Optical studies of the cone photoreceptor synapse

描述（由申请人提供）：视锥光感受器与其突触后靶点（双极和水平细胞（BC和HC））之间的连接是视觉系统中的第一个突触。 视锥细胞的神经递质释放受光的内在调节和HC反馈信号的外在调节。 我们的长期目标是在分子水平上了解这些信号如何调节释放。 视锥细胞末端含有一种称为突触带的特殊结构。 丝带结合突触囊泡，并被认为将它们递送到质膜，在那里它们经历Ca 2+依赖性胞吐作用。 我们的第一个具体目标是了解突触囊泡通过丝带传递的机制，并评估Ca 2+在调节这一过程中的作用。 我们提出了三个步骤，在带介导的囊泡传递：囊泡结合到带，囊泡运动沿着带，囊泡脱离带。 为了解决第一步，我们将询问Rab 3a，一种囊泡相关的小G蛋白，是否负责突触囊泡与带状物的初始结合。 为了解决第二步，我们将使用突触囊泡的荧光标记物，通过光漂白后荧光恢复（FRAP）和荧光相关光谱（FCS）测量囊泡在带上的移动性。 为了解决第三步，我们将使用电子显微镜来评估当细胞质中Ca 2+升高时，囊泡是否腾空带状物。 最后，为了更好地理解Ca 2+如何调节这些事件，我们将用一种新的“带相关Ca 2+指示剂”（RACI）测量沿带沿着的Ca 2+分布。 总之，这些实验将有助于解释控制锥细胞中突触囊泡传递的基本事件。 我们的第二个具体目标是研究HC反馈到视锥细胞终末的机制。 质子被认为是HC负反馈的潜在信号。 我们将用pH敏感的GFP（pHluorin）测量斑马鱼锥体突触的局部pH。 pHluorin探针将被拼接到突触蛋白上，使得能够在HC反馈的非常位点处进行高空间分辨率pH测量。 我们将评估第二个“ephaptic”假说与“笼”谷氨酸受体激动剂局部改变电流流入个别树突的HC。 最后，我们将探讨一个新发现的正反馈系统，从HC锥，调查逆行信号的性质，并确定其作用机制。 这些研究之所以重要，有三个原因：1）它们将提高我们对视觉第一步的基本过程的理解，2）它们可能提供对几种致盲疾病的机制和后果的见解，包括Ushers综合征和常染色体显性视锥-视杆细胞营养不良（CORD 7），其与光感受器突触中的破坏有关;以及3）通过阐明视网膜中突触信息传递的正常机制，它们可以为用于恢复盲人视力的假体装置的设计和编程提供更清晰的模板。 公共卫生相关性：在这个项目中，我们将研究锥状光感受器，负责白天视觉的细胞，如何将信息发送到其他视网膜神经元，并最终发送到大脑，使我们能够看到。我们的首要目标是了解控制视锥细胞释放神经递质的基本分子机制。我们的第二个目标是了解这种机制是如何被来自其他视网膜神经元的反馈信号调节的，从而提高我们检测物体边缘的能力。该项目将提供有关视网膜正常功能的基本信息。这些信息对于理解致盲性疾病，如视网膜色素变性和黄斑变性可能是重要的，并将有助于为盲人患者恢复正常视力的假体装置的设计提供模板。

DESCRIPTION (provided by applicant): The connections between cone photoreceptors and their postsynaptic targets, bipolar and horizontal cells (BCs and HCs), are the first synapses in the visual system. Neurotransmitter release from cones is regulated intrinsically, by light, and extrinsically, by feedback signals from HCs. Our long-term goal is to understand at a molecular level how these signals regulate release. Cone terminals contain a specialized structure called the synaptic ribbon. The ribbon binds synaptic vesicles and is thought to deliver them to the plasma membrane where they undergo Ca2+-dependent exocytosis. Our first specific aim is to understand the mechanism of synaptic vesicle delivery by the ribbon, and to evaluate the role of Ca2+ in regulating this process. We propose three steps in ribbon-mediated vesicle delivery: Vesicle binding to the ribbon, vesicle movement along the ribbon, and vesicle detachment from the ribbon. To address the first step, we will ask whether Rab3a, a vesicle- associated small G-protein, is responsible for the initial binding of synaptic vesicles to the ribbon. To address the second step, we will use fluorescent markers of synaptic vesicles to measure vesicle mobility on the ribbon with Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS). To address the third step, we will use electron microscopy to evaluate whether vesicles vacate the ribbon when Ca2+ is elevated in the cytoplasm. Finally, to better understand how Ca2+ might regulate these events, we will measure the Ca2+ profile along the ribbon with a novel "Ribbon-Associated Ca2+ Indicator" (RACI). Together, these experiments will help explain the fundamental events that control synaptic vesicle delivery in cones. Our second specific aim is to investigate the mechanisms of HC feedback onto cone terminals. Protons have been proposed to be the signal underlying HC negative feedback. We will measure the local pH at the cone synapse of zebrafish with pH-sensitive GFP (pHluorin). The pHluorin probe will be spliced onto synaptic proteins enabling high spatial resolution pH measurement at the very site of HC feedback. We will evaluate a second "ephaptic" hypothesis with "caged" glutamate receptor agonists to locally alter current flow into individual dendrites of HCs. Finally, we will explore a newly-discovered positive feedback system from HCs to cones, investigating the nature of the retrograde signal and determining its mechanism of action. These studies are important for three reasons: 1) they will improve our understanding of the fundamental processes underlying the first steps in seeing, 2) they may provide insights into the mechanisms and consequences of several blinding disorders, including Ushers Syndrome and autosomal dominant cone-rod dystrophy (CORD7), which are associated with disruptions in photoreceptor synapses, and 3) by elucidating normal mechanisms of synaptic information transfer in the retina, they may provide a clearer template for the design and programming of prosthetic devices for restoring vision to blind patients. PUBLIC HEALTH RELEVANCE: In this project we will investigate how cone photoreceptors, the cells responsible for daytime vision, send information to other retinal neurons and ultimately to the brain, enabling us to see. Our first aim is to understand the fundamental molecular machinery that controls the release of neurotransmitter from cones. Our second aim is to understand how this machinery is regulated by feedback signals from other retinal neurons, increasing our ability to detect edges of objects. This project will provide fundamental information about the normal function of the retina. This information may be important for understanding blinding diseases such as retinitis pigmentosa and macular degeneration, and will help provide a template for the design of prosthetic devices for restoring normal vision to blind patients.