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: 8022180
• 负责人: DAVID Paul ZENISEK
• 金额: $ 37.25万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8022180
• 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. PUBLIC HEALTH RELEVANCE: Information in the nervous system is transmitted at the synapse via the release of neurotransmitter. In the retina and inner ear, primary sensory information is transmitted at specialized synapses specially evolved to transmit high rates of neurotransmitter release in a graded manner. We aim to understand, at the molecular level, how these cells accomplish this task. Understanding these synapses will ultimately aid in understanding how visual and auditory information is processed and communicated and 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）：视网膜的感光细胞和双极细胞以分级连续的方式释放神经递质，而不是在爆发中释放神经递质，而不是响应动作电位。为此，这些细胞已经进化出突触带，蛋白质结构，它们在释放位点附近绑定了大量的突触囊泡。突触色带的分子生物学知之甚少。少数蛋白质已定位在突触色带上，这些单个分子的重要性以及它们如何对突触色带的独特功能做出贡献仍然难以捉摸。在这些蛋白质中，最丰富的是Ribeye，一种被认为构成大多数突触色带的蛋白质，并被认为是突触色带的核心。 Ribeye的确切作用仍然未知，该提议的长期目标是确定Ribeye在突触色带中的功能作用。为此，我们将使用两个模型系统：斑马鱼和小鼠使用分子生物学，形态分析和电生理学的组合。在AIM 1中，我们研究了Ribeye自我聚集的特性，并使用Ribeye在斑马鱼中的表达突变体上指向突触。已经提出了汇总是为了构成色带本身的形成，因此在色带形成中可能至关重要。这些实验不仅会告知我们有关Ribeye定位的信息，而且这些实验对于解释功能研究至关重要。在AIM 2中，我们打算研究野生型和突变体肋眼的过度表达对使用电子模拟对光的视网膜反应的影响。在初步实验中，我们已经鉴定出表现出改变ERG B波反应的转基因线。任何引起ERG B波变化的突变都将在AIM 4中使用全细胞电压夹技术进一步分析。在AIM 3中，我们将研究Ribeye突变体对表达对色带形态的影响。由于Ribeye是色带的主要组成部分，并且可能形成构建突触色带的支架，而不是Ribeye或Ribeye的表达，而Ribeye的表达缺乏蛋白质的重要​​功能特征，可能会改变突触色带的形态或数量。我们将使用电子显微镜和刺激的发射排泄显微镜（STED）（一种超分辨率的光成像技术）的组合研究AIM 1中产生的转基因动物的形态特征。在AIM 4中，我们将评估Ribeye突变体对突触释放的影响。为了更好地理解肋眼在突触传播中的作用，我们将研究肋眼转基因在视网膜切片记录中从杆双极细胞上释放到AII无分花细胞的突触释放的过度表达的影响。 AIM 2中鉴定为对斑马鱼中ERG具有影响的转基因的类似小鼠突变将通过体内电穿孔引入小鼠杆双极细胞中，然后用于配对的全细胞记录。配对记录将用于确定对囊泡池大小的影响，连续释放的速率，抑郁症的恢复和多重释放。 了解分子水平的突触色带功能最终将有助于了解如何处理和传达视觉和听觉信息。此外，它可以提供线索，以帮助了解特异性影响视力和听力的疾病，例如Usher综合征。此外，对这些专业神经元中突触前过程的基本理解将对一般的神经元交流具有更广泛的影响，因此，可能有助于我们对心理健康和神经系统疾病的各个方面的理解。 公共卫生相关性：神经系统中的信息通过神经递质的释放传递。在视网膜和内耳内，主要的感官信息是在专门演变的专用突触中传输的，以分级的方式传输高递质的神经递质释放速率。我们的目标是在分子水平上了解这些细胞如何完成这项任务。了解这些突触最终将有助于了解如何处理和传达视觉和听觉信息，并提供线索，以帮助理解特别影响视力和听力的疾病，例如Usher综合征。此外，对这些专业神经元中突触前过程的基本理解将对一般的神经元交流具有更广泛的影响，因此，可能有助于我们对心理健康和神经系统疾病的各个方面的理解。