Rothe F, Brosz M, Storm-Mathisen J
Institute of Medical Neurobiology, Medical Faculty, University of Magdeburg, Germany.
Neuroscience. 1995 Feb;64(4):iii-xvi. doi: 10.1016/0306-4522(94)e0200-n.
Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.
谷氨酸脱氢酶是参与神经递质谷氨酸形成和代谢的主要酶之一。在本研究中，我们通过包埋前免疫细胞化学染色（过氧化物酶 - 抗过氧化物酶）以及包埋后免疫金标记，在富含明确定义的谷氨酸能神经元的小脑皮质区域对该酶进行超微结构研究，包埋后免疫金标记采用了一种新系统，用于在免疫细胞化学程序条件下进行定量和特异性测试。使用针对免疫纯化的牛肝谷氨酸脱氢酶的新抗血清或通过亲和层析从该抗血清中分离的抗体，对经醛灌注固定的大鼠进行实验。包埋前方法显示过氧化物酶反应优先出现在星形胶质细胞（包括伯格曼胶质细胞）的线粒体中。神经组织成分的线粒体通常没有过氧化物酶反应产物。未观察到线粒体外染色。采用包埋后免疫金方法以克服穿透问题，并对定位和特异性进行半定量分析。在星形胶质细胞成分（包括伯格曼胶质细胞）的线粒体上发现了最高密度的金颗粒。伯格曼胶质细胞胞体中的线粒体颗粒密度低于星形胶质细胞突起中的线粒体。在后者中，频率分布分析未发现缺乏谷氨酸脱氢酶的线粒体群体的证据，但表明存在具有不同免疫反应性水平的群体。与包埋的牛肝谷氨酸脱氢酶的标记比较表明，该酶占这些线粒体总基质蛋白的很大比例（10%）。在白质的少突胶质细胞中发现较弱但显著的标记。包括谷氨酸能苔藓纤维终末在内的神经成分中线粒体的标记约为星形胶质细胞线粒体标记的15%。在谷氨酸能神经元（苔藓纤维和平行纤维、颗粒细胞）和非谷氨酸能神经元（浦肯野细胞）之间未检测到差异。非线粒体区域的颗粒密度非常接近空树脂的背景。用不同的组织和抗体制备方法获得的结果一致表明，目前形式的谷氨酸脱氢酶仅限于线粒体，并且优先定位于星形胶质细胞中。
