COMPARATIVE METHODOLOGICAL INVESTIGATIONS ON THE CYTOCHEMICAL-LOCALIZATION OF CALCIUM IN BRAIN AND INNER-EAR OF CICHLID FISH

Four different methods for calcium precipitation are compared in the o ptic tectum and the inner ear of the cichlid fish, Oreochromis mossamb icus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and o xalate-pyroantimonate) produce fine-grained deposits, often flocculent in the latter method. The fourth method (potassium-pyroantimonate) ge nerates predominantly coarse-grained reaction product. The calcium con tent of the deposits is always proven with energy-filtering transmissi on electron microscopy (EFTEM). In both tissues fine-grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and i n addition in some mitochondria and at the cytoskeleton. The coarse-gr ained deposits of the potassium-pyroantimonate method have a more unsp ecific distribution. This is the only method which produces extracellu lar deposits in the inner ear, whereas in the optic tectum extracellul ar precipitates are always present except with the oxalate-pyroantimon ate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of th e fixation time up to 24 hours leads to an increase of the reaction pr oduct, which also becomes coarse-grained. These observations are corro borated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reac tion product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the in vestigated tissue, but it is necessary to optimize a certain method fo r a given tissue. (C) 1995 Wiley-Liss, Inc.