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 ca lcium at the ultrastructural level in the central nervous system (CNS) . The method is based on the use of phosphate buffer in the primary fi xation 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 sele ctivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron e nergy loss spectroscopy (EELS). The technique was found to be reproduc ible, 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 accu mulate in different subcellular compartments such as endoplasmic retic ulum, mitochondria and synaptic vesicles. Interestingly, electron-dens e reaction products were also visualized in smooth endoplasmic reticul um structures localized in presynaptic terminals or post-synaptic spin es as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifica lly with regard to synaptic activity, in the CNS.