Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

NMDA-receptors are involved in Cu<Superscript>2+</Superscript>/paraquat-induced death of cultured cerebellar granule neurons

Rat cultured cerebellar granule neurons (CGNs) were not sensitive to CuCl₂ (1-10 µM, 24 h), whereas paraquat (150 µM) decreased neuronal survival to 79 ± 3% of control level. Simultaneous treatment of CGNs with paraquat and CuCl2 (2, 5, or 10 µM Cu²⁺/paraquat) caused significant copper dose-dependent death, lowering their survival to 56 ± 4, 37 ± 3, or 16 ± 2%, respectively, and stimulating elevated production of free radicals in CGNs. Introduction of vitamin E, a non-competitive antagonist of NMDA subtype of glutamate receptors (MK-801), and also removal of glutamine from the incubation medium decreased toxicity of Cu²⁺/paraquat mixture. However, addition of Cu²⁺ into the incubation medium did not affect CGNs death caused by glutamate. These data emphasize that excessive copper in the brain may trigger oxidative stress, which in turn results in release of glutamate, overstimulation of glutamate receptors, and neuronal death.     

Changes in some enzymic activities of separated neuronal and glial cell-enriched fractions from rat brains during development

Bulk-prepared neuronal perikarya and glial fractions have been used to study developmental changes of some enzymic activities concerning glycolysis and synthesis of neurotransmitters. Somewhat higher pyruvate kinase activity was found in neuronal perikarya than in glial cells, and its rapid rise was observed during early developmental stages. Increased K⁺ concentration ord-glutamate addition to the incubation medium enhanced consumption of phosphoenolpyruvate. This activation of the enzyme was small just after birth, but it increased in parallel with development to adult level, where the activation in glial fractions was over twice that in neuronal fractions. Choline acetyltransferase activity was found in purified neuronal fractions and increased with age; glutamate decarboxylase was also found in high activity in purified neuronal fractions and increased with development. However, some enzymic activities were also found in glial fractions, and possibilities of contamination by synaptosomal, myelin, or other subfractions are discussed.     

Effects of amino acids on calcium uptake by glial and neuroblastoma cells

The uptake of [⁴⁵Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M₁ compared to glial clones. In all cases [⁴⁵Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 μM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [⁴⁵Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [⁴⁵Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Borna disease virus infection impairs synaptic plasticity

The mechanisms whereby Borna disease virus (BDV) can impair neuronal function and lead to neurobehavioral disease are not well understood. To analyze the electrophysiological properties of neurons infected with BDV, we used cultures of neurons grown on multielectrode arrays, allowing a real-time monitoring of the electrical activity across the network shaped by synaptic transmission. Although infection did not affect spontaneous neuronal activity, it selectively blocked activity-dependent enhancement of neuronal network activity, one form of synaptic plasticity thought to be important for learning and memory. These findings highlight the original mechanism of the neuronal dysfunction caused by noncytolytic infection with BDV.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Release of endogenous dopamine from tuberoinfundibular neurons

Release of endogenous dopamine (DA) from arcuate-periventricular nucleus-median eminence fragments has been analyzed in an $\text{in vitro}$ static incubation system.Exposure of these hypothalamic fragments to increasing concentrations of K⁺ ions produced a dose-dependent release of endogenous DA. The highest rate of K⁺-stimulated DA efflux occurred in the first 10 minutes, thereafter it progressively decline reaching prestimulated levels at 30 minutes. If two consecutive depolarizing stimuli of 40 mM KCl were applied to the same hypothalamic fragment, after a 40 minutes rest period, an equivalent release of endogenous DA occurred. Removal of Ca⁺⁺ ions from the incubation medium containing the Ca⁺⁺ chelator EGTA caused a decrease of basal DA efflux and completely prevented the K⁺-induced release of DA.Furthermore when verapamil, a blocker of Ca⁺⁺ entrance, was added to the incubation medium in a concentration of 50 μM, the K⁺-induced DA efflux was completely counteracted, whereas spontaneous release was unmodified.Finally nomifensine, a potent blocker of DA uptake, added $\text{in vitro}$ in a final concentration of 10 μM, significantly reinforced K⁺-induced release of endogenous DA. Since nomifensine did not modify basal DA release, this study confirmed its prevalent uptake blocking property rather than its releasing action on DA.     

Low-frequency stimulation induces stable transitions in stereotypical activity in cortical networks

Reverberating spontaneous synchronized brain activity is believed to play an important role in neural information processing. Whether and how external stimuli can influence this spontaneous activity is poorly understood. Because periodic synchronized network activity is also prominent in in vitro neuronal cultures, we used cortical cultures grown on multielectrode arrays to examine how spontaneous activity is affected by external stimuli. Spontaneous network activity before and after low-frequency electrical stimulation was quantified in several ways. Our results show that the initially stable pattern of stereotypical spontaneous activity was transformed into another activity pattern that remained stable for at least 1 h. The transformations consisted of changes in single site and culture-wide network activity as well as in the spatiotemporal dynamics of network bursting. We show for the first time that low-frequency electrical stimulation can induce long-lasting alterations in spontaneous activity of cortical neuronal networks. We discuss whether the observed transformations in network activity could represent a switch in attractor state.     

Low dose isoflurane exerts opposing effects on neuronal network excitability in neocortex and hippocampus

The anesthetic excitement phase occurring during induction of anesthesia with volatile anesthetics is a well-known phenomenon in clinical practice. However, the physiological mechanisms underlying anesthetic-induced excitation are still unclear. Here we provide evidence from in vitro experiments performed on rat brain slices that the general anesthetic isoflurane at a concentration of about 0.1 mM can enhance neuronal network excitability in the hippocampus, while simultaneously reducing it in the neocortex. In contrast, isoflurane tissue concentrations above 0.3 mM expectedly caused a pronounced reduction in both brain regions. Neuronal network excitability was assessed by combining simultaneous multisite stimulation via a multielectrode array with recording intrinsic optical signals as a measure of neuronal population activity.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.     

Effects of dopamine on spontaneous activity generated by isolated spinal cord in 16- to 20-day-old chick embryos

The effects were investigated of applying L-DOPA, dopamine (DA), and noradrenaline (NA) on spontaneous activity (cyclic fluctuations in electrotonic dorsal and ventral root (DR and VR) potentials generated by a section of spinal cord isolated from 16 to 20-day-old chick embryos. A low concentration of L-DOPA (30–150 µm) intensified operation of the spinal generator, giving rise to above-threshold rhythm (i.e., spike activity in the DR and the VR). At a high concentration, L-DOPA produced inhibition of generator operation, although spontaneous activity did intensify during subsequent washout of the substance, with the onset of above-threshold rhythm. Both DA and NA failed to affect spontaneous activity in the VR and the DR at a concentration to 50 µM but a concentration of 100 µM produced inhibition. Application of 20 µM 2-amino-5-phosphonovaleric acid blocked the reinforced spontaneous activity produced by low L-DOPA concentrations. Activity generated by the neuronal network of the isolated dorsal horn rose under the effects of low L-DOPA concentrations; rhythmic activity was observed neither before nor after applying this substance in isolated ventral horn. Findings obtained would point to the occurrence of a direct (i.e., non-catecholamine dependent) excitatory influence of L-DOPA on the neuronal network of the chick embryo dorsal horn.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 338–343, May–June, 1991.     

Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down's syndrome

It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50-100 microM glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8-110.8%; Ts16, 83.1-96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate-induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria-mediated, cyclosporin A-sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.     

Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments

Cd accumulation, its effects on elongation growth of maize coleoptile segments, pH changes of their incubation medium and the membrane potential of parenchymal cells were studied. The Cd content increased significantly with exposure to increasing cadmium concentrations. Coleoptile segments accumulated the metal more efficiently in the range 10–100 μM Cd, than in the range 100–1000 μM Cd. Cd at concentrations higher than 1.0 μM produced a significant inhibition of both growth and proton extrusion. 100 μM Cd caused depolarization of the plasma membrane (PM) potential in parenchymal cells. The simultaneous treatment of maize coleoptile segments by indole-3-acetic acid (IAA) and Cd, counteracted the toxic effect of Cd on growth. Moreover, our data also showed that 100 μM Cd suppressed the characteristic IAA-induced hyperpolarization of the membrane potential, causing membrane depolarization. These results indicate that the toxic effect of Cd on growth of maize coleoptile segments might be, at least in part, caused via reduced PM H⁺-ATPase activity.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Factors affecting adventitious regeneration from in vitro leaf explants of 'Improved French' plum, the most important dried plum cultivar in the USA

An adventitious shoot regeneration protocol from in vitro leaves of the most important dried plum cultivar in the USA, ‘Improved French’, has been established. Factors affecting regeneration were studied in order to optimise regeneration. The proliferation medium in which the shoots, used as the source of leaf explants, were cultured had a strong influence on subsequent regeneration. Shoot regeneration was observed at a mean frequency of 52% when a Murashige-based and Skoog-based shoot culture medium with 3 μM N⁶-benzylaminopurine and 0.25 μM indole-3-butyric acid (IBA) was employed compared with shoot regeneration frequencies of less than 5% for a Quoirin-based and Lepoivre-based shoot culture medium, with 8.9 μM N⁶-benzylaminopurine and 0.49 μM IBA. The shoot regeneration medium contained α-naphthaleneacetic acid at 2.0–6.0 μM and thidiazuron at 4.5–15.0 μM. 2,4 Dichlorophenoxy-acetic acid at 9.0 μM was included in the medium but only for the first 4 days of culture. Shoot regeneration frequencies were positively related to thidiazuron concentration and significantly greater (P < 0.05) for 9–15 μM thidiazuron than for the media with 4.5 μM thidiazuron. Leaf explants, incubated in a 16-h-light/8-h-dark photoperiod or in the dark for 1 week followed by exposure to light, showed significantly more organogenic activity (P < 0.01) than was observed for leaves cultured in the dark for 2 or 3 weeks before they were transferred to the light. The utilisation of Bacto agar (0.7%) as the gelling agent increased organogenesis compared with media gelled with TC Agar (0.7%), or an agar–gellan gum blend (Agargel™) (0.45%). The addition of the ethylene inhibitor silver thiosulphate at 60–120 μM also improved organogenesis. When all the studied factors were optimised, a regeneration rate of 65% was achieved. Rooting frequency of regenerated shoots was significantly increased (P < 0.05) by the use of full-strength Murashige and Skoog salts (40%) or 100 mg L⁻¹ phloroglucinol (53%) to the rooting medium.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Levetiracetam protects hippocampal neurons in culture against hypoxia-induced injury

Many experimental studies indicate that some antiepileptic drugs possess neuroprotective properties in varied models of neuronal injury. Levetiracetam is a second-generation antiepileptic drug with a novel mechanism of action. In the present study, we evaluated the putative neuroprotective effect of levetiracetam on primary hippocampal cultures at seven day in vitro. Cell death was induced by incubation of neural cultures in hypoxic conditions over 24 hours. Neuronal injury was assessed by morphometric investigation of death/total ratio of neurons in light microscopy using Trypan blue staining and by evaluation of lactate dehydrogenase (LDH) release in the culture medium. Our results indicate that pre-conditioning of hippocampal cultures with high concentrations of levetiracetam (100 μM and 300 μM) protects neurons against hypoxia-induced death. Two-fold higher number of neurons remained viable as compared to control cultures without drug. Lack of neuroprotective action of the drug on hippocampal neural cultures was observed, when a low concentration (10 μM) of levetiracetam was used.     

Expression of alternatively spliced variants of Na–Ca-exchanger-1 in experimental colitis: role in reduced colonic contractility

In an earlier article, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) inhibits electron transport in the respiratory chain and uncouples oxidative phosphorylation, and postulated that these effects are probably involved in its antitumor activity. We now report the effect of SYD-1 on certain macrophage functions, considering the important role of these cells in inflammatory response and also the relevant anti-inflammatory activity reported for some sydnones. Incubation of macrophages with SYD-1 (5-100 μM) for 48 h did not affect the cell viability up to a concentration of 50 μM. However, at the highest concentration (100 μM), the compound decreased macrophage viability by ~20%. In assays involving 2 h and 24 h of incubation, SYD-1 (5-100 μM) did not affect the cell viability. The incubation of macrophages with the compound for 2 h promoted a dose-dependent reduction of phagocytic activity of up to ~65% (100 μM). SYD-1 (100 μM) was also able to increase the production of superoxide anion (~50%). In the absence of LPS, SYD-1 decreased NO production dose-dependently by up to ~80% (100 μM). When SYD-1 and LPS were incubated concomitantly, the decrease of NO promoted by SYD was the most pronounced, reaching up to ~98% at the same concentration (50 μM). SYD-1 dose-dependently suppressed IL-6 secretion by LPS-stimulated macrophages, reaching up to ~90% of inhibition at the highest concentration (100 μM). These results indicate that SYD-1 promotes effects similar to those described for anti-inflammatory and immunosuppressive drugs, thus motivating further studies to clarify the mechanisms involved in this activity.     

Excitotoxicity triggered by Neurobasal culture medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca²⁺ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.