Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in efficiency of therapeutic strategies against post-ischemic pathology.

The effect of neuraminidase blocker on gabazine-induced seizures in rat hippocampus.

Concentration of neuraminidase (NEU), an enzyme which cleaves negatively charged sialic acids from carbohydrate moieties of the cellular membrane, could vary depending on physiological conditions. Multiple evidences suggest that fluctuations of NEU extracellular concentrations can influence neuronal activity. In the present study we examined the effect of down regulation of endogenous NEU activity on seizure-like activity (SLA) induced by gabazine (specific blocker of inhibitory synaptic transmission) in the hippocampal CA1 pyramidal region of cultured slices. We show that in slices pretreated with the blocker of endogenous NEU, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), duration of synchronous oscillations induced by gabazine was considerably increased comparatively to control untreated slices. This study adds further information that changes in the level of NEU activity is an important factor, which can affect neuronal network excitability.

[Morphological plasticity of hippocampal CA1 neurons in vitro after short-term anoxia/hypoglycemia followed by reoxygenation].

Oxygen and glucose are known to modulate the neuronal plasticity. Their fluctuations have ability to induce cell damage, the degree of which is thought to depend on the intensity and duration of pathological events. Experimental investigations have shown that a short-term anoxia-hypoglycemia results in delayed cell destruction through multiple cellular and molecular mechanisms and these processes are accompanied by certain morphological alterations. The aim of the present study was to evaluate the neuronal, synaptic and glial plasticity in hippocampal CA I area after short-term (10 min) oxygen-glucose deprivation (OGD) followed by reoxygenation during 1 h. The experiments were carried out on hippocampal slice cultures. We estimated the following parameters: viability and condensation of CA1 neurons, the number and volume of the simple and complex asymmetric synapses, postsynaptic density (PSD) thickness, total vesicle number and the state of vesicle pools, the glial volume contacted with CA1 synapses. It is revealed that 10 min OGD has no influence on cell viability and condensation but the synapse number and volume have the tendency for reduction. Though, we found some changes in synaptic remodeling (an increase in PSD thickness, a decrease in the total vesicle number and the density of the active and reserve vesicle pools) and significant increase of the glial volume contacted with CA1 synapses. Thus, it was assumed that the early structure changes could be the reason of the functional abnormality and tissue damage. Our data confirm also the glial ability to modulate the neuronal function.

[Spatial distribution of synaptic vesicles in CA1 hippocampal synapses under conditions of induced long-term potentiation in vitro].

Prolonged activation of excitatory glutamatergic synapses causes modifications in their functioning and ultrastructural organization. While postsynaptic activity-induced changes have been relatively well studied, the data on spatial dynamics of synaptic vesicles (SV) under conditions of synaptic activation are still lacking. Using organotypic hippocampal slice cultures as a model system and electron microscopy as a technique, we analyzed changes in SV numbers and their spatial distribution in spine synapses ofhippocampal CA1 area. Two approaches were used to activate synapses: a protocol of brief oxygen-glucose deprivation known to induce so-called anoxia-hypoglycemic long-term potentiation (LTP), as well as high frequency stimulation of Schaffer collaterals inducing LTP of evoked postsynaptic potentials in CA1 synapses. Observations during the first hour after stimulation (30 and 60 min time-points) have shown that in both cases active functioning of synapses leaded to decrease in the total SV number as well as to depletion of the readily releasable SV pool. Both experimental protocols caused a decrease in spatial clustering of SV which was more pronounced after anoxia-hypoglycemic LTP. Possible mechanisms and functional consequences of these phenomena are discussed.

[Functional activity of hippocampal neurons after short-term oxygen-glucose deprivation in vitro].

The deficiency of oxygen and glucose induces neuronal cell damage and death through multiple cellular and molecular mechanisms. The degree of the damage is thought to depend on the intensity and duration of pathological event. In vivo and in vitro experimental investigations have shown that short-term oxygen-glucose deprivation (OGD) results in the delayed cell destruction. It allows studying of the neurodegenerative mechanisms induced by OGD for possible corrections. The aim of the present study was to evaluate the morpho-functional changes in cultured hippocampal neurons after short-term OGD followed by reoxygenation during 1, 4 or 24 h. It is revealed that 20 min OGD has no influence on the cell viability, but metabolic activity, synaptic quantity and activity are decreased at 1 h at 4 h after OGD. These functional characteristics are normalized at 24h. The results expand a concept about different aspects of hippocampal neurons injury under deficiency of oxygen and glucose. A suggested experimental model could be used for testing different neuroprotective tools.

Estimating transmitter release rates and quantal amplitudes in central synapses from postsynaptic current fluctuations.

Several approaches recently introduced to analyze release rates in central synapses advanced our understanding of synaptic neurotransmission, however, leaving many questions still unresolved. In this work we present evidence that a new method recently developed by Sakaba and Neher to study neurotransmission in calyx of Held, a giant glutamatergic synapse, could be also applied for estimating release rate functions and averaged quantal sizes in small central synapses. By means of different simulation approaches applied to reproduce GABAergic neurotransmission in the hippocampus we have shown that possible problems with a spatial voltage clamp which can occur in synaptic connections distributed over a large area of dendritic tree are not crucial for applicability of the method when synapses are compactly distributed or located proximally and when release rates are below 1 ms(-1). In another set of simulations we have also shown that at above mentioned release rates desensitization and/or saturation of postsynaptic GABAA receptors does not prevent accurate estimates of release rate and averaged quantal size. Thus, we conclude that the new approach based on analysis of fluctuations of postsynaptic currents under conditions of stationary release or moderately nonstationary conditions might be applicable to studies of small central synapses.

Morphological and functional changes in rat hippocampal slice cultures after short-term oxygen-glucose deprivation.

To study effects of short-term cerebral ischemia, hippocampal slice cultures were subjected to oxygen and glucose deprivation (OGD) followed by a period of normoxic reoxygenation. Propidium iodide staining, and MTT/formazan-assay were used to evaluate cell viability and metabolic activity. CA1 pyramidal cells were analyzed at the light- and electron microscopic levels. Cell damage was found to be insignificant during the first hour after 10 min OGD but profound following 4 h, showing delayed neuronal cell damage caused by short-term OGD. Our model can be used to characterize the mechanisms of cell damage caused by mild cerebral ischemia. These data might apply to further development of neuroprotective tools for the treatment of brain diseases.

[Effect of oxygen-glucose deprivation of different duration on rat hippocampal slice cultures]. / Vplyv kysnevo-hliukoznoï depryvatsiï riznoï tryvalosti na kul'tyvovani zrizy hipokampa shchuriv.

We studied morphofunctional changes in organotypic hippocampal slice cultures (OHSC) subjected to oxygen-glucose deprivation (OGD) for 10, 30, and 60 min followed by normoxic reoxygenation for 1, 4, and 24 h. Cell viability was estimated using trypan blue (TB) staining, lactate dehydrogenase (LDH) assay, and MTT/formazan assay. Structural changes in CA1 area of OHSC were analyzed by light and electron microscopy. No significant signs of destruction were found in the cultures 1 h following 10 min OGD; moreover, clear increase in cell metabolic activity was determined by MTT/formazan assay. Ultrastructural analysis of CA1 stratum radiatum revealed an increase in the number of glial processes and the number of perforated and multiple synapses as compared to the control, where simple synapses were relatively more numerous. 4 h following 10 min OGD, manifestations of cell damage in the cultures appeared and become profound at 24 h after OGD. We suppose that mild (10 min) OGD leads to plastic changes in neurons and delayed cell damage. 30 and 60 min OGD resulted in more early and pronounced cell damage as compared to 10 min OGD. To conclude, clear dependence of cell damage on the duration of deprivation as well as reoxygenation was observed. The model of ischemic damage in the slice cultures appears convenient to be used to analyze properties and mechanisms of neural cell response to OGD and to test neuroprotective tools.

[Lipoxygenase inhibitors induce rat hepatocyte apoptosis in primary culture]. / Blokatory lipoksyhenaz indukuiut' apoptoz hepatotsytiv shchuriv u pervynnii kul'turi.

Arachidonic acid metabolites have been shown to have a wide range of effects on cell proliferation and viability. In this study, the effects of lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) on the viability of cultured rat hepatocytes (HC) were investigated. As a result, treatment with NDGA and CA for 4 h and 24 h decreased ALT release from HC and increased a number of apoptotic cells. Apoptosis inducing effects of general LO inhibitor NDGA were more pronounced, than those of 5-LO inhibitor CA. The results suggest that lipoxygenase pathway of arachidonic acid metabolism, in particular 5-LO, is essential regulator of hepatocyte survival and apoptosis.

[Effect of exogenous leukotrienes and lipoxygenase inhibitors on apoptosis and necrosis in cultured rat hepatocytes]. / Vplyv ekzohennykh leikotryieniv i blokatoriv lipoksyhenaz na apoptoz ta nekroz hepatotsytiv shchuriv u pervynnii kyl'turi.

Liver cell death by apoptosis and necrosis occurs upon the liver injury. Lipoxygenase pathway of arachidonic acid metabolism is known to regulate the viability and apoptosis in some cell types, but its role in hepatocyte cell death is not fully understood. We studied the influence of leukotrienes (LT) and lipoxygenase inhibitors on apoptosis and necrosis in rat hepatocyte primary culture by double staining with Hoechst 33342 and propidium iodide and electron microscopy. Treatment with general lipoxygenase inhibitor nordihydoguaiaretic acid and 5-lipoxygenase inhibitor caffeic acid (2. 10(-5) M) for 4 and 24 h induced hepatocyte apoptosis. LTB4 and LTC4 (10(-8) M) decreased the number of living cells and increased the number of necrotic cells. LTs exerted the same necrotic effect on hepatocytes, treated with lipoxygenase inhibitors. It is important that LTs decreased apoptosis induced by inhibitors treatment. These data suggest that lipoxygenase pathway of arachidonic acid metabolism is important regulator of hepatocytes viability and apoptosis The increase of lipoxygenase product formation, in particular LTs, may diminish apoptosis and increase necrosis in hepatocytes upon the liver injury.

Effects of exogenous leukotrienes B4 and C4 on the viability of cultured rat hepatocytes.

Leukotrienes (LTs) are thought to be extensively involved in a liver damage in vivo through different mechanisms. In this study we used different doses (10(-7)-10(-12) M) of the dehydroxilated LTB4 and the cysteinyl LTC4 to estimate their direct injurious effects on cultured rat hepatocytes (HC). Our experiments demonstrated that exogenous LTB4 and LTC4 caused a rapid and transient increase in alanine aminotransferase release from HC and a slight, but significant decrease of mitochondrial electron transport chain activity in HC. Significant increases in ALT release were observed with LTs doses as low as 10(-12) M, but the loss of mitochondrial function was significant only at the two higher doses (10(-7) and 10(-8) M). HC were treated with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to inhibit the possible synthesis of endogenous LTs. The effects of exogenous LTB4 and LTC4 on NDGA-treated HC tended to be similar to those indicated in the absence of inhibitor, but were more pronounced. These data suggest that LTs may be involved in the direct damage of liver cells under pathological conditions associated with enhanced LTs formation.

Effects of nordihydroguaiaretic acid and indomethacin on the viability and functional activities of normal and carbon tetrachioride-injured rat hepatocytes cultured alone and with Kupffer cells.

In order to study the contribution of eicosanoids to the regulation of the functions of normal and carbon tetrachloride (CCl4)-injured liver cells, primary cultures of hepatocytes (HC) either alone or in coculture with Kupffer cells (KC) were exposed for 4 and 24 h to lipoxygenase inhibitor (nordihydroguaiaretic acid-NDGA) or cyclooxygenase inhibitor (indomethacin-IND) in the presence and in the absence of CCl4. Treatment with CCl4 resulted in increased ALT release and a decreased mitochondrial respiration (MR) in HC and their cocultures with KC. The addition of NDGA decreased ALT levels and increased MR in control and CCL4-injured cells. Urea production (UP) was not significantly affected by NDGA. In contrast, addition of IND) decreased UP by HC (4 h), and did not alter ALT release and MR in control and CCl4-treated cells. These results indicate that arachidonic acid metabolites are involved in the regulation of HC flinctions. There is also evidence that a protective action of lipoxygenase inhibitors on CCl4-injured liver is mediated, at least partly, by their direct effects on HC and KC, in particular by increasing the mitochondrial respiration.

[The action of specific antimembrane antibodies on the tonic tension and contractile phases of the heart muscle in rats]. / Diia spetsyfichnykh antymembrannykh antytil na tonichnu napruhu ta fazni skorochennia sertsevoho m'iaza shchura.

Antibodies specific to rat myocardial sarcolemma inhibit phasic contractions and promote an increase in tonic tension of isolated strips of the cardiac muscle in rat. These results indicate that antibodies induce accumulation of intracellular free Ca2+. Analysis of the authors' data and those from literature suggests that specific antibodies inhibit Na(+)-K(+)-pump activity of the cardiac muscle in rat. This conclusion is confirmed by analogy of effects of ouabaine, the well-known inhibitor of Na(+)-K(+)-pump, on the rat cardiac muscle.