TRANSMITTER/RECEPTOR-SPECIFIC CIRCUITRY IN THE RETINA

编码许多神经递质受体的cDNA已经被 最近克隆的。因此，我们现在可以开始识别 含有递质的视网膜细胞和那些承载 传送器的受体。一个基本的观察结果是， 电离型受体是相当多样化的；对于每种神经递质 有许多不同的受体亚基，甚至更多的 潜在的亚单位复合体。有可能每个发射机 用于视网膜的受体有许多不同的亚型特异性 突触。拟议工作的目标是探索这种异质性。 通过定义红藻氨酸/AMPA型谷氨酸受体亚基是如何 由视网膜细胞表达和结合。谷氨酸是一种 视网膜功能的基础神经递质，以及原位 我们实验室的杂交实验表明，这些mRNAs 表达了编码7个克隆的亚基(GluR1至GluR7)的基因 在视网膜上。 1)。工具：a)谷氨酸受体亚单位特异性的产生 抗血清和b)亚基承载细胞：亚基特异性抗体 必须创建来研究实际的受体亚单位蛋白质。至 这样做，每个编码的cDNA中最不相似的部分 GluR1至GluR7将被亚克隆到细菌的质粒中 过度表达。由此产生的融合蛋白将被用作 用来免疫兔子和山羊的抗原。西方人的印迹 融合蛋白将用于识别免疫反应。肾 每个受体亚基都将被导入细胞中以 提供亚基的独立验证和确认- 抗血清在以下每一项实验中的特异性。 2)。解剖：谷氨酸的免疫组织化学定位 视网膜中的受体亚基：亚基特异性抗血清 用于免疫组织化学定位受体亚基。 将使用光学和电子显微镜来定义 受体免疫反应性和TO的细胞定位 尽最大可能识别携带受体的细胞。双倍 将使用标注策略来确定哪些集合 受体亚基是共表达的。 3)。化学：受体亚单位蛋白的特性 以及它们形成的复合体：首先，西方印迹将被用来 粗膜中受体亚单位蛋白的分析 视网膜的一部分。然后，将兔抗血清用于 免疫沉淀视网膜上溶解的受体复合体。 然后这些将被分级在凝胶上，吸上印迹并用 山羊亚单位特异性抗血清。目标将是定义 相互关联的多组子单元。

The cDNAs encoding many of the neurotransmitter receptors have been cloned recently. Consequently, we can now begin to identify both the retinal cells that contain a transmitter and those that bear the receptors for the transmitter. A fundamental observation is that the ionotropic-type receptors are quite diverse; for each neurotransmitter there are many different receptor subunits and an even greater number of potential subunit complexes. It is possible that for each transmitter used in the retina there are many different receptor-subtype specific synapses. The goal of the proposed work is to explore this heterogeneity by defining how the Kainate/AMPA-type glutamate receptor subunits are expressed and combined by the cells of the retina. Glutamate is a neurotransmitter fundamental to retinal function, and in situ hybridization experiments in our laboratory have revealed that the mRNAs encoding the seven cloned subunits (GluR1 through GluR7) are expressed in the retina. 1). Tools: the generation of a) glutamate receptor subunit-specific antisera and b) subunit-bearing cells: Subunit-specific antibodies must be created to study the actual receptor subunit proteins. To do this, the most dissimilar portions of each of the cDNAs encoding GluR1 through GluR7 will be subcloned into plasmids for bacterial overexpression. The resulting fusion proteins will be used as antigens to immunize rabbits and goats. Western blots of the fusion proteins will be used to identify immune responses. Kidney cells will be transfected with each of the receptor subunits to provide independent verification and validation of the subunit- specificity of the antisera in each of the following experiments. 2). Anatomy: the immunohistochemical localization of the glutamate receptor subunits in the retina: Subunit-specific antisera will be used to immunohistochemically localize the receptor subunits. Light and electron microscopy will be employed to define the cellular localization of the receptor immunoreactivity and to identify as best as is possible the receptor-bearing cells. Double labeling strategies will be used to determine which sets of receptor subunits are co-expressed. 3). Chemistry: the characterization of the receptor subunit proteins and the complexes they form: First, Western blots will be used to analyze the receptor subunit proteins in the crude membrane fraction of retina. Then, the rabbit antisera will be used to immune precipitate solubilized receptor complexes from the retina. These will then be fractionated on gels, blotted and probed with the goat subunit-specific antisera. The goal will be to define the sets of subunits that associate with one another.