5 SUBTYPES OF TYPE-A GAMMA-AMINOBUTYRIC-ACID RECEPTORS IDENTIFIED IN NEURONS BY DOUBLE AND TRIPLE IMMUNOFLUORESCENCE STAINING WITH SUBUNIT-SPECIFIC ANTIBODIES
5 SUBTYPES OF TYPE-A GAMMA-AMINOBUTYRIC-ACID RECEPTORS IDENTIFIED IN NEURONS BY DOUBLE AND TRIPLE IMMUNOFLUORESCENCE STAINING WITH SUBUNIT-SPECIFIC ANTIBODIES
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DOI：
10.1073/pnas.89.15.6726
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发表时间：
1992-08-01
影响因子：
11.1
通讯作者：
MOHLER, H
中科院分区：
文献类型：
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作者：
FRITSCHY, JM;BENKE, D;MOHLER, H
脑中形成A型γ-氨基丁酸（GABA（A））受体的亚基的非凡结构多样性预期引起GABA能突触抑制的不同模式和在抗焦虑、催眠和抗癫痫治疗中有效的调节药物的不同特征。为了在原位鉴定受体亚型，通过大鼠脑中的双重和三重免疫荧光染色，使用α-1、α-3和γ-2亚基的多克隆抗体和定位β-2和β-3亚基的单克隆抗体，观察最普遍的亚基。在细胞和亚细胞水平上，鉴定了五种不同的亚基共定位模式：I，α-1-β-2，3-γ-2; II，α-3-β-2，3-γ-2; III，α-1-α-3-β-2，3-γ-2; IV，α-3-γ-2;和V，α-1-α-3-γ-2。如通过共聚焦激光显微镜分析的，不同的亚基显示相同的局部变化的染色强度（“热点”）沿质膜沿着。因此，共可视化的亚基似乎在受体亚型中共组装。大多数神经元只表达一种主要的受体亚型，突触和突触外位点之间没有明显的区别。然而，在一些神经元，最明显的是在浦肯野细胞，亚基组成之间的索马和树突，指出在单个神经元内的受体异质性的存在。此外，不同的神经元群体可以通过特定的受体亚型来表征。显示α-1-亚单位免疫反应的细胞大多被鉴定为GABA能，而单胺能神经元显示强烈的α-3-亚单位免疫反应，但几乎没有α-1-亚单位免疫反应。将确定的GABA（A）受体亚型分配给已鉴定的神经元为受体异质性的功能分析开辟了道路。
The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABA(A)) receptors in the brain is expected to give rise to different modes of GABAergic synaptic inhibition and different profiles of modulatory drugs effective in anxiolytic, hypnotic, and antiepileptic therapy. To identify receptor subtypes in situ, the most prevalent subunits were visualized by double and triple immunofluorescence staining in rat brain, using polyclonal antibodies to the alpha-1, alpha-3, and gamma-2 subunits and a monoclonal antibody to locate both the beta-2 and the beta-3 subunit. At both cellular and subcellular levels five distinct patterns of subunit colocalization were identified: I, alpha-1-beta-2,3-gamma-2; II, alpha-3-beta-2,3-gamma-2; III, alpha-1-alpha-3-beta-2,3-gamma-2; IV, alpha-3-gamma-2; and V, alpha-1-alpha-3-gamma-2. As analyzed by confocal laser microscopy, different subunits displayed the same local variations of staining intensity ("hot spots") along the plasma membrane. The covisualized subunits appear therefore to be coassembled in receptor subtypes. Most neurons expressed only a single major receptor subtype with no apparent distinction between synaptic and extrasynaptic sites. However, in some neurons, most notably in Purkinje cells, the subunit composition varied between the soma and the dendrites, pointing to the existence of receptor heterogeneity within single neurons. Furthermore, different populations of neurons may be characterized by particular receptor subtypes. Cells displaying alpha-1-subunit immunoreactivity were mostly identified as GABAergic, whereas monoaminergic neurons displayed intense alpha-3-subunit immunoreactivity but virtually no alpha-1-subunit immunoreactivity. The allocation of defined GABA(A) receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.