MICROARRAY ANALYSIS OF SINGLE RETINAL GANGLION CELLS

描述（申请人的摘要）：视网膜神经节细胞（RGC）过程 眼睛中的视觉信息并将其传输到大脑。所有RGC共享密钥 特征，但已确定不同的亚型，在形态上不同， 生物物理特性，视觉反应，突触输入和突触 目标的这些RGC亚型已被深入研究， 生理方法，因为它们是视觉的关键决定因素， 因为它们提供了一个可访问的系统， 神经元多样化和分类的问题。然而不幸的是， 如果有的话，也只有很少的分子标记是已知的， 哺乳动物的RGC亚类，因此不可能回答基本的问题， 关于有多少亚型真正存在，当他们出现，他们如何获得他们的 确定的身份，或者是什么分子构成了其亚型特异性的基础 特性.在这里，我们建议确定两对RGC的分子标记， 已经在结构上和生理上定义的子集，以及 具有明确的功能重要性：α-与β-样（其在索马中不同 大小、树突状扩散和反应的敏捷度）以及ON与OFF（不同 在输入、输出、树突分层和对发作的反应性与 光的终结）。为此，我们已经生成并表征了 转基因小鼠，其中绿色荧光蛋白（GFP）标记的RGC为 多种类型，使我们能够识别和分离它们。我们 将从单个转基因阳性RGC制备和表征cDNA， 使用我们最近成功用于表征小鸡RGC的方法。 然后将探针与市售的微阵列杂交 （AffysseGeneChips），使我们能够评估约27，000个基因的表达 在四种类型（α-ON，α-OFF，β-样-ON， β-样-OFF）。将分析输出以鉴定表达的基因， 定义的RGC子集或定义新的子集。最后，候选标记 将通过原位杂交进行验证。我们相信这些实验 (a)为发育研究提供标记，（B）揭示基因， 本身在亚型多样化或功能中发挥作用，和（c） 有助于开发普遍适用的基因表达方法 从单个细胞进行分析。

DESCRIPTION (Applicant's abstract): Retinal ganglion cells (RGCs) process visual information in the eye and transmit it to the brain. All RGCs share key features, but distinct subtypes have been identified that differ in morphology, biophysical properties, visual responsiveness, synaptic inputs and synaptic targets. These RGC subtypes have been studied intensively with anatomical and physiological methods, both because they are critical determinants of visual perception and because they provide an accessible system for addressing general issues of neuronal diversification and categorization. Unfortunately, however, few if any molecular markers are known that distinguish functionally relevant mammalian RGC subclasses, so it has been impossible to answer basic questions about how many subtypes really exist, when they arise, how they acquire their definitive identities, or what molecules underlie their subtype-specific properties. Here, we propose to identify molecular markers for two pairs of RGC subsets that have already been defined structurally and physiologically, and are of clear functional importance: alpha- vs. beta-like (which differ in soma size, dendritic spread and briskness of response) and ON vs. OFF (which differ in inputs, outputs, dendritic stratification, and responsiveness to onset vs. termination of light). To this end, we have already generated and characterized transgenic mice in which the Green Fluorescent Protein (GFP) labels RGCs of multiple types in their entirely, allowing us to identify and isolate them. We will prepare and characterize cDNA from individual transgene-positive RGCs, using methods we have recently used successfully to characterize chick RGCs. The probes will then be hybridized to commercially available microarrays (Affymetrix GeneChips), allowing us to assess expression of about 27,000 genes in cells of each of four classes (alpha-ON, alpha-OFF, beta-like-ON, beta-like-OFF). The output will be analyzed to identify genes expressed by defined RGC subsets or that define novel subsets. Finally, candidate markers will be validated by in situ hybridization. We believe these experiments will (a) provide markers for developmental studies, (b) uncover genes that themselves play roles in subtype diversification or function, and (c) contribute to development of generally applicable methods for gene expression profiling from single cells.