Ultrastructural localization of calcium in the CNS of vertebrates

The ultrastructural localization of calcium in synaptic areas of the CNS of fish was investigated. Prefixation with phosphate-buffered glutaraldehyde followed by post-fixation with osmium/potassium-bichromate was used to precipitate and visualize endogenous calcium without the addition of external calcium. The presence of calcium in the electron-dense precipitates produced using this method was demonstrated by electron spectroscopic imaging using a Zeiss EM-902 transmission electron microscope, and in various control experiments using the calcium chelator EGTA. In the optic tectum of fish, electron dense precipitates containing calcium were found not only in intracellular compartments, e.g. the smooth endoplasmic reticulum, mitochondria and synaptic vesicles, but also at extracellular locations, particularly in synaptic clefts. In the extracellular sites, only chelate complexes of ionic calcium were found. This would seem to be in agreement with electrophysiological and biochemical data reported in earlier studies. Thus, using the present method, it should be possible to obtain further ultrastructural information concerning the mechanisms of synaptic transmission.     

Ultrastructural localization of endogenous calcium in the teleost retina

Summary The ultrastructural localization of endogenous calcium in the retina of adult cichlid fishOreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electronspectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.     

Ultrastructural and cytochemical observation of calcium ion localization in the black-beetle pleural muscles

The authors examined the infrastructure and distribution of calcium ions in the plexural muscles of a black-beetle (Blatta orientalis L.) after being fixed in a solution of osmiumtetroxide buffered with a cacodylate buffer (pH = 7.4) and according to the method of Carasso-Favard (1966). The infrastructure of these muscles differs from the muscles of other insects, first of all, in the amount and distribution of the sarcoplasmic net (SR) and mitochondria and also in the amount and topography of the location of lead precipitations which mark the calcium ions. The authors ascertained an intensive and permanent positive reaction to the presence of calcium in the mitochondria and sarcotubular systems of the sarcoplasmic reticulum, and in the intrafibrillar spaces and in the Z and M lines of the sarcomeres calcium concentrations are not detectable. The authors checked the results by using the method of Carasso-Favard (1966).     

Ultrastructural localization of calcium deposits during in vitro culture of pig oocytes

Calcium deposits were localized using the combined oxalate-pyroantimonate technique in follicle-enclosed oocytes fixed in situ. These deposits can be observed within vacuoles, mitochondria, and on the surface of yolk granules as well as in the caryoplasm, but are absent from the endoplasmic reticulum. Isolation of the oocyte from the follicle resulted in the immediate depletion of these calcium deposits. Replenishment of these deposits started during the first 8 hr of in vitro culture of the oocyte and they were gradually replenished to the levels observed before the liberation of oocytes during in vitro maturation to the stage of metaphase II.     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.     

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet

Summary The localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO₄. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.This investigation was supported in part by the Mitsubishi-Foundation, 1976 to Professor V. Mizuhira     

Ultrastructural localization of acid protein polysaccharides and calcium in the vacuoles of the chicken thrombocyte.

The coexistence of acid protein polysaccharides and calcium in the vacuoles of chicken thrombocytes were studied by means of ultrahistochemical methods and electron probe X-ray microanalysis. The thrombocytes possessed large vacuoles of a surface connected membrane system. After both ruthenium red staining and tannic acid fixation the innersurface coat of vacuoles was always strongly and continously visualized. Electron microscopic X-ray microprobe analysis of antimonate precipitates in thrombocytes fixed in K-antimonate-OsO4 revealed calcium localization on the innersurface of vacuoles. From these facts it seems likely that the vacuoles of the surface connected membrane system may take part in the pool or the transport of calcium.     

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet.

The localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO4. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.     

Tritrichomonas foetus: Ultrastructural localization of calcium in the plasma membrane and in the hydrogenosome

The osmium tetroxide-potassium pyroantimonate technique was used to localize Ca²⁺-containing sites in the protozoan Tritrichomonas foetus. Reaction product was seen in association with the plasma membrane and with a membrane-bound organelle, the hydrogenosome. Reaction product was also seen in some cytoplasmic vesicles and in lysosomes. Treatment of the ultrathin sections with EGTA resulted in removal of the pyroantimonate precipitate. These results suggest that the hydrogenosome may be involved in the control of the intracellular concentration of Ca²⁺ in T. foetus.     

Ultrastructural localization of acetylcholinesterase

Summary A method is described allowing localization of acetylcholinesterase (AChE) by both light and electron microscopy. During the reaction lead thio-diacetyl is decomposed, and therefore precipitated as PbS in the presence of native-SH group produced by the hydrolysis of acetylthiocholine perchlorate. The reaction takes place at neutral pH, since improves the sensitivity of AChE localizations. Application of the method to parasympathetic neurons showed that AChE was mainly localized in the rough endoplasmic reticulum of the perikaryons. No reaction was visible in glial cells. AChE was also localized on the plasma membrane of parasympathetic neurons. In mouse embryo muscles AChE activity was seen to be high and was not yet restricted to the synaptic area. The well developed Schwann cells accompanying the neurites displayed constant AChE activity on their plasma membrane.Supported by a grant of INSERM C.R.L. N⁰ 79-5-318-6     

Ultrastructural localization of calcium pyroantimonate in Mauthner neurons of goldfish fry adapted to natural stimulation

The localization of Ca2+ in control and adapted goldfish fry Mauthner cells (M-cells) revealed by sedimentation with potassium pyroantimonate technique was investigated. It has been shown the following. 1. In the control M-cells electron dense precipitates are present in the extracellular space, commonly within the active zone clefts of chemical synapses, throughout the whole apposition of the mixed synapses and in the synaptoplasm of both type afferent boutons. No precipitates were seen in the cytoplasm of M-cells. 2. After long term natural (vestibular) stimulation (LTNS), resulting in a strong functional suppression of M-cells, precipitates disappeared entirely from active zones but remained numerous in the cytoplasm of M-cells. The distribution of precipitates within the cytoplasm was non-uniform, the highest density was observed on the surfaces of intracellular organelles and elements of the cytoskeleton. 3. In fatigued M-cells after LTNS and after a subsequent one day rest the distribution of precipitates was less intensive, while in the whole it resembled that of fatigued M-cells. 4. In adapted M-cells the distribution of precipitates was similar to that observed in control. M-cells after LTNS, but the amount and size of the precipitated grains were noticeably increased. 5. The most numerous precipitates were seen in adapted M-cells after LTNS. They were localized throughout the postsynaptic cytoplasm and in a lesser order in the presynaptic cytoplasm. 6. After one day rehabilitation the intensitivity of cytochemical reaction of Ca2+ ion precipitation restored to the initial stage characteristic of adapted M-cells before LTVS. The results obtained suggest that the total concentration of Ca2+ ions in adapted M-cells and the dynamics of their exchanges between cytosole and intracellular depots, such as the smooth endoplasmic reticulum, may increase to keep a normal or even increased functional activity of M-cells, both before and after the LTNS.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.