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 视网膜细胞，包含一个发射器和那些承担 接收器 一个基本的观察是， 离子型受体是相当不同的;对于每一个神经递质 有许多不同的受体亚单位， 潜在的亚基复合物。 对于每个发射机， 在视网膜中有许多不同的受体亚型特异性 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型谷氨酸受体亚单位是如何 由视网膜细胞表达和组合。 谷氨酸是一种 视网膜功能的基本神经递质， 我们实验室的杂交实验表明， 表达编码7个克隆亚基（GluR 1至GluR 7）的基因 在视网膜上。 1）。 工具：a）谷氨酸受体亚基特异性的产生 抗血清和B）携带亚单位的细胞：亚单位特异性抗体 来研究真正的受体亚基蛋白。 到 这样做，每个cDNA编码的最不相似的部分， GluR 1至GluR 7将被亚克隆到用于细菌的质粒中。 过度表达 所得融合蛋白将用作 抗原免疫兔子和山羊。 蛋白质印迹法 融合蛋白将用于鉴定免疫应答。 肾 细胞将用每种受体亚单位转染， 提供子单元的独立验证和确认- 在以下每个实验中测定抗血清的特异性。 2）。 解剖学：谷氨酸的免疫组织化学定位 视网膜中的受体亚单位：亚单位特异性抗血清将被 用于化学定位受体亚单位。 将采用光学和电子显微镜来确定 受体免疫反应性的细胞定位， 尽可能好地识别带有受体的细胞。 双 标签策略将用于确定哪些组 受体亚单位共表达。 3）。 化学：受体亚基蛋白的表征 以及它们形成的复合物：首先，蛋白质印迹将用于 分析粗膜中的受体亚基蛋白 视网膜部分。 然后，兔抗血清将用于 免疫沉淀溶解的受体复合物从视网膜。 然后将这些在凝胶上分级分离，印迹并用 山羊亚基特异性抗血清。 我们的目标是定义 彼此关联的子单元集合。

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.