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G G Skibo

Publications and source records attributed to G G Skibo.

At least 19 recordsLinked to original sources
PubMed2003

The influence of Hericium erinaceus extract on myelination process in vitro.

Myelin sheaths, wrapping axons, perform the following important functions: support, protection, feeding and isolation. Injury of myelin compact structure leads to an impairment and severe illness of the nerve system. Exact mechanisms underlying the myelination process and myelin sheaths damage have not established yet. Therefore search for substances, which provide regulatory and protective effects on the normal myelination as well as stimulating action on the remyelination after myelin damage, is of special interest. Recently it was shown that extract from mushroom Hericium erinaceus had activating action on the nerve tissue. So the aim of the present work was to study an influence of an extract from H. erinaceus on the cerebellar cells and the process of myelination in vitro. Obtained data revealed the normal growth of the nerve and glial cells with extract at cultivating. No pathologic or toxic action of the extract has been found. The cell ultrastructure was intact and similar to that observed in vivo. The process of myelination in the presence of the extract began earlier as compared to controls and was characterised by a higher rate. Thus, extract of H. erinaceus promoted normal development of cultivated cerebellar cells and demonstrated a regulatory effect on the process of myelin genesis process in vitro.

Animals↗
PubMed2003

Myelination and demyelination processes in the rat cerebellum cell culture: an electron microscopic study.

We observed manifestations of the myelination process in dissociated culture of the cerebellar tissue of newborn rats and modifications of the structure of myelin sheaths after treating the culture with a demyelinating factor, blood serum of patients suffering from multiple sclerosis (MS). On day in vitro (DIV) 26, in the control myelin sheaths of the axons demonstrated the closest resemblance to those observed in vivo, and we selected this term for inducing demyelination. Addition of the serum of MS patients to the culturing medium evoked rapid (in 3-6 hours) dramatic changes in the ultrastructure of myelin sheaths; these were a decrease in the number of the lamellae, their splitting and invagination, formation of vesicles, etc. The serum of MS patients in an acute stage of the disease exerted more intensive demyelination effects than that of patients in a remission stage.

Animals↗
PubMed2000

Computer simulation approach to the quantification of immunogold labelling on plasma membrane of cultured neurons.

Cell culture is a convenient model system to study the expression of plasma membrane-bound proteins in nerve cells. Analysing it with an ultrastructural detail researchers often apply transmission electron microscopy together with immunogold labelling. Plasma membrane profiles are one-dimensional (1D) and provide little information about the topography of membrane-bound proteins. In order to convert 1D estimates of spatial arrangement for preembedding immunogold labelled proteins into two-dimensional (2D) quantities, namely the 2D pattern and density of labelling, this paper presents a simple computer simulation technique. This technique is based on a mathematical model permitting a simulated immunogold labelled membrane to be sampled in a way similar to microtome sectioning. An interlabel distance (ILD) estimate is used to define the position of immunogold particles in membrane profiles. In order to interpret experimental ILD measurements the simulated distribution best fit to the experimental data is selected and the corresponding 2D density and pattern of particle scattering are considered to explain the real situation. Various parameters including a cell section thickness, immunogold particle size etc can be adjusted to suit the demands of a particular experiment. The technique was applied to quantify the NCAM preembedding immunogold labelling in the plasma membrane of cultured rat hippocampal neurons.

Animals↗
PubMed2000

Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity.

The distribution of glial cells (microglia and astrocytes) in different regions of normal adult rat brain was studied using immunohistochemical techniques and computer analysis. Lipocortin 1, phosphotyrosine, and lectin GSA B(4), were used for identification of microglia, while S100beta and glial fibrillary acidic protein identified astrocytes. Bioquant computerized image analysis was used to quantify and map the immunostained cells in sections from adult rat brain. If lipocortin 1 was used as a marker, more microglial cells were detected than with phosphotyrosine or lectin. The lipocortin 1-positive microglial population was most numerous (on average, 130+/-5 cells/mm(2) of the brain section area) in neostriatum, and least (51+/-4 cells/mm(2)) in cerebellum and medulla oblongata. In general, the density of lipocortin 1 microglia was higher in the forebrain, and lower in the midbrain, and the least in the brainstem and cerebellum. The number of S100beta astrocytes was two to three times larger than the number of microglial cells, and approximately two times greater than glial fibrillary acidic protein cells. A high density of astrocytes was found in the hypothalamus and hippocampus (more than 260 cells/mm(2)); they were more numerous in the white matter than in the gray matter. Fewer astrocytes were observed in the cerebral cortex, neostriatum, midbrain, medulla oblongata and cerebellum (less than 200 cells/mm(2)). Thus lipocortin 1 and S100beta were shown to be the most specific and reliable markers for microglia and astrocytes, respectively. The regional population differences demonstrated for lipocortin 1 microglia and S100beta astrocytes presumably reflect structural and functional specializations of the certain brain regions.

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PubMed2000

Microglia in organotypic hippocampal slice culture and effects of hypoxia: ultrastructure and lipocortin-1 immunoreactivity.

Lipocortin-1 immunocytochemistry was used to study the various cell forms of microglia that appear during organotypic hippocampal tissue culture, as well as in the in vitro toxic hypoxia model. Antibodies against lipocortin-1 identified activated and phagocytic cells that were abundant in a slice after the plating of a culture: cells of the intermediate form at the later time-points of culturing, resting ramified microglia beginning from the seventh day of culturing, as well as activated and phagocytic cells that appeared in the slice after experimental toxic hypoxia induced by potassium cyanide treatment. Lipocortin-1-positive microglia cell forms corresponded well to the description of the microglia in vivo, and the morphology of microglia corresponded to the circumstances under which these cells were observed in slice cultures. Electron microscopic studies have demonstrated, for the first time, that microglia in organotypic slice culture preserve morphological features typical of different microglial forms in vivo, as well as specific contacts and interactions with the other neural tissue elements. After experimental toxic hypoxia, rapid changes in microglial ultrastructure and localization were observed, reminiscent of in vivo models of ischaemia. In conclusion, observations of microglial morphology and behaviour allow us to suggest that microglia in the organotypic culture preserve their essential characteristic features and properties, thus providing an important model system for studying the structure and function of these cells.

Animals↗
PubMed1999

[Changes in the topography of various proteins in cultured neurons after selective injury of various cytoskeletal elements with neurotoxins].

The spinal cord and hippocampal primary cultures were incubated with three neurotoxins (separately) known to impair the main components of the cytoplasmic cytoskeleton: 1) colchicine blocking the repolymerization of microtubules, 2) cytochalasin preventing elongation of actin filaments, and 3) beta, beta'-iminodipropionitrile (IDPN), causing disorganisation of neurofilaments. The distribution of surface membrane molecules on the surface of the neurons was evaluated in the ultrastructural study after treatment with the neurotoxins on the 5th, 12th, and 15th days in vitro (DIV). On the 12 DIV, the density of immunogold labelled neural cell adhesion molecules (NCAM) on IDPN-treated hippocampal neurons increased 1.45 times comparing to the controls. On the 5 DIV, the density of WGA (wheat germ agglutinin)-binding membrane glycoproteins increased 2.09 times on colchicine-treated neurons, and 3.98 times on cytochalasin-treated ones, whereas on the 12 DIV, the increase was 3.28 and 2.72 times, respectively, as compared to the control cultures of the same age. These data provide insights into the mechanisms of neurodegenerative changes in the nerve cells and into the relationship between the cytoskeletal elements and the surface molecules on the neuronal plasmatic membrane.

Animals↗
PubMed1998

Increased immunogold labelling of neural cell adhesion molecule isoforms in synaptic active zones of the chick striatum 5-6 hours after one-trial passive avoidance training.

An area of the chick striatum, the lobus parolfactorius plays an important role in one-trial passive avoidance learning tasks. In the present study we report evidence that 5-6 h post-training, a significantly higher proportion of synaptic active zones in this area contain labelled epitopes of the neural cell adhesion molecule, with the greatest occurrence of labels at the edges of active zone profiles (in both control and trained groups). This suggests that there is a period after training when expression of the neural cell adhesion molecule in synaptic membranes almost doubles, and that events at active zone edges may play a specific role in mechanisms of synaptic adhesion. Cellular mechanisms of long-term memory formation are believed to include alterations in neural circuitry at the synaptic level. The involvement of the neural cell adhesion molecule (NCAM) in functional synaptic modifications has been demonstrated using a number of physiological models. Performance of rats in the Morris water maze, a spatial learning paradigm which requires the hippocampus, is impaired by either intraventricular injection of NCAM antibodies, or injection into the hippocampus of an enzyme which increases homophilic adhesion of the molecule, due to the removal of polysialic acid residuals from extracellular NCAM domains. In addition, intraventricular injections of anti-NCAM antibodies 6-8 h post-training were shown to impair memory for a one-trial passive avoidance task in the rat. An avoidance training model in the one-day-old chick indicates a similar time window, 5-6 h post-training during which memory for the task can be impaired by intraventricular injection of NCAM antibodies. In the hyperstriatum ventrale, a chick forebrain area involved in the passive avoidance task. subtle changes in the distribution pattern, but not density of NCAM molecules in synaptic membranes were revealed 5-6 h post-training. However, on the basis of studies of synaptic morphometry, a region of striatum, the lobus parolfactorius (LPO), appears to play a more important role in longer term memory storage for the task.

Animals↗
PubMed1995

Growth related changes in sugar determinants on the surface of C6 glioma cells in culture: a cytochemical lectin-binding study.

The cell surface sugar determinants (CSSD) were examined in C6 glioma cells in cultures at different conditions of growth by peroxidase conjugates of the lectins: peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Helix pomatia agglutinin (HPA), wheat germ agglutinin (WGA), lentil agglutinin (LCA), laburnum bork agglutinin (LABA), and lotus agglutinin (TPA). It was found that the cells bound more intensively WGA, LCA, and RCA compared to PNA, HPA; the weakest staining was provided by LABA and TPA. Binding intensity for PNA significantly increased after pretreatment of the cells with neuraminidase. This indicates that a part of the beta-D-galactose residues on the surface membrane of C6 glioma cells is covered by sialic acid. The process of sialization was increased during the culturing of C6 glioma cells. Addition of cis-DDP or dBcAMP to cultures growing in medium with 10% of CS increased the number of Gal residues which are not covered by sialic acid. The expression of beta-D-galactose (Gal), N-acetyl-D-galactosamine (NAcDGal), and fucose (Fuc) residues appeared to be most responsive to changes in growth conditions and degree of cell differentiation. The expressions of N-acetyl-D-glucosamine (NAcDGlc) and mannose (Man) residues were high and seems did not depend on changing of the conditions of culturing. In C6 glioma cells cultures in which the rate of cell division, formation of the cell processes, and adhesiveness of the cells to the substratum were reduced by growing cells in MEM+, expression of beta-Gal, NAcDGal, and Fuc was considerably reduced. The decrease of expression of beta-Gal, NAcDGal, and Fuc on the surface of cell membrane was more pronounced in MEM+ with 1% of CS than in MEM+ with 10% of CS. In DbcAMP and cis-DDP treated cultures, grown in medium with 1% serum, in which cell division was inhibited without obvious changes in cell adhesiveness to the substratum, binding of PNA and HPA was increased due to higher expression of beta-Gal and NAcDGal. From these observations it was concluded that the pattern of expression of sugar residues on the cell surface varies according to the biological state of the cells and are easily affected by tissue culture conditions.

Animals↗
PubMed1995

Neural cell adhesion molecule (N-CAM) distribution may predict the effect of neurotoxins on the brain.

The neural cell adhesion molecule (N-CAM) is a convenient neurospecific marker for investigating the effects of neurotoxins on cell migration, cell recognition and differentiation of neurons during development. In this report, we discuss the developmental toxicity of valproic acid studied by two different approaches (the immunochemical detection of N-CAM content and polypeptide composition, and immunohistochemical analysis of N-CAM topography). Immunohistochemical analysis of distribution of N-CAM as a surface marker on the neural cells predicted the effect of the neurotoxin.

Animals↗
PubMed1993

Changes in the neural cell adhesion molecule patterns on the rat glial cell surfaces with development and contact formation in vitro.

In monolayer cultures of newborn rat hippocampal cells, immunogold-labelling at the electron microscope level was used to study quantitatively the neural cell adhesion molecule (N-CAM) arrangement on the surface of glial soma and processes on 5 and 12 days in vitro (DIV). Four corresponding samples of micrographs were formed. To quantify the labelling, a stochastic geometry approach was used. Spectra of lateral distances between labels as well as simulated images of the surface label arrangement (invisible in micrographs) were derived and compared. The data show that, on both 5 and 12 DIV, N-CAM density on the surface of processes is approximately 2 times higher than that in somata; 12-DIV cells showing a lower (approximately 25%) N-CAM surface density as compared with the 5-DIV cells. This suggests that N-CAM expression in glia surfaces decreases while the cells form contacts, and N-CAM sorting between soma and processes remains stable. The simulated topographies of the lateral N-CAM arrangement might highlight fundamental mechanisms that underlie formation of the neural network.

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PubMed1993

Cytoskeleton-mediated, age-dependent lateral topography of lectin-gold-labelled molecules on the plasma membrane of cultured neurons: a statistical view.

In dissociated spinal cord neurons (12-day-old mouse embryo, monolayer culture), an electron microscopic study was carried out to examine quantitatively the rearrangement of wheat-germ agglutinin-gold-labelled molecules on the neuronal somatic surface at two developmental stages (on the fifth and 15th days in vitro), and after cytoskeletal interruptions. In tests, before labelling the cultures were incubated with colchicine or cytochalasin in order to affect microtubules or mostly actin filaments, respectively. Samples of electron micrographs that display soma membrane (profile) fragments were quantified. A set of stochastic geometry approaches was accomplished, which allowed statistical and stereological analysis of labelling. Images that illustrate the lateral (surface) patterns of label were simulated. On the fifth day in vitro, both colchicine and cytochalasin were found to cause an increase in the surface density and aggregation of wheat-germ agglutinin label relative to controls, the effect of cytochalasin being significantly more profound. By the 15th day in vitro, treatment with both drugs led to a similar tendency towards heavy aggregation of wheat-germ agglutinin labels. In contrast, neuron processes showed an opposite tendency of label rearrangement, which suggests lateral migration of labelled molecules, as a result of drug action. Possible molecular mechanisms involved in the phenomena are discussed.

Aging↗
PubMed1993

Lateral patterns of the neural cell adhesion molecule on the surface of hippocampal cells developing in vitro.

In monolayer cultures of hippocampal neurons from newborn rats, an immunocytochemical quantitative study was carried out to investigate age-dependent arrangement of the neural cell adhesion molecules in different parts of cell membranes. On the fifth and 12th day in vitro, neural cell adhesion molecules were labelled with specific antibodies and protein A conjugated to colloidal gold particles. Samples of randomly selected electron micrographs that displayed labelled membrane fragments of cell bodies, growth cones, and axons were numerically analysed for the five- and 12-day in vitro neurons. Neural cell adhesion molecules surface topography was quantitatively described and compared, using a statistical stereological approach. The mean surface density of labelled neural cell adhesion molecules was found to be approximately 2.5 times higher in growth cone membranes relative to somatic and axonal membranes in five-day in vitro neurons. By the 12th day in vitro, this density decreases in somatic membranes (approximately 18%) and increases in axonal membranes (approximately 60%). Representative spectra of lateral intervals between labels as well as images that show typical topography of label on membrane surfaces were simulated. The results revealed regular patterns of neural cell adhesion molecules on the somatic surface and allowed consideration of neural cell adhesion molecules arrangement in a view of membrane adhesion properties. Participation of cytoskeleton in neural cell adhesion molecules rearrangement is discussed.

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PubMed1991

The spatial pattern of the synaptic vesicular apparatus as a correlate of transmitter storage models.

A statistical stereological approach which allows one to derive a three-dimensional pattern of synaptic vesicle accumulation in relation to the active zone from an analysis of electron micrograms (random sections) of synapses is described. This approach is illustrated with a study of presynaptic terminals from the dorsal horn of the cat spinal cord, based on the morphometrical treatment of 105 micrographs containing 5190 synaptic vesicles. The spatial pattern obtained was found to have a bimodal shape, which can be considered a possible structural correlate of the two-pool model of transmitter storage. The connection of similar quantitative estimates with physiological data is discussed.

Animals↗
PubMed1991

Blocking effect of intraperitoneal injection of phenylalanine on high-threshold calcium currents in rat hippocampal neurones.

Calcium currents were recorded in cultured (5-7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of L-phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in L-phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at Vt = +20 mV became 40 +/- 30% as contrasted to 416 +/- 130% in neurones from control animals (the amplitude of the calcium currents at Vt = -10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of L-tyrosine to the cultivation medium (50 microM) restored the high-voltage calcium current, its relative amplitude reaching 280 +/- 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.

Animals↗
PubMed1991

[Restoration of the potential-dependent L-phenylalanine-inhibited calcium current by L-tyrosine in cultured hippocampal neurons].

Calcium currents were recorded in cultured (5-7 days) hippocampal neurons isolated from one-day rats. The animals were intraperitoneally injected L-phenylalanine which induced in their brain biochemical changes typical of phenylketonuria. It has been found that in neurons from injected animals the amplitude of the high-threshold calcium current was substantially inhibited and amounted to 40 +/- 30% at Vt = +20 mV (amplitude of calcium currents at Vt = -10 mV taken as 100%). Addition of L-tyrosine to the cultivation medium (50 mumol/l) restored the high-voltage calcium current, its relative amplitude reaching 280 +/- 57%.

Animals↗
PubMed1991

[Quantitative characteristics and reconstruction of the surface topography of histochemical markers on the nerve cell plasmalemma].

An electron microscopic study of topography of colloidal gold-bound lectins conjugating to glycocalix carbohydrate residues on the somatic membrane surface of the cultivated spinal neurons has been carried out. The quantitative procedures are suggested for analyzing the surface pattern of the markers: two stochastic functions are considered to correspond properly to the particle distribution observed in the electron micrograms. The analysis of these functions permits obtaining required numerical characteristics. The Monte Carlo reconstructing model is described, and results of its work (on the basis of the above experimental data) are demonstrated in the form of "averaged" surface topography of the studied markers within the membrane fragments bordered. Possible connection of the obtained data with cooperative properties of the membrane is discussed.

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