Brückner G, Bringmann A, Köppe G, Härtig W, Brauer K
Department of Neurochemistry, Paul Flechsig Institute for Brain Research, University of Leipzig, Germany.
Brain Res. 1996 May 13;720(1-2):84-92. doi: 10.1016/0006-8993(96)00152-7.
Previous lectin-histochemical and immunocytochemical investigations using fixed tissue revealed perineuronal nets as lattice-like accumulations of extracellular matrix proteoglycans at the surface of several types of neurons. In the present study, perineuronal nets in the rat brain were labelled for the first time in vivo by stereotaxic injections of biotinylated Wisteria floribunda agglutinin (Bio-WFA), as well as in vitro, by incubation of unfixed brain slices with the same lectin. Six days after Bio-WFA injections into the parietal cortex, medial septum, reticular thalamic nucleus and red nucleus, the lectin remaining bound to perineuronal nets was detected by streptavidin/biotinylated peroxidase complexes or red fluorescent Cy3-streptavidin, respectively. Double-fluorescence labelling showed that Bio-WFA applied in vivo reacted with the chondroitin sulphate proteoglycan immunoreactive perineuronal nets in the injection zone. Labelling of perineuronal nets in unfixed slices was obtained with either Cy3-tagged WFA or Bio-WFA and subsequent visualization by Cy3-streptavidin which confirmed the region-dependent distribution patterns and the structural characteristics of perineuronal nets known from histochemical studies. These results provide support for the role of extracellular matrix proteoglycans to maintain a considerable chemical and, probably, spatial heterogeneity of the extracellular space in vivo. The ability of in vivo and in vitro labelling may promote the functional characterization of the extracellular matrix in various brain structures including its species-dependent neuronal association patterns.
以往利用固定组织进行的凝集素组织化学和免疫细胞化学研究显示，在几种类型神经元的表面，神经周网是细胞外基质蛋白聚糖的晶格状积聚物。在本研究中，首次通过立体定向注射生物素化的紫藤凝集素（Bio-WFA）在大鼠脑内对神经周网进行标记，并且在体外，通过将未固定的脑片与相同的凝集素孵育进行标记。向顶叶皮质、内侧隔核、丘脑网状核和红核注射Bio-WFA六天后，分别通过链霉亲和素/生物素化过氧化物酶复合物或红色荧光Cy3-链霉亲和素检测到仍与神经周网结合的凝集素。双荧光标记显示，体内应用的Bio-WFA与注射区域中硫酸软骨素蛋白聚糖免疫反应性神经周网发生反应。用Cy3标记的WFA或Bio-WFA对未固定切片中的神经周网进行标记，随后通过Cy3-链霉亲和素进行可视化，证实了组织化学研究中已知的神经周网的区域依赖性分布模式和结构特征。这些结果支持了细胞外基质蛋白聚糖在体内维持细胞外空间相当大的化学异质性以及可能的空间异质性方面的作用。体内和体外标记的能力可能会促进对包括其物种依赖性神经元关联模式在内的各种脑结构中细胞外基质的功能表征。
