Investigating the role of ribeye in retinal ribbon function

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，而不是响应动作电位的爆发。为此，这些细胞进化出突触带，这是一种蛋白质结构，可以在释放位点附近拴住大量突触囊泡。突触带的分子生物学尚不清楚。一些蛋白质已经定位于突触带，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是难以捉摸的。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。ribeeye的确切作用尚不清楚，本研究的长期目标是确定ribeeye在突触带中的功能作用。为此，我们将结合分子生物学、形态学分析和电生理学，使用两种模型系统：斑马鱼和老鼠。在Aim 1中，我们利用斑马鱼的ribeye过表达突变体研究ribeye自聚集和定向到突触的特性。这种聚集被认为是带状本身形成的基础，因此可能是带状形成的关键。这些实验不仅将告诉我们关于肋眼定位，这些实验对解释功能研究至关重要。在目的2中，我们打算利用视网膜电图研究野生型和突变型ribeye过表达对视网膜对光反应的影响。在初步实验中，我们已经发现转基因品系表现出改变的ERG b波反应。任何引起ERG b波变化的突变将在目标4中使用全细胞电压箝位技术进一步分析。在Aim 3中，我们将研究ribeye突变体过表达对条带形态的影响。由于ribeye是带状的主要组成部分，并可能形成突触带状构建的支架，缺乏重要功能特征的ribeye的过度表达或突变版本的ribeye可能会改变突触带状的形态或数量。我们将利用电子显微镜和超分辨率光成像技术受激发射损耗显微镜（STED）的结合来研究目标1中产生的转基因动物的形态学特征。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转基因基因过表达对杆双极细胞向AII无突细胞释放突触的影响。在目标2中鉴定的对斑马鱼ERG b波有影响的类似转基因小鼠突变将通过体内电穿孔引入小鼠杆状双极细胞，然后用于成对全细胞记录。配对记录将用于确定对囊泡池大小、持续释放率、抑郁恢复和多囊泡释放的影响。在分子水平上理解突触带的功能将最终有助于理解视觉和听觉信息是如何被处理和交流的。此外，它可能提供线索，帮助了解疾病，特别是影响视力和听力，如Usher综合征。此外，对这些特殊神经元突触前过程的基本理解将对一般的神经元交流产生更广泛的影响，因此，可能有助于我们对精神健康和神经系统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.