Investigating the role of ribeye in retinal ribbon function

描述(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，而不是以突发性的方式响应动作电位。为了做到这一点，这些细胞进化出突触带，这是一种蛋白质结构，将释放部位附近的大量突触小泡捆绑在一起。突触带的分子生物学还知之甚少。少数蛋白质被定位在突触带上，这些单个分子的重要性以及它们如何对突触带的独特功能做出贡献仍然是个谜。在这些蛋白质中，最丰富的是Ribeye，一种被认为构成突触带大部分的蛋白质，并假设形成突触带的核心。Ribeye的确切作用尚不清楚，该提议的长期目标是确定Ribeye在突触带中的功能作用。为此，我们将结合分子生物学、形态分析和电生理学，使用两个模型系统：斑马鱼和老鼠。在目标1中，我们研究了在斑马鱼中利用核眼过表达突变体进行自聚集并定向到突触的特性。聚集被认为是带状结构本身形成的基础，因此在带状结构的形成中可能是关键的。这些实验不仅将告诉我们关于核眼定位的信息，这些实验对于解释功能研究也是必不可少的。在目标2中，我们打算用视网膜电描记术研究野生型和突变型Ribeye的过度表达对视网膜光反应的影响。在初步实验中，我们已经确定了表现出改变的ERG b波反应的转基因株系。任何引起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，我们将研究核眼突变体过表达对带状结构的影响。由于核眼是突触带的主要组成部分，并可能形成构建突触带的支架，过度表达核眼或缺乏重要功能特征的突触眼突变版本可能会改变突触带的形态或数量。我们将使用电子显微镜和受激发射耗尽显微镜(STED)相结合的超分辨率光成像技术来研究在AIM 1中产生的转基因动物的形态特征。在目标4中，我们将评估核眼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，我们将在视网膜切片记录中研究Ribeye转基因过表达对视杆双极细胞到AII无长突细胞突触释放的影响。目标2中发现的对斑马鱼ERG b波有影响的转基因的类似小鼠突变将通过体内电穿孔导入小鼠杆状双极细胞，然后用于配对全细胞记录。配对记录将被用来确定对囊泡池大小、持续释放速率、从抑郁中恢复和多囊释放的影响。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交流的。此外，它还可能提供线索，帮助理解特定影响视力和听力的疾病，如亚瑟综合征。此外，对这些特殊神经元突触前过程的基本了解将对神经元的总体交流产生更广泛的影响，从而可能有助于我们对心理健康和神经疾病的各个方面的理解。 与公共健康相关：神经系统中的信息通过神经递质的释放在突触中传递。在视网膜和内耳，初级感觉信息通过专门进化的专门突触传递，以分级的方式传递高速率的神经递质释放。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理解视觉和听觉信息是如何处理和交流的，并提供线索帮助理解具体影响视觉和听力的疾病，如亚瑟综合征。此外，对这些特殊神经元突触前过程的基本了解将对神经元的总体交流产生更广泛的影响，从而可能有助于我们对心理健康和神经疾病的各个方面的理解。

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.