Combined effects of ethanol and manganese on cultured neurons and glia

Manganese is essential for normal development and activity of the nervous tissue. Mn²⁺ ions are involved in protein synthesis and may prevent free radical damage. Since it is now established that alcohol degradation may produce free radicals, we studied the effect of Mn²⁺ on ethanol induced alterations using cultured nerve cells as an experimental model of the central nervous system. Neurons and glial cells were cultured from rat brain cortex; a tumoral rat glial cell line (C6) was also examined. We measured enzymatic markers of nerve cell maturation (enolase, glutamine synthetase) and superoxide dismutase, a scavenger of free radicals; all these enzymes being activated by Mn²⁺ ions. Only for the glial cell types an alcohol antagonizing effect was found when Mn²⁺ was combined with ethanol. Neurons were not sensitive to that Mn²⁺ effect.     

Proliferation and cell death of hepatocytes in regenerating rat fetal liver

Proliferation and death of hepatocytes in regenerating liver of 17-day white rat fetuses were investigated. During 2 days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply in 15 h after liver resection. In all experimental and control groups, the ratio of the metaphase, the longest phase of mitosis, and index to mitotic index remained unchanged, indicating identical duration of hepatocytes mitoses in regenerating liver. In the regenerating and intact liver hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% rat fetal liver did not contribute to increased apoptosis of hepatocytes.     

Proliferation and cell death of hepatocytes in regenerating fetal rat liver

Proliferation and death of hepatocytes in regenerating liver were studied in 17-day-old fetal white rats. Two days after liver resection (20%), animals were sacrificed every 3 h. In experimental groups, the index of Ki67-positive hepatocytes increased sharply 15 h after liver resection. In all experimental and control groups, the ratio of the index of the metaphase, the longest phase of mitosis, to the mitotic index remained unchanged, indicating the same duration of hepatocyte mitoses in regenerating liver. In regenerating and intact liver, hepatocytes labeled with antibodies to caspase 3 were not detected. Thus, resection of 20% fetal rat liver did not promote enhancement of apoptosis of hepatocytes.     

Differential effects of ethanol on the expression of cyclo-oxygenase in cultured cortical astrocytes and neurons

The developing central nervous system is a primary target of ethanol toxicity. The teratogenic effect of ethanol may result from its action on prostaglandins. Prostaglandins are generated through the release of arachidonic acid (AA) by the action of cytosolic phospholipase A(2) (cPLA(2)) on membrane-bound phospholipids and the catalytic conversion of AA to prostaglandin E(2) (PGE(2)) by cyclo-oxygenase (COX). COX is expressed in two isoforms, constitutive COX1 and inducible COX2. Cultured astrocytes and neurons from immature cerebral cortex were used as in vitro models to investigate the effect of ethanol on PGE(2) synthesis. In both cell types, neither the activity nor the expression of cPLA(2) was affected by ethanol. PGE(2) was synthesized by astrocytes and neurons. Ethanol (200-400 mg/dL for 24 h) significantly increased PGE(2) production in both cell types and the ethanol-induced increase in PGE(2) accumulation in astrocytes was significantly greater than in neurons. These increases resulted from the effects of ethanol on COX. Overall COX activity was up-regulated by ethanol in astrocytes and neurons, and indomethacin, a nonselective blocker for COX, eliminated the ethanol-induced increases of COX activity in both cell types. Increased COX activity in astrocytes resulted from an increase in COX2 expression. NS-398, a selective COX2 blocker, completely inhibited ethanol-induced alterations in COX activity. In neurons, however, ethanol had a direct effect on COX activity in the absence of a change in COX expression. NS-398 only partially blocked ethanol-induced increases in neuronal COX activity. Thus, astrocytes are a primary target of ethanol and ethanol-induced increases in glial PGE(2) synthesis are mediated by COX, principally COX2. Ethanol toxicity may be mediated through PGE(2) in immature cortical cells.     

Inhibition of apoptosis-inducing factor translocation is involved in protective effects of hepatocyte growth factor against excitotoxic cell death in cultured hippocampal neurons

Although hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain, their effects and mechanism of action under pathological conditions remain to be determined. Over-activation of the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor, has been implicated in a variety of neurological and neurodegenerative disorders. We investigated the effects of HGF on the NMDA-induced cell death in cultured hippocampal neurons and sought to explore their mechanisms. NMDA-induced cell death and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were prevented by HGF treatment. Although neither the total amounts nor the mitochondrial localization of Bax, Bcl-2 and Bcl-xL were affected, caspase 3 activity was increased after NMDA exposure. Treatment with HGF partially prevented this NMDA-induced activation of caspase 3. Although the amount of apoptosis-inducing factor (AIF) was not altered, translocation of AIF into the nucleus was detected after NMDA exposure. This NMDA-induced AIF translocation was reduced by treatment with HGF. In addition, increased poly(ADP-ribose) polymer formation after NMDA exposure was attenuated by treatment with HGF. These results suggest that the protective effects of HGF against NMDA-induced neurotoxicity are mediated via the partial prevention of caspase 3 activity and the inhibition of AIF translocation to the nucleus.     

Onset dynamics of action potentials in rat neocortical neurons and identified snail neurons: quantification of the difference

The generation of action potentials (APs) is a key process in the operation of nerve cells and the communication between neurons. Action potentials in mammalian central neurons are characterized by an exceptionally fast onset dynamics, which differs from the typically slow and gradual onset dynamics seen in identified snail neurons. Here we describe a novel method of analysis which provides a quantitative measure of the onset dynamics of action potentials. This method captures the difference between the fast, step-like onset of APs in rat neocortical neurons and the gradual, exponential-like AP onset in identified snail neurons. The quantitative measure of the AP onset dynamics, provided by the method, allows us to perform quantitative analyses of factors influencing the dynamics.     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Capsaicin-induced death of cultured neurons of the rat hippocampus

We studied the effects of an agonist of vanilloid receptors (VRs), capsaicin, and of an antagonist of VRs of type 1, capsazepin, on cultured neurons of the rat hippocampus. In cultures incubated for 1 day in a medium containing 10 μM capsaicin, the numbers of cytologically normal cells and those with manifestations of necrosis and apoptosis were, on average, 46.4 ± 3.3, 30.7 ± 2.4, and 22.9 ± 5.4%. The latter two values were more than three times greater than the respective indices under control conditions (P < 0.05). Coincubation of the cells with 10 μM capsaicin and 25 μM capsazepin decreased the normalized number of apoptotic units by about one-third, while the number of cells with necrotic changes showed nearly no changes. Using confocal microscopy and staining the cells with a fluorescent dye, JC-1, we found that incubation with capsaicin resulted in a dramatic drop in the mitochondrial potential in the great majority of cultured cells, while capsazepin somewhat smoothed this effect. Thus, our data show that the cytotoxic effect of capsaicin is related to changes in the mitochondrial potential and is at least partially mediated by activation of type-1 VRs. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 13–20, January–February, 2008.     

Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons

The actions of cholecystokinin octapeptide (CCK) on the membrane properties of mouse spinal neurons grown in monolayer culture were examined using intracellular recording techniques. In a subpopulation of cells, application of CCK (0.2-100 micron) by pressure ejection from micropipettes produced a small (approximately 2 mV) membrane depolarization that was accompanied by a decrease in membrane conductance (approximately 11 percent). These effects were associated with an enhanced tendency of the cells to generate action potentials when stimulated with intracellular depolarizing current. The unsulfated analog of CCK, which possesses weak biological activity in the gut, had little or no effect on cultured spinal neurons. A number of differences were noted between the responses to CCK and the excitatory amino acid glutamate. First, the effects of CCK were more delayed in onset (approximately 17 sec) and prolonged in duration (approximately 124 sec). Second, the depolarizations produced by glutamate were of larger magnitude and associated with variable effects on membrane conductance. Third, the response to CCK showed tachyphylaxis with repeated applications whereas glutamate remained effective as often as it was applied. It is concluded that CCK facilitates the excitability of spinal neurons in a manner distinct from that of the conventional excitant glutamate.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Frequency-dependent and cell-specific effects of electrical activity on growth cone movements of cultured Helisoma neurons

The present experiments address the question of how stimulation parameters, which evoke action potentials in neuronal cell bodies, influence growth cone movements of different identified neurons. The motility of growth cones of Helisoma buccal neurons B19 and B4 was monitored while somata were stimulated simultaneously via an intracellular microelectrode. The findings show that the responses of growth cones of B19 and B4 contain components that are common as well as unique to each neuron. Whereas rates of growth cone advance were suppressed in a graded fashion by stimulus frequencies beyond a threshold of 2 s-1 for both neurons, B4 was more sensitive to electrical stimulation and exhibited a new response, namely, growth rates were enhanced during the poststimulation recovery period after stimulation at specific frequencies. Thus, electrical activity can result in enhancement as well as in inhibition of growth cone movement. Changes in number of filopodia on B19 and B4 were graded also, with B4 again displaying greater sensitivity. The frequency dependence of filopodia compared to growth rate changes was different and suggests a possible dissociation between filopodial activity and growth cone motility. Patterned electrical activity produced effects similar to constant stimulation for B19 growth cones, whereas it decreased the threshold frequency and eliminated the growth enhancement effect for B4. Taken together, these data demonstrate that the quantitative features of electrical activity as well as intrinsic properties of neurons both determine the growth cone response to changes in neuronal activity.     

Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death

The role of the enteric nervous system in intestinal inflammation is not fully understood and the plethora of cellular activities concurrently ongoing in vivo renders intelligible studies difficult. In order to explore possible effects of bacterial lipopolysaccharide (LPS) on enteric neurons we utilised cultured myenteric neurons from rat small intestine. Exposure to LPS caused markedly reduced neuronal survival and increased neuronal expression of vasoactive intestinal peptide (VIP), while the expression of Toll-like receptor 4 (TLR4) was unchanged. TLR4 was expressed in approximately 35% of all myenteric neurons irrespective of if they were cultured in the presence or absence of LPS. In neurons cultured in medium, without LPS, 50% of all TLR4-immunoreactive neurons contained also VIP. Addition of LPS to the neuronal cultures markedly increased the proportion of TLR4-immunoreactive neurons also expressing VIP, while the proportion of TLR4 neurons devoid of VIP decreased. Simultaneous addition of LPS and VIP to the neuronal cultures resulted in a neuronal survival comparable to controls. CONCLUSIONS: LPS recognition by myenteric neurons is mediated via TLR4 and causes neuronal cell death. Presence of VIP rescues the neurons from LPS-induced neurodegeneration.     

Effect of ethanol on cell growth and cholesterol metabolism in cultured Hep G2 cells.

The Hep G2 human hepatoma cell line has been recognized as an excellent in vitro human model system. For this reason, this line was used to study the effect of ethanol on HMG-CoA reductase activity concerning cell growth and cholesterol metabolism. Cells were incubated in ethanol-containing medium (0-400 mmol/L) for up to 102 h. Ethanol caused an inhibition in the growth rate and in HMG-CoA reductase activity that could be reverted by the removal of ethanol from the culture medium, indicating no cellular damage. These changes cannot be ascribed to the regulatory effect of cholesterol levels, since its content was not modified either in the cells or in the medium. The addition of mevalonate to the culture medium could not revert the growth rate inhibition evoked by ethanol. Moreover, ethanol produced an increment in the cholesterol efflux in [3H]cholesterol-prelabeled cells. We conclude that the decrease in HMG-CoA reductase activity evoked by ethanol treatment on Hep G2 cells would not be the cause but the consequence of the impairment in cellular growth, since this impairment could not be reverted by the addition of mevalonate to the culture medium.     

Guanine nucleotides inhibit NMDA and kainate-induced neurotoxicity in cultured rat hippocampal and neocortical neurons

Previous evidence suggests that guanine nucleotides can directly inhibit N-methyl-d-aspartate (NMDA) and AMPA/kainate receptors and antagonize a variety of cellular functions elicited by these glutamate receptor agonists. We investigated the possibility that the guanine nucleotides GTP, GDP, and GMP exert a neuroprotective effect on cultured rat hippocampal or neocortical neurons exposed to the excitotoxicants NMDA (30 microM) or kainate (300 microM). On co-application with NMDA all three nucleotides revealed a comparable rescue effect from 100 microM nucleotide concentrations onwards, with a higher inhibitory potential in hippocampal than in neocortical cultures. Similarly, kainate-induced neurotoxicity was inhibited by all three nucleotides but the inhibitory potential was lower than after application of NMDA. Guanosine had no effect on either culture system. GTP and GDP where hydrolyzed by hippocampal and cortical cultures with GMP accumulating in the medium, suggesting that hydrolysis of GTP had no effect on the effective nucleotide concentration. Our results show that GTP, GDP, and GMP inhibit NMDA- and kainate-mediated neurotoxicity in cultured hippocampal and neocortical neurons. They suggest that guanine nucleotides may be candidates for broadly antagonizing glutamate receptor-mediated neurotoxicity.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Influence of ethanol on fatty acid composition of phospholipids in cultured neurons

Animals chronically exposed to ethanol show changes in neural membrane lipids which may underlie the development of tolerance and physical dependence. The object of this study was to investigate changes in the fatty acid composition of neuronal phospholipids cultured in the presence of ethanol (55 or 110 mM) for periods up to 7 days. Decreases were observed in the percentage of individual and total saturated fatty acids, while the double bond index: total saturated fatty acid ratio, increased. These changes do not support the hypothesis that neural membrane lipid composition changes to counteract the fluidizing action of ethanol.     

Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.