Investigating the role of ribeye in retinal ribbon function

研究 ribeye 在视网膜功能中的作用

• 批准号: 8518337
• 负责人: DAVID Paul ZENISEK
• 金额: $ 35.59万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518337
• 关键词: Action Potentials; Adult; Affect; Amacrine Cells; Animals; Auditory; Binding; Biological Models; Breeding; Cell membrane; Cells; Communication; DNA; Disease; Electron Microscopy; Electrophysiology (science); Electroporation; Electroretinography; Evaluation; Exhibits; Generations; Goals; Hearing; Imaging Techniques; Individual; Labyrinth; Light; Lighting; Mental Depression; Mental Health; Microscopy; Molecular; Molecular Biology; Morphology; Mus; Mutation; Nervous system structure; Neurons; Oxidoreductase; Photoreceptors; Preparation; Principal Investigator; Process; Property; Protein Isoforms; Proteins; RNA Splicing; Recovery; Resolution; Retina; Retinal; Role; Sensory; Site; Slice; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Transgenes; Transgenic Animals; Transgenic Organisms; Usher Syndrome; Vesicle; Vision; Visual; Voltage-Clamp Technics; Whole-Cell Recordings; Zebrafish; in vivo; mutant; nervous system disorder; neurotransmitter release; overexpression; presynaptic; programs; research study; response; retinal rods; ribbon synapse; scaffold; transcription factor; voltage clamp

DESCRIPTION (provided by applicant): Photoreceptors and bipolar cells of retina release neurotransmitter in a graded-continuous manner rather than in bursts in response to action potentials. To do so, these cells have evolved synaptic ribbons, proteinaceous structures that tether large numbers of synaptic vesicles near release sites. The molecular biology of the synaptic ribbon is poorly understood. A handful of proteins have been localized to the synaptic ribbon and the importance of these individual molecules as well as how they contribute to the unique functions of the synaptic ribbon remains elusive. Of these proteins, the most abundant is ribeye, a protein thought to constitute most of the synaptic ribbon and hypothesized to form the core of the synaptic ribbon. The precise role of ribeye remains unknown and the long-term goal of this proposal is to determine the functional role of ribeye in the synaptic ribbon. To do so, we will employ a combination of molecular biology, morphological analysis and electrophysiology using two model systems: zebra fish and mouse. In Aim 1 we investigate the property of ribeye to self-aggregate and be directed to the synapse using ribeye over expression mutants in zebra fish. The aggregation has been proposed to underlie the formation of the ribbon itself and thus may be critical in ribbon formation. Not only will these experiments inform us about ribeye localization, these experiments are essential to interpreting functional studies. In Aim 2, we intend to investigate the effects of over expression of wild-type and mutant ribeye on retinal responses to light using electroretinography. In preliminary experiments, we have identified transgenic lines that exhibit altered ERG b-wave responses. Any mutations that give rise to changes in the b-wave of the ERG will be further analyzed in aim 4 using whole-cell voltage clamp technique. In Aim 3, we will investigate the effect of ribeye mutant over expression on ribbon morphology. Since ribeye is the major constituent of the ribbon and may form the scaffold upon which the synaptic ribbon is built, over expression of ribeye or mutant versions of ribeye that lack important functional features of the protein may alter the morphology or number of synaptic ribbons. We will investigate the morphological features of transgenic animals generated in aim 1 using a combination of electron microscopy and stimulated emission-depletion microscopy (STED), a super-resolution light imaging technique. In Aim 4, we will evaluate the effects of ribeye mutants on synaptic release. To better understand the role of ribeye in synaptic transmission, we will investigate the effects of over expression of ribeye transgenes on synaptic release from rod bipolar cells on to AII amacrine cells in retinal slice recordings. The analogous mouse mutations of transgenes identified in aim 2 as having effects on b-wave of the ERG in zebra fish will be introduced into mouse rod bipolar cells by in vivo electroporation and then used for paired whole-cell recordings. Paired recordings will be used to determine the effects on vesicle pool size, rates of continuous release, recovery from depression and multivesicular release. Understanding synaptic ribbon function at the molecular level will ultimately aid in understanding how visual and auditory information is processed and communicated. In addition, it may provide clues to help understand diseases that specifically affect vision and hearing, such as Usher syndrome. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.

描述（由申请人提供）：视网膜的光感受器和双极细胞以分级连续的方式释放神经递质，而不是响应于动作电位而爆发。为此，这些细胞进化出了突触带，这是一种蛋白质结构，将大量突触小泡束缚在释放位点附近。人们对突触带的分子生物学知之甚少。少数蛋白质已定位于突触带，这些单个分子的重要性以及它们如何促进突触带的独特功能仍然难以捉摸。在这些蛋白质中，最丰富的是肋眼蛋白质，这种蛋白质被认为构成了突触带的大部分，并被假设形成了突触带的核心。肋眼的确切作用仍然未知，该提案的长期目标是确定肋眼在突触带中的功能作用。为此，我们将使用两种模型系统：斑马鱼和小鼠，结合分子生物学、形态学分析和电生理学。在目标 1 中，我们研究了 ribeye 自我聚集的特性，并使用斑马鱼中 ribeye 过度表达突变体定向到突触。已提出聚集是带本身形成的基础，因此可能对于带形成至关重要。这些实验不仅能让我们了解肋眼定位，而且对于解释功能研究至关重要。在目标 2 中，我们打算使用视网膜电图研究野生型和突变型 ribeye 的过度表达对视网膜对光反应的影响。在初步实验中，我们已经鉴定出表现出改变的 ERG b 波反应的转基因品系。任何导致 ERG b 波变化的突变都将在目标 4 中使用全细胞电压钳技术进行进一步分析。在目标 3 中，我们将研究 ribeye 突变体过度表达对带形态的影响。由于 ribeye 是带的主要成分，并且可能形成构建突触带的支架，因此缺乏蛋白质重要功能特征的 ribeye 或突变版本的 ribeye 的过度表达可能会改变突触带的形态或数量。我们将结合使用电子显微镜和受激发射损耗显微镜 (STED)（一种超分辨率光成像技术）来研究目标 1 中产生的转基因动物的形态特征。在目标 4 中，我们将评估 ribeye 突变体对突触释放的影响。为了更好地了解 ribeye 在突触传递中的作用，我们将研究 ribeye 转基因的过度表达对视网膜切片记录中从视杆双极细胞到 AII 无长突细胞的突触释放的影响。目标 2 中鉴定出的对斑马鱼 ERG b 波有影响的类似小鼠转基因突变将通过体内电穿孔引入小鼠视杆双极细胞，然后用于配对全细胞记录。配对记录将用于确定对囊泡池大小、连续释放速率、抑郁恢复和多囊泡释放的影响。 在分子水平上了解突触带功能最终将有助于理解视觉和听觉信息是如何处理和传达的。此外，它还可以提供线索来帮助了解特别影响视力和听力的疾病，例如亚瑟综合症。此外，对这些特殊神经元突触前过程的基本理解将对一般神经元通讯产生更广泛的影响，因此可能有助于我们对心理健康和神经系统疾病各个方面的理解。