Immunohistochemical analysis of neuronal networks in brain using antibody for neurotransmitter receptors

使用神经递质受体抗体对大脑神经元网络进行免疫组织化学分析

• 批准号: 02557011
• 负责人: KURIYAMA Kinya
• 金额: $ 3.71万
• 依托单位: Kyoto Prefectural University of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557011/
• 关键词: GABA_Areceptor antibody; Receptor purification; GABA_B_receptor antibody; Monoclonal antibody; Baclofen affinity column; Immunoaffinity column; Immunohistochemistry; Electron Microscopic immuno-cytochemistry; GABA_A受容体; GABA_B受容体; インミュノアフィニティ精製

Immunohistochemical procedures for cerebral GABA_A and GABA_B receptors using specific monoclonal antibodies have been developed. The results obtained are as follow. 1)Albin o rabbits were immunized against partially purified GABA_A receptor complex from the bovine brain using baclofen-affinity column, and the specific antibody for GABA_A receptor complex was obtained. 2) Mice were immunized against partially purified GABA_B receptor protein using baclofen-affinity column, and the monoclonal antibody which recognizes 80KDa GABA_B receptor was obtained. 3) The immunoaffinity column using GABA_B receptor monoclonal antibody was obtained, and complete purification of the GABA_B receptor protein(80KDa) was achieved using this immunoaffinity column. 4) The purified GABA_B receptor possessed pharmacological properties as GABA_B receptor and exhibited the inhibition of cAMP formation when it was reconstituted in phospholipid vesicles with purified adenylate cyclase and Gi/Go type of GTP binding protein. 5) Immunohistochemical studies using above GABA_A receptor and GABA_B receptor antibodies revealed that the localization of GABA_A receptor and GABA_B receptors at GABAergic synapses was different. 6) It has been confirmed that these immunohistochemical procedures are useful for the analysis of GABAergic pathways in the brain.

用特异性单克隆抗体对脑GABA_A和GABA_B受体进行了免疫组织化学研究。所得结果如下。1)用纯化的牛脑GABA_A受体复合物免疫阿尔宾0兔，获得了GABA_A受体复合物的特异性抗体。2)用部分纯化的GABA_B受体蛋白免疫小鼠，获得了识别80 KDa GABA_B受体的单克隆抗体。3)制备了GABA_B受体单克隆抗体免疫亲和层析柱，用此亲和层析柱可完全纯化GABA_B受体蛋白（80 KDa）。4)纯化的GABA_B受体具有GABA_B受体的药理学特性，当它与纯化的腺苷酸环化酶和Gi/Go型GTP结合蛋白重组在磷脂囊泡中时，表现出抑制cAMP形成的作用。5)用上述GABA_A受体和GABA_B受体抗体进行免疫组织化学研究，发现GABA_A受体和GABA_B受体在GABA能突触上的定位不同。6)已经证实，这些免疫组织化学方法可用于分析大脑中的GABA能通路。

项目成果

J.Taguchi: "Immunohistchemistry of GABA_A receptor complex in the central nervous system." Neuroscience Research. Suppl.14. S.80- (1991)

J.Taguchi：“中枢神经系统中 GABA_A 受体复合物的免疫组织化学。”

H.Nakayasu: "Immunoaffinity purification and characterization of gamma-aminobutyric acid(GABA)_B receptor from bovine cerebral cortex" Journal of Biological Chemistry. (1993)

H.Nakayasu：“来自牛大脑皮层的γ-氨基丁酸（GABA）_B 受体的免疫亲和纯化和表征”生物化学杂志。

K.Kuriyama: "Immunohistochemistry of GABA receptors using monoclonal antibody: special emphasis on GABA_B receptor" Folia Pharmacologia Japonica. (1993)

K.Kuriyama：“使用单克隆抗体的 GABA 受体的免疫组织化学：特别强调 GABA_B 受体”Folia Pharmacologia Japonica。

Y.Suzuki: "Effect of γ-vinyl-γ-aminobutyric acid on the γ-aminobutyric acid receptor-coupled chloride ion channel in vesicles from the brain of rat." Neuropharmacology. 30. 423-427 (1991)

Y.Suzuki：“γ-乙烯基-γ-氨基丁酸对大鼠脑囊泡中 γ-氨基丁酸受体偶联氯离子通道的影响。” 30. 423-427 (1991)。

H.Mizumani Y.Ohmori K.Kuriyama: "Neurochemical Studies on GABA receptor (XXVIII) Purification of cerebral GABA_B receptor by affinitycolumn chromatography and its characterization." Japanese Journal of Pharmacology. 55. Suppl.I-165p (1991)

H.Mizumani Y.Ohmori K.Kuriyama：“GABA 受体的神经化学研究 (XXVIII) 通过亲和柱色谱法纯化脑 GABA_B 受体及其表征。”