Buchs P A, Stoppini L, Párducz A, Siklós L, Muller D
Department of Pharmacology, Centre Médical Universitaire, Geneva, Switzerland.
J Neurosci Methods. 1994 Sep;54(1):83-93. doi: 10.1016/0165-0270(94)90162-7.
We have developed a new cytochemical method for the localization of calcium at the ultrastructural level in the central nervous system (CNS). The method is based on the use of phosphate buffer in the primary fixation followed by a mixture of a complex of chromium(III)-trisoxalate and osmium tetroxide (OsO4) which precipitates calcium and results in the formation of a high electron-dense reaction product. Calcium selectivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The technique was found to be reproducible, yielding similar results in acutely prepared hippocampal slices or organotypic cultures fixed by immersion and in brain areas fixed by perfusion. In hippocampal slices, calcium deposits were found to accumulate in different subcellular compartments such as endoplasmic reticulum, mitochondria and synaptic vesicles. Interestingly, electron-dense reaction products were also visualized in smooth endoplasmic reticulum structures localized in presynaptic terminals or post-synaptic spines as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifically with regard to synaptic activity, in the CNS.
我们开发了一种新的细胞化学方法，用于在中枢神经系统（CNS）的超微结构水平上定位钙。该方法基于在初次固定中使用磷酸盐缓冲液，随后使用铬（III）-三草酸配合物和四氧化锇（OsO4）的混合物，该混合物沉淀钙并导致形成高电子密度的反应产物。通过试管反应、组织的乙二醇双四乙酸（EGTA）处理、电子光谱成像（ESI）和电子能量损失谱（EELS）验证了钙的选择性。发现该技术具有可重复性，在急性制备的海马切片或通过浸入固定的器官型培养物以及通过灌注固定的脑区中产生相似的结果。在海马切片中，发现钙沉积物积聚在不同的亚细胞区室中，如内质网、线粒体和突触小泡。有趣的是，在位于突触前终末或突触后棘以及突触间隙和活性区的滑面内质网结构中也可见到电子致密反应产物。因此，这种新方法可能对研究中枢神经系统中钙的代谢，特别是与突触活动有关的钙代谢具有重要意义。
