CHEMICAL ARCHITECTURE OF RETINAL CIRCUITS

视网膜回路的化学结构

• 批准号: 6518536
• 负责人: NOGA VARDI
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6518536
• 关键词: G protein; GABA receptor; amacrine cells; chemical structure; chloride channels; confocal scanning microscopy; dopamine receptor; electron microscopy; gap junctions; genetically modified animals; glutamate receptor; human tissue; immunocytochemistry; in situ hybridization; laboratory mouse; neuroanatomy; phosphodiesterases; polymerase chain reaction; retinal bipolar neuron; second messengers; visual photoreceptor; yeast two hybrid system

DESCRIPTION (Adapted from applicant's abstract): The proposed study is designed to examine the G protein and its downstream effectors activated by the metabotropic glutamate receptors (mGluR6), which are present on the dendrites of the rod and ON cone bipolar cells. More specifically, the investigator intends to: (1) identify mRNAs in retina enriched for bipolar cells to initially identify the GC (GC) and phosphodiesterase (PDE) with RT-PCR; (2) use in situ hybridization to identify the subset of isoforms and splice-variants that localize to ON bipolar cells, and carry out double immunostaining with confocal and electron microscopy to identify the subset of isoforms and proteins that co-localize postsynaptically with mGluR6; (3) test the function of the selected proteins in "knock-out" mice by examining the ERG b-wave; (4) test for the binding between mGluR6 and Go and between Go and selected protein effectors by using the two-hybrid interaction trap, GTS pulldown, and co-immunoprecipitation; and (5) identify Go's effector using a constitutively-active Go-alpha as bait in two-hybrid screening, which will be performed in parallel with aims 1-4 because it may identify novel effectors. The molecular approach outlined may characterize multiple isoforms and interactions irrelevant to the mGluR6 cascade, but they may be relevant to other aspects of retinal signaling. To determine the relevant isoforms, the study is designed to examine the broad picture with a highly specific microscopic assay and then to test the ultimate candidate with a simple physiological assay.

描述（根据申请人的摘要改编）：拟议的研究是设计的 检查G蛋白及其下游效应子 代谢性谷氨酸受体（MGLUR6），存在于树突上 杆和锥双极细胞上的。更具体地说，调查员 打算：（1）识别富含双极细胞的视网膜中的mRNA 最初用RT-PCR鉴定GC（GC）和磷酸二酯酶（PDE）； （2）使用 原位杂交以识别同工型和剪接变差的子集 该位置在双极细胞上，并与 共聚焦和电子显微镜，以鉴定同工型的子集和 与mglur6共定位的蛋白质； （3）测试功能 通过检查ERG B波中的“敲除”小鼠中选定的蛋白质； （4） 测试MGLUR6和GO之间以及GO和选定蛋白之间的结合 通过使用两个杂交相互作用陷阱，GTS下拉和效应子 共免疫沉淀； （5）使用 组成型活跃的go-alpha作为两种杂交筛查中的诱饵，这将是 与目标1-4并行进行，因为它可以识别新颖的效应子。 概述的分子方法可能表征多种同工型，并且 与MGLUR6级联无关的相互作用，但它们可能与 视网膜信号的其他方面。为了确定相关的同工型， 研究旨在检查广泛的图景 微观测定，然后用简单的 生理测定。