Optical studies of the cone photoreceptor synapse

视锥细胞与其突触后靶点、双极细胞和水平细胞的联系 和HCS)，是视觉系统中的第一个突触。视锥细胞的神经递质释放受到调节 从本质上说，是通过光，从外部，是通过来自HCS的反馈信号。我们的长期目标是在一个 分子水平这些信号如何调节释放。锥形端子包含一种特殊结构，称为 突触带。突触带结合突触小泡，被认为是将它们输送到质膜 在那里它们会经历依赖于钙离子的胞吐作用。我们的第一个具体目标是了解 突触囊泡的传递，并评价钙离子在这一过程中的调节作用。我们建议 丝带介导的囊泡传递的三个步骤：囊泡与丝带的结合，囊泡沿丝带的运动 丝带，以及水泡从丝带上脱落。为了解决第一步，我们将询问Rab3a，一种囊泡- 相关的小G蛋白负责突触小泡与带状突触的初始结合。要解决这个问题 第二步，我们将使用突触小泡的荧光标记来测量带上的小泡的流动性 光漂白后的荧光恢复(FRAP)和荧光相关光谱(FCS)。至 解决第三步，我们将使用电子显微镜来评估囊泡是否腾出丝带 胞质内钙离子含量升高。最后，为了更好地了解钙离子如何调节这些事件，我们将 使用一种新型的“功能区相关钙指示器”(RACI)测量功能区内的钙离子分布。一起， 这些实验将有助于解释控制锥体内突触小泡传递的基本事件。我们的 第二个具体目的是研究HC反馈到锥体末端的机制。质子已经被 被认为是HC负面反馈背后的信号。我们将测量锥体突触局部的pH值 在斑马鱼中加入对pH敏感的绿色荧光蛋白(PHluorin)。PHLOORIN探针将被拼接到突触蛋白上，使其能够 在HC反馈位置进行高空间分辨率的pH测量。我们将评估第二个“触觉” “笼式”谷氨酸受体激动剂局部改变流入单个树突的电流的假说 HCS。最后，我们将探索新发现的从HCS到锥体的正反馈系统，调查 逆行信号的性质和确定其作用机制。这些研究对三个方面都很重要 理由：1)它们将提高我们对以下第一步所依据的基本过程的理解 看到，2)它们可能提供对几种失明障碍的机制和后果的洞察， 包括引座症和常染色体显性遗传性视锥-视杆细胞营养不良(CORD7)，这与 光感受器突触的破坏，以及3)通过阐明突触信息的正常机制 在视网膜中转移，它们可能为假体设备的设计和编程提供更清晰的模板 用于恢复失明患者的视力。

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.