Fritschy J M, Benke D, Mertens S, Oertel W H, Bachi T, Möhler H
Institute of Pharmacology, University of Zürich, Switzerland.
Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6726-30. doi: 10.1073/pnas.89.15.6726.
The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABAA) 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 GABAA receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.
大脑中构成A型γ-氨基丁酸（GABAA）受体的亚基具有非凡的结构多样性，预计这会产生不同模式的GABA能突触抑制以及在抗焦虑、催眠和抗癫痫治疗中有效的不同调节药物谱。为了原位鉴定受体亚型，使用针对α1、α3和γ2亚基的多克隆抗体以及一种用于定位β2和β3亚基的单克隆抗体，通过大鼠脑内的双重和三重免疫荧光染色来观察最常见的亚基。在细胞和亚细胞水平上，鉴定出了五种不同的亚基共定位模式：I，α1β2,3γ2；II，α3β2,3γ2；III，α1α3β2,3γ2；IV，α3γ2；以及V，α1α3γ2。通过共聚焦激光显微镜分析，不同亚基沿质膜显示出相同的局部染色强度变化（“热点”）。因此，共可视化的亚基似乎在受体亚型中共同组装。大多数神经元仅表达单一的主要受体亚型，突触和突触外位点之间没有明显区别。然而，在一些神经元中，最显著的是浦肯野细胞，胞体和树突之间的亚基组成有所不同，这表明单个神经元内存在受体异质性。此外，不同的神经元群体可能以特定的受体亚型为特征。显示α1亚基免疫反应性的细胞大多被鉴定为GABA能神经元，而单胺能神经元显示出强烈的α3亚基免疫反应性，但几乎没有α1亚基免疫反应性。将特定的GABAA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。
