptic proteins with roles in the neurotransmission have been implicated in various retinopathies.