Optical studies of the cone photoreceptor synapse

视锥细胞与其突触后靶细胞--双极细胞和水平细胞之间的联系 和HC），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 内在地，通过光，和外在地，通过来自HC的反馈信号。 我们的长期目标是了解 分子水平上这些信号如何调节释放。 Cone终端包含一个专门的结构，称为 突触带 丝带结合突触囊泡，并被认为是将它们传递到质膜 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 通过丝带传递突触囊泡，并评估Ca 2+在调节这一过程中的作用。 我们提出 带介导的囊泡递送中的三个步骤：囊泡结合到带，囊泡沿带的沿着运动， 带状物和囊泡从带状物脱离。 为了解决第一步，我们将问Rab 3a，一个囊泡- 相关的小G蛋白，负责突触囊泡与带状物的初始结合。 解决 第二步，我们将使用突触囊泡的荧光标记物来测量带状物上的囊泡移动性， 光漂白后的荧光恢复（FRAP）和荧光相关光谱（FCS）。 到 解决第三步，我们将使用电子显微镜来评估囊泡是否腾空带时， Ca 2+在细胞质中升高。 最后，为了更好地了解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。 在一起， 这些实验将有助于解释控制视锥细胞中突触囊泡传递的基本事件。 我们 第二个具体目标是研究HC反馈到视锥细胞终末的机制。 质子已经 提出是HC负反馈的潜在信号。 我们将测量锥状突触的局部pH值 斑马鱼与pH敏感的绿色荧光蛋白（pHluorin）。 荧光素探针将被拼接到突触蛋白上， 在HC反馈的位置进行高空间分辨率pH测量。 我们将评估第二个“ephaptic” 用“笼状”谷氨酸受体激动剂局部改变流入单个树突的电流的假说， HC。 最后，我们将探讨一个新发现的从HC到锥细胞的正反馈系统， 逆行信号的性质并确定其作用机制。 这些研究对三个 原因：1）它们将提高我们对第一步的基本过程的理解， 看到，2）它们可以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），它们与 破坏感光突触，3）阐明突触信息的正常机制 转移在视网膜，他们可能会提供一个更清晰的模板，设计和编程的假体设备 帮助盲人恢复视力

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.