Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

Electron microscopic research on the mitochondria-rich bladder cells of the frog

The ultrastructural peculiarities of mitochondria-rich cells of the frog urinary bladder are analysed using three electron microscopic methods: ultrathin sections, scanning electron microscopy, freeze fracture. The mitochondria and tubular and vesicular structures are most abundant in the apical region of cytoplasm. The P-face (PF) of the apical plasma membrane is characterized by the presence of rod-shaped intramembrane particles (IMP), whereas the E-face (EF) possesses complementary pits. Depending on the distribution density of the rod-shaped IMP, three types of cells are described. The apical plasma membrane has an invert distribution of the globular IMP: a great quantity of IMP on the EF and a few particles on the PF. This structure of the apical plasma membrane is supposed to correlate with its very low water permeability. Using filipin as a marker of cholesterol localization, it has been shown that the mitochondria-rich cell apical membrane contains more cholesterol than that of the granular cells. The nature of the rod-shaped IMP and their role in the transmembrane ion transport have been discussed.     

Electron microscopic cytochemical localization of Ca-ATPase in toad urinary bladder

The calcium-regulating enzyme calcium adenosine triphosphatase (Ca-ATPase) was localized in the epithelium of amphibian urinary bladder by the one-step electron microscopic cytochemical procedure. The enzyme was identified along the basolateral border of the epithelial cells that comprise the bladder mucosa. The electron-dense precipitate indicating Ca-ATPase activity was seen in association with the outer leaflet of the basolateral plasmalemmae. Intracellularly, Ca-ATPase activity was seen in association with the mitochondrial matrix of the mitochondria-rich cells. Ca-ATPase was not seen along the apical microvillated border. Enzyme activity was also not seen after incubation in substrate-free media, calcium-free media, or incubation in the presence of vanadate. However, Ca-ATPase activity was evident when the calcium in the standard reaction medium was deleted in favor of magnesium. Addition of antidiuretic hormone (ADH; vasopressin) increased both the basolateral Ca-ATPase reaction and the mitochondrial reaction. Such data appear to indicate further that changes in cytosolic calcium ion concentration take place during the response of amphibian urinary bladder to the polypeptide hormone vasopressin.     

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods

Summary Two histochemical methods for visualization of zine in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata.In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining.Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type, However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.     

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods

Two histochemical methods for visualization of zinc in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata. In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining. Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type. However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.     

Electron microscopic cytochemical and morphometric study of the cerebral cortex synapses in postmortem autolysis

Brain tissue staining with phosphotungstic acid was performed to assay neurofilament accumulations in synapses in the molecular layer of the rat cerebral cortex at different intervals after the animals' death. It was found that autolysis began in the dense projections of presynaptic grid. Within 30 min autolysis developed in mature and very young (immature) synapses. By the 90th min autolysis in asymmetric synapses was considerably enhanced. 6 hours later autolysis involved mature and indefinite synapses.     

Electron microscopic research on the mechanism of intestinal secretion

Electron microscopic investigation of the rat small intestine revealed a great number of vesicles 50-75 nm in diameter with enterocyte microvilli. The number of vesicles increased with the increase of digestive activity in the small intestine. Vesicles were formed by gemmation of enterocyte microvilli from the lateral membrane in contraction of microvillous actin skeleton. Simultaneously with the production of exocytotic vesicles, the formation of pinocytotic vesicles in the base of microvilli was observed. There is a supposition that the vesicle gemmation is a natural process of the intestinal secretion to fulfil numerous important function: it promotes the penetration of enterocyte hydrolases into the parietal layer; equilibrates an increase in the enterocyte volume during absorption. This is a possible way of translocation of synthesized enzymes into the cytoplasm and of transport proteins on the apical surface of epithelial cells.     

Electron microscopic cytochemical localization of nucleoside phosphatases in normal and chronic granulocytic leukaemic human neutrophils

Summary Using electron microscope cytochemistry and cells separated on Ficoll-Hypaque, Mg²⁺-dependent ATPase, ADPase and 5-nucleotidase were predominantly localized as ectoenzymes on normal human granulocytes. Large deposits of ATPase final reaction product and more finely granular deposits of 5-nucleotidase final reaction product were firmly attached to the outer surface of cell plasma membranes. The final reaction product from ecto-ADPase was, however, only loosely associated with the plasma membrane. In addition, finer deposits of ADPase final reaction product were seen in specific granules and in background cytoplasm. No nucleotidase phosphatase activity was localized to the alkaline phosphatase-containing granules (phosphasomes) recently described by Rustinet al.In granulocytes from patients with chronic granulocytic leukaemia, ecto-ATPase had a patchy distribution on the plasma membranes. There was considerable heterogeneity between cells with regard to ADPase and 5-nucleotidase localization. In some cells, ADPase was seen only as an ectoenzyme and in a few it was present in specific granules, but in others it was seen at both sites, while in some cells no activity was detected. 5-Nucleotidase localization was normal in some cells but lacking from many. No correlation was found between enzyme heterogeneity and the degree of morphological cell maturity.     

Electron microscopic demonstration of adenylate cyclase activity in rat cortical synaptosomes

Summary The electron cytochemical demonstration of adenylate cyclase activity was carried out in rat cortical synaptosomes. Reaction product was found in 60–70% of the synaptosomes in three predominant localizations: (i) on the postsynaptic density; (ii) on the outer aspect of the synaptosomal membrane; (iii) inside the synaptosome. Results suggest that in addition to postsynaptic localization adenylate cyclase activity is cytochemically demonstrable also at presynaptic sites.     

Roles of P2 receptors in glial cells: focus on astrocytes

Central nervous system glial cells release and respond to nucleotides under both physiological and pathological conditions, suggesting that these molecules play key roles in both normal brain function and in repair after damage. In particular, ATP released from astrocytes activates P2 receptors on astrocytes and other brain cells, allowing a form of homotypic and heterotypic signalling, which also involves microglia, neurons and oligodendrocytes. Multiple P2X and P2Y receptors are expressed by both astrocytes and microglia; however, these receptors are differentially recruited by nucleotides, depending upon specific pathophysiological conditions, and also mediate the long-term trophic changes of these cells during inflammatory gliosis. In astrocytes, P2-receptor-induced gliosis occurs via activation of the extracellular-regulated kinases (ERK) and protein kinase B/Akt pathways and involves induction of inflammatory and anti-inflammatory genes, cyclins, adhesion and antiapoptotic molecules. While astrocytic P2Y₁ and P2Y_2,4 are primarily involved in short-term calcium-dependent signalling, multiple P2 receptor subtypes seem to cooperate to astrocytic long-term changes. Conversely, in microglia, exposure to inflammatory and immunological stimuli results in differential functional changes of distinct P2 receptors, suggesting highly specific roles in acquisition of the activated phenotype. We believe that nucleotide-induced activation of astrocytes and microglia may originally start as a defence mechanism to protect neurons from cytotoxic and ischaemic insults; dysregulation of this process in chronic inflammatory diseases eventually results in neuronal cell damage and loss. On this basis, full elucidation of the specific roles of P2 receptors in these cells may help exploit the beneficial neuroprotective features of activated glia while attenuating their harmful properties and thus provide the basis for novel neuroprotective strategies that specifically target the purinergic system.     

Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli

The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E. coli spheroplasts. With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved. The majority of DNA macromolecules were released in the form of single and connected rosettes. Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm). The mean length of the rosette loops was 1.06 +/- 0.4 micron. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops. Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. coli reflect one of the levels of organization of prokaryotic genetic material.     

Electron microscopic studies on mouse Leydig cells in vitro

The ultrastructure of cultured Leydig cells isolated from mice testes was studied in the early and late phases of culture. Cells were cultured in Leighton tubes on glass evaporated with carbon and covered with Formvar films. Additionally a histochemical test was carried out in order to evaluate the delta 5, 3 beta-hydroxysteroid dehydrogenase activity of Leydig cells in vitro. Levels of androgen released into the culture medium were measured by radioimmunoassay. Both the histochemical and radioimmunological analyses showed high activity of the enzyme studied and a higher androgen level in the first 4 days of culture. During culture a progressive decrease of the steroidogenic function of Leydig cells in vitro as well as some degenerational changes of the cells were observed. The ultrastructural studies showed the difference between Leydig cells in vitro and in vivo and proved the occurrence of the degenerational modifications of cells in the late phase of culture.     

Electron microscopic morphometric analysis of small cortical and medullary thymocytes from the rat

Electron microscopic morphometric analysis of rat small thymocytes reveals quantitative differences between small cortical and medullary thymocytes. The unit gravity velocity sedimentation technique was used to obtain a cellular pool composed mainly of small sized thymocytes. Stimulation "in vitro" with phytohemagglutinin followed by cell size separation was employed to separate cortical small thymocytes. Furthermore, isolation of medullary small thymocytes was carried out by treatment "in vivo" with hydrocortisone. Our results show that the majority of the quantitative changes correspond to differences in the distribution of chromatin and the density of perichromatin granules. They demonstrate the importance of chromatin pattern analysis for the identification of small cortical and medullary thymocytes.