A new cytochemical method for the ultrastructural localization of calcium in the central nervous system

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.