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Background Tetrabromobisphenol A (TBBPA) is a toxic brominated flame retardant. Previous studies have demonstrated that exposure of primary cultures of rat cerebellar granule cells (CGC) to ≥ 10 μM TBBPA induces toxicity and excitotoxicity, and the underlying mechanism may involve calcium imbalance and oxidative stress. Here we examined whether the application of TBBPA at subtoxic concentrations may exacerbate acute damage of CGC challenged with oxygen-glucose deprivation (OGD), and evaluated with fluorescent indicators the involvement of calcium imbalance, mitochondrial depolarization and oxidative stress. Methods Survival of CGC was assessed 24 h after OGD/TBBPA using fluorescent dyes. An OGD challenge lasting for 45, 60 or 75 min induced a duration-dependent injury to the neurons. Results Application of 2.5, 5 or 7.5 μM TBBPA for 45 min to normoxic and glucose-containing incubation medium did not reduce the viability of cultured CGC, but this compound exacerbated the toxic effects of OGD in a concentration-dependent way. Moreover, TBBPA had a slight effect on calcium homeostasis and mitochondrial membrane potential, but significantly activated the production of reactive oxygen species in CGC. The application of H 2 O 2 at 5, 10 and 25 μM mimicked the effects of TBBPA on OGD toxicity, while 0.1 mM ascorbic acid or 1 mM glutathione ameliorated this toxicity. Conclusion These results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD.

The aim of this study was to establish the antagonistic effects of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) on NMDA receptors and its neuroprotective abilities on primary cultures of rat cerebellar granule cells exposed for 30 min to 250 or 100 μM glutamate. Neuronal viability was tested after 24 h with propidium iodide or calcein/ethidium homodimer-1 staining. The neuroprotective potential of 100, 250 or 500 μM 1MeTIQ was compared with established uncompetitive NMDA receptor antagonists, 0.5 Μm MK-801, or 5 μMmemantine. These substances were applied for 30 min either together with glutamate, 24 or 48 h before glutamate, or 0.5 h, 1 h and 3 h after exposure to the excitotoxin. The results demonstrated that MK-801, memantine and 500 μM 1MeTIQ induced an almost complete neuroprotection when co-applied with glutamate, but lower concentrations of 1MeTIQ were slightly less effective. Similar effects for 1MeTIQ and the established NMDA receptor antagonists were observed in the pretreatment experiments, even with a 48-h lag between the application of the tested substances and the excitotoxic challenge. In the post-treatment experiments, MK-801 and memantine and 500 μM 1MeTIQ applied up to 3 h after the exposure to glutamate significantly reduced the excitotoxic lesion, but 1MeTIQ in lower concentrations was ineffective. These results indicate that 1MeTIQ shares neuroprotective abilities with established uncompetitive NMDA receptor antagonists, which suggests that its inhibitory effect on NMDA receptors plays a key role in its anti-excitotoxic activity. Moreover, our data disclose a new mechanism of 1MeTIQ-evoked neuroprotection based on the induction of neuronal tolerance to excitotoxicity.

Previous studies have demonstrated that repeated submission of rats to mild hypobaric hypoxia reduces the persistent behavioral and hormonal depressive symptoms induced by exposure to footshock in the learned helplessness paradigm. The aim of this study was to determine whether hypoxic preconditioning of mice can also induce antidepressant- and anxiolytic-like effects that are detectable with the other commonly used behavioral tests, and to determine whether these effects are accompanied by an increase in neuropeptide Y (NPY) in the hippocampus, which may suggest the involvement of NPY in these mechanisms. The intermittent mild hypobaric hypoxia was generated by 2-h exposure of mice to 0.47 atm for 3 consecutive days. In the tail suspension test a significant decrease in the duration of immobility was observed 24 h, but not 48 h after the last hypobaric session. The elevated plus maze trials performed 48 h after preconditioning showed a significant increase in the frequency of open arm entries, a reduction in the duration of closed arm occupancy and substantially more time spent in the open arms in comparison to the control groups. The open field test demonstrated the absence of increases in general activity or unspecific exploratory behavior in hypoxia-preconditioned mice. The EIA test detected a statistically significant but relatively weak increase in the NPY content in the hippocampus 24 h after preconditioning. Together, our data demonstrate that preconditioning of mice with intermittent mild hypobaric hypoxia induces anxiolytic- and antidepressant-like effects. They are accompanied by up-regulation of NPY which may suggest its mechanistic role.

In the present study, we examined the effects of negative and positive allosteric modulators of metabotropic glutamate receptor 5 (mGluR5), fenobam and ADX47273, respectively, on brain damage induced by hypoxia-ischemia (H-I) in 7-day-old rats. The test drugs were administered intraperitoneally 10 min after H-I. Rectal body temperature was measured for 2.5 h after the insult. The number of apoptotic neurons in the immature rat brain was evaluated after 24 h. The wet weight of both hemispheres was determined 14 days after H-I, and its loss was used as an indicator of brain damage. In the vehicle-treated groups, H-I reduced the weight of the ipsilateral (ischemic) hemisphere by approximately 33% and sixfold increased the number of apoptotic cells in the cortex. Fenobam (10 mg/kg) and ADX47273 (5, 10, and 30 mg/kg) had no significant effect on brain damage, although application of fenobam at this dose significantly reduced the number of apoptotic cells. In contrast, fenobam (20 mg/kg) potentiated ischemic brain damage to 57.4% and had no effect on H-I-induced apoptosis. In all of the experimental groups, we detected no significant changes in the weight of the contralateral (control) hemisphere or the rectal temperature. In conclusion, in 7-day-old rats, the bidirectional modulation of mGluR5 by fenobam (10 mg/kg) and ADX47273 (all doses tested) did not result in significant changes in H-I-evoked brain damage, supporting our previous data indicating that also the antagonists of mGluR5 MPEP and MTEP, which reduce neuronal lesions in adult animals submitted to brain ischemia, were ineffective in 7-day-old rat pups.

Calretinin (CR) is an EF-hand calcium binding protein expressed in a tissue-specific manner. CR gene is active in some neurons, such as cerebellar granule neurons and in some tumor cells such as colon adenocarcinoma or epithelial mesothelioma. Our aim was to understand the mechanism(s) involved in tissue-specific regulation of CR gene transcription. Our recent paper showed that an “AP2-like” sequence located between −90/−80 bp of the CR gene promoter is important for specific expression of this gene in neurons differentiating from DEV multipotent cells [Biochim. Biophys. Acta (1577) 2000 412]. We now show by electrophoretic mobility shift assay (EMSA) that in cerebellar granule neurons, there is a nuclear protein that interacts with the “AP2-like” sequence. This interaction results in an increased CR gene promoter activity. In contrast, the “AP2-like” sequence does not play such a role in regulation of the CR gene transcription in adenocarcinoma and mesothelioma cancer cells. In these cells mutation in the “AP2-like” element did not affect transcriptional activity of the CR gene promoter and no protein binding to this element was observed by EMSA. These data clearly show that expression of CR gene in neurons and cancer cells is regulated by different mechanisms. On the other hand, in both cell types, the activity of CR gene promoter can be stimulated by zinc ions.

Dantrolene is an inhibitor of a skeletal muscle subtype of ryanodine receptors that stabilizes intracellular calcium concentrations and exerts neuroprotective effects in neurons submitted to excitotoxic challenges. The mechanisms of dantrolene-induced neuroprotection are not clear. In this study, using a model of cultured rat cerebellar granule neurons, we demonstrated that dantrolene inhibits NMDA-evoked 45 Ca uptake, indicating that this drug may inhibit the activity of NMDA receptor channels. Primary neuronal cultures were incubated for 10 min in Mg2+-free ionic medium with NMDA and 45 Ca in the presence of different concentrations of dantrolene, then radioactivity in neurons was measured by liquid scintillation spectroscopy. The results demonstrated that dantrolene, applied at micromolar concentrations, inhibits NMDA-evoked 45 Ca uptake in neurons in a dose-dependent manner. DMSO, a vehicle to dantrolene, in concentrations used in this study had no effect on NMDA-evoked 45 Ca uptake. These results, indicating that dantrolene inhibits activation of the NMDA receptors, might at least partially explain the mechanisms of a dantrolene-evoked protection of neurons against excitotoxicity mediated by agonists of NMDA receptors.

Preconditioning is an experimental strategy for reducing ischemic brain damage. There are reports that brief exposure of neurons to NMDA-receptor antagonists may be an adequate preconditioning stressor. We studied effects of preconditioning of the cerebellar granule cells (CGC) in primary culture by 30-minute exposure to NMDA receptor antagonists 0.5 μM MK-801 or 5 μM memantine. CGC were challenged with oxygen and glucose deprivation (OGD) or excitotoxic glutamate and cell viability was tested 24 h later using calcein/ethidium homodimer-1 staining. We studied glutamate-induced increases in 45Ca uptake and in the intracellular Ca2+ level assessed with the fluorescent probe fluo-3. The number of living cells in OGD-treated cultures decreased by 42%. Preconditioning with MK-801 or memantine 24 h earlier reduced cell death to 8% and 30% and 48 h earlier to 27% and 33%, respectively. Pretreatment with MK-801 followed by the standard MK-801 wash out was slightly cytoprotective in a glutamate excitotoxicity test performed immediately; the protection increased significantly 24 h after preconditioning. In both cases the extensive wash out of MK-801 after preconditioning resulted in loss of cytoprotection. The increase in the intracellular Ca2+ level evoked by glutamate was decreased 24 h after preconditioning and even halved in the neuronal cultures 48 h after preconditioning with MK-801 and memantine. Glutamate-induced 45Ca uptake in these cells was decreased by 18%, irrespective of the time laps after preconditioning. These results demonstrate that preconditioning of CGC with NMDA receptor antagonists induces prolonged tolerance to OGD, which is accompanied by the reduction of glutamate-evoked calcium fluxes. The causal relationship between these effects may be suggested.

In this article we present evolutionary aspects of the dual role of Ca2+ as signaling molecules and cytotoxic cations. We discuss the mechanisms of calcium homeostasis in neurons, taking into account the specific features of excitable cells and the mechanisms of generation and transduction of calcium signals. Based on this information we outline the role of Ca2+ ions in specific functions of the nerve cell, such as excitability, propagation of the action potential, synaptic transmission, neuronal plasticity and various forms of mobility. Then we discuss the role of disturbances of calcium homeostasis and signaling function in the mechanisms of injury and death of neurons in acute diseases with special regard to cerebral ischemia, and in chronic neurodegenerative disorders.

Several in vivo and in vitro studies have demonstrated the neuroprotective potential of pretreatment with 1alpha,25-dihydroxyvitamin D3 (calcitriol). The aim of the present study was to determine the effectiveness of calcitriol administered in vivo after a brain ischemic episode in the rat model of perinatal asphyxia, or when co-applied with or without delay during 24-h exposure of mouse hippocampal, neocortical and cerebellar neuronal cultures to glutamate on their 7th and 12th day in vitro (7 DIV and 12 DIV, respectively). Calcitriol was also administered after acute exposure of rat cerebellar neurons to glutamate. In 7-day-old rat pups subjected to hypoxia-ischemia, acute application of calcitriol in a single dose of 2 microg/kg, 30 min after termination of the insult, or subchronic, 7-day post-treatment with calcitriol, effectively reduced brain damage. The level of neuroprotection exceeded that achieved by hypoxic preconditioning used as the reference neuroprotective method. The results of in vitro experiments revealed the ability of calcitriol to reduce excitotoxicity in a manner dependent on the origin of the neuronal cells, their stage of maturation in culture and the duration of exposure to the excitotoxic insult before calcitriol application. Calcitriol was neuroprotective when it was administered together with glutamate or even after a delay of up to 6h during 24-h excitotoxic challenge of hippocampal and neocortical, but not cerebellar neuronal cultures. Application of calcitriol to cultured cerebellar granule neurons after acute exposure to glutamate was ineffective. In 12 DIV hippocampal cell cultures, 50 nM calcitriol inhibited glutamate-induced caspase-3 activity, while only 100 nM concentrations were effective in 7 DIV cultures. We ascribe the protective effects of calcitriol to the rapid modulation of mechanisms that are instrumental in the direct anti-apoptotic, neuroprotective action of this compound.

45CaCl2 uptake at low (5 mM), and high, depolarizing (65 mM) KCl concentration was measured in a fraction enriched in astrocytes, a crude mitochondrial-synaptosomal (P2) preparation and in purified synaptosomes derived from normal rats and from rats with thioacetamide-induced acute hepatic encephalopathy (HE). HE was found to reduce the potassium-evoked component of the astroglial uptake to 50% of its control level, without affecting the uptake into the P2 fraction or synaptosomes. The results are in keeping with the view that astrocytes are the cells whose metabolism and functions are predominantly affected during HE.

A body of evidence suggests that the mechanisms of excitotoxic neuronal damage evoked by excessive or prolonged activation of the excitatory amino acid receptors may be involved in pathogenesis of brain damage in acute insults and in chronic neurodegenerative diseases. In this review we briefly discuss several selected mechanisms of the excitotoxicity, focusing attention on the role of ionotropic glutamate receptors, calcium transients and calcium-mediated cell injury. In the second part of this paper we provide information on elements of excitotoxicity in brain diseases.

The neuroprotective potential of mGluR1 and mGluR5 antagonists (group I), EMQMCM and MTEP, respectively was studied using the 3 min forebrain ischemia model in Mongolian gerbils and the hypoxia-ischemia model in 7-day-old rats. Hypoxia-ischemia was induced by unilateral carotid occlusion followed by 75 min exposure to hypoxia (7.3% O(2) in N(2)), forebrain ischemia in gerbils was evoked by bilateral common carotid artery occlusion. The postischemic rectal body temperature in rat pups or brain temperature of gerbils was measured. The drugs were administered i.p. three times every 2 h after the insult, each time in equal doses of 1.25, 2.5 or 5.0 mg/kg. After 2 weeks brain damage was evaluated as weight decrease of the ipsilateral hemisphere in the rat pups or damage to CA1 pyramids in the gerbil hippocampus. The results demonstrated a dose dependent neuroprotection in both ischemic models by EMQMCM, while MTEP was neuroprotective only in the gerbil model of forebrain ischemia. EMQMCM reduced postischemic hyperthermia in gerbils. Thus, the antagonists of mGluR1 and mGluR5 show differential neuroprotective ability in two models of brain ischemia. Postischemic hypothermia may be partially involved in the mechanism of neuroprotection following EMQMCM in gerbils.

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl- d -aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg2+-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24 h later using propidium iodide staining. d,l -Homocysteine given alone appeared to be a weak neurotoxin, with EC50 of 17.4 mM, whereas EC50 for l -glutamate was 0.17 mM. Addition of 50 μM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 μM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45 Ca uptake by granule cells incubated in the presence of 25 mM d,l -homocysteine with 50 μM glycine was only 3% of the net uptake evoked by 1 mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 μM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca2+ to neurones.

In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca(2+) concentration (Ca(i)) and in the level of Ca(2+) bound to intracellular hydrophobic domains (Ca(b)) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Ca(i) and Ca(b), reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca(2+) pools and prevents Ca(2+) overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.

To evaluate the participation of excitatory amino acid receptors sensitive to N-methyl-D-aspartate (NMDA) in ischemia-evoked redistribution of Ca2+ ions from the extra- to the intracellular compartment of the hippocampus, 2-amino-5-phosphonovalerate (APV), a specific antagonist of NMDA receptors, was administered to the rabbit hippocampus through a dialysis probe before, during, and after complete reversible 15-min cerebral ischemia. Microdialysis of the hippocampus allowed us to determine the changes in extracellular calcium and amino acid concentrations and to monitor the permeability of the blood-brain barrier (BBB) to fluorescein. Moreover, EEG and general physiological parameters were registered. APV significantly reduced the ischemic drop of calcium and increased the taurine and phosphoethanolamine content in the extracellular compartment, whereas changes in concentrations of other amino acids and BBB permeability were not modified. Local administration of APV also improved the recovery of EEG activity after ischemia. Inhibition by APV of ischemia-induced calcium redistribution in the hippocampus suggests a major role of NMDA receptors in the influx of calcium to hippocampal neurons during cerebral ischemia.

Dantrolene – an inhibitor of ryanodine receptors and calcium stabilizer – prevents ischemia- and excitotoxicity-evoked neurodegeneration. To elucidate the mechanisms of this phenomenon, we investigated effects of dantrolene on the NMDA- and glutamate-induced lesion and stimulation of 45Ca uptake in primary cultures of rat cerebellar granule neurons. Neurodegeneration was evaluated after 24 h with the propidium iodide staining. Bcl-2 immunoreactivity in cell homogenates was measured by immunoblotting. The results demonstrated that dantrolene applied at micromolar concentrations inhibits in a dose-dependent manner NMDA- and glutamate-evoked 45Ca uptake in neurones and induces neuroprotection. This effect was additive to known effects of DMSO, a vehicle to dantrolene. Dantrolene failed to induce changes in Bcl-2 immunoreactivity. Thus, dantrolene-induced neuroprotection against excitotoxicity may be at least partially mediated by its inhibitory effect on the NMDA receptors.

Homocysteine (HCY) neurotoxicity is known to be mediated by disturbances in methylation processes and by the NMDA receptor-mediated excitotoxicity. In this study we examined the role of group I metabotropic glutamate receptors (mGlyRs) in HCY-induced toxicity in cultured rat cerebellar granule neurons exhibited for 30 min, or cultured in the presence of HCY for 3 days. Acute neurodegeneration induced by >10 mm D,L-HCY, or subchronic damage evoked by 75–500 µm HCY were enhanced by 50 µm glycine and moderately inhibited by 0.5 µm MK-801, revealing the NMDA receptor-mediated portion of HCY neurotoxicity. Also group I mGluRs antagonists 25 µm LY367385 or MPEP given alone only moderately inhibited acute and subchronic HCY neurotoxicity, but given together with MK-801 completely prevented neurotoxicity. These results demonstrate that acute homocysteine-induced neurotoxicity is mediated cooperatively by group I mGluRs and NMDA receptors.

Pretreatment of isolated brain synaptic endings with commercial phospholipase A2 isolated from venom of Apis mellifera, followed by a BSA washing, selectively inhibited the depolarization-dependent portion of 45Ca uptake. Phospholipase A2 initially caused an increase of synaptosome respiration and subsequently inhibited their oxygen uptake, but this effect was completely abolished in BSA-containing media. The classical uncoupler of oxidative phosphorylation, DNP, produced release of 45Ca or [3H]GABA from superfused synaptosomes previously loaded with radioactive calcium or GABA. The treatment of synaptosomes with phospholipase A2 had no effect on the spontaneous efflux of 45Ca or [3H]GABA. However, depolarization-dependent release of [3H]GABA from synaptosomes treated with phospholipase A2 was significantly inhibited. We suggest that the inhibition of depolarization-dependent influx of 45Ca into synaptosomes treated with phospholipase A2 may be attributed to the lesion of the specific function of plasma membrane rather than to the impairment of the calcium-sequestrating function of intraterminal structures.

The mitochondrial fraction obtained from brains of animals submitted to ischemia shows a decrease of phospholipid level, especially plasmalogens in the fraction of ethanolamine phospholipids and choline phospholipids. THere appears simultaneously an increase of free radical oxidation processes of unsaturated fatty acids from these phospholipids. The peroxidation processes of mitochondrial lipids are stimulated by calcium ions and, to a smaller extent, by magnesium ions. Barbiturate anesthesia inhibits the peroxidation of fatty acids and increases the antioxidant abilities of the nervous tissue. Nembutal added in vitro remains without effect on the above processes. The effect of acetylcholine and the antioxidant ability of nervous tissue under barbiturate anesthesia with respect to ischemia are discussed.

The effect of cerebral ischemia on extracellular amino acids and calcium content and on the permeability of the blood-brain barrier was studied by in vivo dialysis of rabbit hippocampus. This was combined with physiological and neurophysiological measurements. It was found that immediately after 15-min ischemia extracellular concentrations of glutamate, aspartate and taurine increased 3-, 2- and 6-fold, respectively, whereas a maximal, 7-fold increase of phosphoethanolamine and persistent elevation of glutamate were observed 45 min after ischemia. Extracellular calcium concentration, monitored with 45Ca2+, increased by 10% during the initial phase of ischemia, and decreased to approx. 74% of the basal level 10 min after ischemia. Recovery of extracellular calcium content was not attained until 45 min of recirculation, at which time the first signs of return of bioelectric activity were noted. Increased permeability of the blood-brain barrier to fluoresceine developed immediately after ischemia and persisted up to 2 h of recirculation. The obtained results are discussed in reference to the noted simultaneity of changes in extracellular excitatory amino acids and calcium concentrations and of brain bioelectric activity during and after ischemia. Causal relations between these effects are suggested.