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

使用特异性单克隆抗体的脑GABA_A和GABA_B受体的免疫组织化学方法已经开发出来。所得结果如下：1)利用巴氯芬亲和柱对白兔进行部分纯化的牛脑GABA_A受体复合物免疫，获得GABA_A受体复合物特异性抗体。2)利用巴氯芬亲和柱对部分纯化的GABA_B受体蛋白进行免疫，获得识别80KDa GABA_B受体的单克隆抗体。3)获得GABA_B受体单克隆抗体免疫亲和柱，利用该免疫亲和柱完全纯化了GABA_B受体蛋白（80KDa）。4)纯化后的GABA_B受体具有与GABA_B受体相同的药理特性，并与纯化的腺苷酸环化酶和Gi/Go型GTP结合蛋白在磷脂囊泡中重组后，表现出对cAMP形成的抑制作用。5)利用上述GABA_A受体和GABA_B受体抗体进行免疫组化研究，发现GABA_A受体和GABA_B受体在gaba能突触的定位是不同的。6)已经证实，这些免疫组织化学方法对于分析脑内gaba能通路是有用的。

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.