Optical studies of the cone photoreceptor synapse

锥体感光器突触的光学研究

• 批准号: 7805368
• 负责人: RICHARD H KRAMER
• 金额: $ 37.77万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7805368
• 关键词: Address; Agonist; Binding; Biological Assay; Brain; Cell membrane; Charge; Consensus; Cytoplasm; Darkness; Dendrites; Disease; Electron Microscopy; Event; Exocytosis; Feedback; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence Spectroscopy; Fluorescent Probes; Funding; Glutamate Receptor; Goals; Imaging Techniques; Individual; Lateral; Light; Macular degeneration; Measurement; Measures; Mediating; Mental Depression; Microscopic; Molecular; Monitor; Monomeric GTP-Binding Proteins; Movement; Nature; Neurons; Neurotransmitters; Optical Methods; Optics; PHluorin; Patients; Photobleaching; Photoreceptors; Physiologic pulse; Process; Prosthesis; Proteins; Protons; RNA Splicing; Recovery; Research Personnel; Resolution; Retina; Retinal Cone; Retinitis Pigmentosa; Role; Signal Transduction; Site; Spectrum Analysis; Structure; Synapses; Synaptic Vesicles; System; Testing; Usher Syndrome; Vertebrate Photoreceptors; Vesicle; Vision; Visual system structure; Zebrafish; analog; blind; controlled release; design; extracellular; gamma-Aminobutyric Acid; horizontal cell; improved; information processing; insight; light intensity; neurotransmitter release; novel; postsynaptic; presynaptic; programs; public health relevance; research study; response; retinal neuron; ribbon synapse; voltage

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.

描述（由申请人提供）：锥形光感受器及其突触后目标，双极和水平细胞（BCS和HCS）之间的连接是视觉系统中的第一个突触。 神经递质从锥体从锥体释放，通过光线和外在的反馈信号本质地调节。 我们的长期目标是在分子级别了解这些信号如何调节释放。 锥端包含一种称为突触色带的专业结构。 色带结合突触囊泡，被认为可以将它们输送到质膜上，在那里它们经历了Ca2+依赖性胞吐作用。 我们的第一个具体目的是了解丝带通过色带传递突触囊泡的机制，并评估Ca2+在调节这一过程中的作用。 我们提出了三个步骤的带状囊泡递送：囊泡结合与色带，囊泡沿色带的运动以及与色带的囊泡脱离。 为了解决第一步，我们将询问Rab3a是一种囊泡 - 相关的小G蛋白，负责突触囊泡与色带的初始结合。 为了解决第二步，我们将使用突触小囊泡的荧光标记物测量色带上的囊泡迁移率，并在光漂白（FRAP）和荧光相关光谱（FCS）后荧光恢复。 为了解决第三步，我们将使用电子显微镜来评估当Ca2+在细胞质中升高时囊泡是否会渗入色带。 最后，为了更好地了解CA2+如何调节这些事件，我们将使用新型的“带有相关的Ca2+指标”（RACI）沿色带测量Ca2+曲线。 这些实验将共同解释控制锥体中突触囊泡递送的基本事件。 我们的第二个具体目的是研究HC反馈对锥端的机制。 质子已被认为是HC负反馈基础的信号。 我们将用pH敏感的GFP（Phluorin）在斑马鱼的锥突触处测量局部pH。 Phluorin探针将被剪接到突触蛋白上，以便在HC反馈的位置实现高空间分辨率测量。 我们将用“笼”的谷氨酸受体激动剂评估第二个“绝击”假设，以局部将电流流动到HCS的单个树突中。 最后，我们将探索从HCS到锥体的新发现的正反馈系统，研究逆行信号的性质并确定其作用机理。 这些研究很重要，有三个原因：1）他们将提高我们对观察第一步的基本过程的理解，2）他们可能会提供一些有关几种盲目疾病的机制和后果的见解，包括与光的跨性别机制相关的，包括综合基因疾病的综合症和常染色体显性统治性疾病，以及3）的传播，以及3），以及3）。视网膜，它们可能会为假肢设备的设计和编程提供更清晰的模板，以恢复盲人的视力。 公共卫生相关性：在这个项目中，我们将调查锥形感光器，负责白天愿景的细胞如何将信息发送给其他视网膜神经元，并最终向大脑发送，使我们能够看到。我们的第一个目的是了解控制从锥体中释放神经递质的基本分子机制。我们的第二个目的是了解该机械如何受到其他视网膜神经元的反馈信号的调节，从而提高了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。这些信息对于理解视网膜炎色素和黄斑变性等盲目疾病可能很重要，并将有助于为设计假肢设计的设计提供模板，以恢复盲人的正常视力。