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Electrophysiological and molecular identification of glutamate receptor subunits expressed in hippocampal neurons

海马神经元表达的谷氨酸受体亚基的电生理学和分子鉴定

基本信息

批准号：
05404088
负责人：
OZAWA Seiji
金额：
$ 12.54万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05404088/
关键词：
Glutamate receptor Hippocampal neuron Patch clamp Reverse transcription Polymerase chain reaction AMPA receptor GluR2 subunit Ca^<2+> permeability 単一ニューロンホールセルパッチクランプ PCR パッチクランプ法 PCR法サブユニット

项目摘要

The glutamate receptor channels in the mammalian central nervous system are classified into three subtypes : NMDA,AMPA and kainate receptors. Among them, AMPA receptors are the principal mediators of fast excitatory neurotransmission. AMPA receptors are formed by various combinations of four subunit proteins, GluRl, GluR2, GluR3 and GluR4. To trace the molecular basis underlying differences in the functional properties of AMPA receptors in rat hippocampal neurons, we coupled whole-cell patch-clamp recordings with reverse transcription (RT) followed by polymerase chain reaction amplification (patch-clamp RT-PCR method).We have previously found two distinct types of AMPA receptors in cultured rat hippocampal neurons. In type I neurons, AMPA receptors displayd an outward rectification and little Ca^<2+> permeability. In contrast, type II neurons had AMPA receptors that displayd strong inward rectification and high Ca^<2+> permeability. The patch-clamp RT-PCR method revealed that only the … More GluRl and GluR4 subunits were expressed and that no GluR2 subunit was detected in type II neurons. In contrast, GluR2 was expressed at a high level together with GluR1, and occasionally with GluR3 or GluR4, in type I neurons. We further applied this technique for analyzing subunit compositions of AMPA receptors in various types of neurons in rat hippocampal slices. Type II neurons carrying AMPA receptors with strong inward rectification and high Ca^<2+> permeability were found in non-pyramidal interneurons in the hippocampus. In all type II neurons tested (16 neurons) in slices, either GluR1, GluR3 or GluR4 was expressed, but no GluR2 expression was detected.Thus, It is likely that the presence or absence of GluR2 determines rectification properties and Ca^<2+> permeability in native AMPA receptors in hippocampal neurons both in cultures and slices. The patch-clamp RT-PCR method will be useful for a wide range of physiological studies to elucidate the molecular basis of functional properties at the single cell level. Less

哺乳动物中枢神经系统中的谷氨酸受体通道分为三种亚型：NMDA、AMPA和红藻氨酸受体。其中，AMPA受体是快速兴奋性神经传递的主要介质。 AMPA受体由四种亚基蛋白GluR1、GluR2、GluR3和GluR4的各种组合形成。为了追踪大鼠海马神经元 AMPA 受体功能特性差异的分子基础，我们将全细胞膜片钳记录与逆转录 (RT) 结合，然后进行聚合酶链反应扩增（膜片钳 RT-PCR 方法）。我们之前在培养的大鼠海马神经元中发现了两种不同类型的 AMPA 受体。在I型神经元中，AMPA受体表现出向外整流和很小的Ca 2+ 通透性。相反，II型神经元具有表现出强向内整流和高Ca 2+ 渗透性的AMPA受体。膜片钳RT-PCR方法显示，II型神经元中仅表达GluR1和GluR4亚基，未检测到GluR2亚基。相比之下，在 I 型神经元中，GluR2 与 GluR1 一起高水平表达，偶尔与 GluR3 或 GluR4 一起表达。我们进一步应用该技术来分析大鼠海马切片中各种类型神经元中 AMPA 受体的亚基组成。在海马的非锥体中间神经元中发现了携带具有强内向整流和高Ca 2+ 通透性的AMPA受体的II型神经元。在切片中测试的所有II型神经元(16个神经元)中，表达GluR1、GluR3或GluR4，但未检测到GluR2表达。因此，GluR2的存在或不存在很可能决定培养物和切片中海马神经元中天然AMPA受体的整流特性和Ca^2+通透性。膜片钳 RT-PCR 方法可用于广泛的生理学研究，以阐明单细胞水平功能特性的分子基础。较少的