AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures.

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+]i. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA-induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca(2+)-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents.     

AR-R15896AR, a low affinity, use-dependent, NMDA antagonist, is protective in a primate model of stroke

The low affinity, use-dependent, N-methyl- -aspartate (NMDA) antagonist, AR-R15896AR, is neuroprotective against transient focal cerebral ischemia in rats. We have examined the effect of AR-R15896AR, administered at a plasma level that is tolerated in acute stroke patients, on both functional and histopathologic measures in marmoset monkeys with permanent middle cerebral artery (MCA) occlusion. The M1 segment of the right MCA was permanently occluded (pMCAO) by bipolar coagulation in 11 marmosets. Five minutes later, the monkeys received either saline or AR-R15896 (4.5 mg/kg) intravenously, infused over 30 seconds. Also, osmotic minipumps were implanted subcutaneously to provide continuous drug or saline infusion for 48 hours. Drug-filled pumps released AR-R15896 at a rate of 1.1 mg/kg/h. The monkeys had been trained and tested preoperatively on a number of behavioral tasks and were retested 3 and 10 weeks after surgery. Three weeks after surgery, all the monkeys had a severe motor deficit, in that they were impaired at reaching with their contralesional arms, and perceptual neglect of contralesional space. AR-R15896AR-treated monkeys, however, had significantly less neglect than saline-treated monkeys, although the AR-R15896AR treatment had no effect on the motor deficit. By 10 weeks, the neglect had recovered, but not the motor deficit, and there were no differences between the 2 groups. Histopathologic analysis showed a reduction in the size of the infarction at several stereotaxic levels of the AR-R15896AR-treated monkeys. This study has shown that AR-R15896AR protected a select region of the brain against permanent focal cerebral ischemia in a primate species and attenuated the ensuing spatial neglect.     

Treatment of acute ischaemic stroke with the low-affinity,use-dependent NMDA antagonist AR-R15896AR. A safety and tolerability study

BACKGROUND AND PURPOSE: A low-affinity, use-dependent N-Methyl-D-Aspartate (NMDA) antagonist AR-R15896AR has neuroprotective properties in animal models of ischaemic stroke. The aim of the present study was to examine the safety and tolerability of a new and higher dosage regimen that would enable acute stroke patients to achieve and maintain neuroprotective plasma concentrations. METHODS: A randomised, multi-centre, double-blind, placebo-controlled, parallel group study was carried out at 19 centres in France, Germany and the Netherlands in patients with a clinical diagnosis of acute ischaemic stroke, and onset of symptoms within 12 hours before start of study drug administration. Two loading doses of 3.5 mg/kg of AR-R15896AR over 60 minutes, followed by a 2.5 mg/kg infusion over the next 120 minutes were given. Eight hours after the start of the loading dose infusion, the first maintenance dose (120 mg) was administered over 60 minutes. Eight further maintenance infusions were administered at intervals of 8 hours over a total treatment period of 3 days. Main variables were safety, tolerability and pharmacokinetics. Follow-up assessments also included the Barthel Index (BI) and the NIH Stroke Scale (NIHSS) at 4-7 days after the end of the last infusion and at 30 days after the onset of stroke. RESULTS: 103 patients with acute ischaemic stroke were randomised to either treatment with AR-R15896AR (70 patients) or placebo (33 patients). Mortality was not significantly different in the AR-R15896AR group compared with the placebo group (10 % vs. 6 %). Serious adverse events during treatment due to psychiatric conditions were associated with AR-R15896AR (3 vs. 0). Other side effects were more common in the group treated with AR-R15896AR: vomiting (29 % vs. 9 %), nausea (23 % vs. 12 %), fever (17 % vs. 12 %), agitation (7 % vs. 3 %), dizziness (7 % vs. 0 %), and hallucinations (6 % vs. 0 %). No significant difference between the two groups (with respect to the proportions of patients with favourable outcome) was detected in either the analysis of the BI or the NIHSS. Pharmacokinetic data showed that plasma concentrations of AR-R15896AR were in the expected neuroprotective range. CONCLUSION: In most of the patients with acute stroke receiving AR-R15896AR the intended high plasma levels were reached within a short time period. However, active treatment produced more side effects than placebo, thus indicating safety concerns and tolerability issues for use in high doses in an acute stroke population.     

Comparison of the neuroprotective effect of clomethiazole,AR-R15896AR and NXY-059 in a primate model of stroke using histological and behavioural measures

Three experimental neuroprotective agents (clomethiazole, AR-R15896AR and NXY-059) have recently been tested in a primate model of acute ischaemic stroke. As the experimental techniques used in all three studies were similar and the compounds were administered at clinically relevant doses, a comparative analysis of the functional benefits of these drug-treatments has now been performed. Furthermore a more detailed histological analysis of the neuroprotection afforded by the drugs has also been made. NXY-059 produced almost twice the degree of neuroprotection than that seen following clomethiazole or AR-R15896AR. Protection by NXY-059 was seen in measurements of damage to cortex and white matter. Clomethiazole and AR-R15896AR provided less protection of cortex and white matter than NXY-059. Conspicuously, AR-R15896AR was without effect in sub-cortical regions. NXY-059 was the only compound to produce a major, statistically significant improvement in the motor deficit induced by the stroke. All three drugs also reduced the degree of spatial neglect 3 weeks after pMCAO, and 10 weeks later only NXY-059 still provided significant additional functional benefit to the spontaneous improvement seen in stroked control animals not receiving treatment. The overview of the behavioural effects and these new histological findings suggest that NXY-059 was by far the most effective neuroprotective agent of the three examined.     

Lack of evidence for direct involvement of NMDA receptors or polyamines in blood–brain barrier injury after cerebral ischemia in rats

It is hypothesized that after various types of brain injury, blood–brain barrier (BBB) opening and vasogenic edema result from excessive neuronal release of glutamate and stimulation of capillary N-methyl-d-aspartate (NMDA) receptors linked to polyamine (putrescine) synthesis in endothelial cells. We produced cerebral ischemia in rats and measured BBB opening 6 h later as the increase in regional transfer constants (K_i) for blood to brain diffusion of []sucrose. Such BBB opening was not mitigated by drugs which block NMDA receptors (MK801 or AR-R 15896AR) or polyamine synthesis (difluoromethylornithine). These results question generality of the capillary NMDA receptor/polyamine hypothesis.     

Efficacy of AR-R15896AR in the rat monofilament model of transient middle cerebral artery occlusion

The monofilament technique of transient middle cerebral artery occlusion (MCAO) was used in 3 separate studies to evaluate the efficacy of the low-affinity, use-dependent N-methyl-d-aspartate receptor antagonist, AR-R15896AR. First, a dose-response curve was attempted. Wister Kyoto rats received 2 hours of MCAO. Five minutes later, a 30-minute intravenous infusion of AR-R15896AR was given, followed by subcutaneous implantation of Alzet minipumps that were calibrated to maintain specified plasma levels (approximately 682, 1885, or 2682 ng/mL) of AR-R15896 (free base) for 1 week. The highest plasma level attained significantly decreased the percentage of damage to the subcortex, cortex, and total brain. Second, the high-dose, 1-week treatment regimen was repeated to determine if neuroprotection would extend to 8 weeks after MCAO. Indeed, in separate groups of animals, significant reduction in the percentage of damage, which was generally confined to the cortex and subcortex, was observed at 1, 2, 4, and 8 weeks. Third, verification was achieved in another laboratory. Lister Hooded rats received 60 minutes of transient MCAO. At 70 minutes, an acute dose of AR-R15896AR (20.3 mg/kg) was injected intraperitoneally and the rats were killed 23 hours later. This treatment group also exhibited significant reduction in the volume of infarction in the subcortex, cortex, and total brain. The outcome of these investigations supports the ongoing Phase II clinical trials in patients with acute stroke.     

N-methyl-d-aspartate and hypoxia induced Ca-changes in the CA1 region of the hippocampal slice

Hypoxic neuronal depolarization was accompanied by a large decrease in extracellular [Ca²⁺]. After reoxygenation, the time at which [Ca²⁺] normalized was correlated with the extent of recovery of N-methyl-d-aspartate (NMDA) and synaptic responses. There was no evidence that the NMDA receptor system was more disrupted following hypoxia than the receptors involved in synaptic transmission. The Na⁺/K⁺ pump appeared to be better able to recover from hypoxia than the NMDA responses or synaptic transmission.     

Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Carnosine protects a primary cerebellar cell culture from acute NMDA toxicity

Activation of extrasynaptic NMDA receptors by high glutamate concentrations is one of the key pathogenic factors following a stroke. For this reason, the search for efficient neuroprotective agents that could reduce glutamate toxicity is a pressing need. Ca²⁺ overload in response to glutamate leads to activation of signaling cascades in the cell and the development of oxidative stress, which ultimately leads to apoptosis. Using a model system of acute excitotoxicity caused by 50 μM NMDA, which was used as a specific NMDA receptor activator, we demonstrated that during 2 hours of incubation the viability of the primary neuronal culture decreased by 30–50%. To demonstrate that the observed effect is associated not only with the Ca²⁺ influx into the cytoplasm through the activated NMDA receptors, we decreased the Ca²⁺ concentration in the medium. The lowered Ca2+ concentration, as well as its complete absence, did not affect NMDA toxicity. We tested carnosine, a naturally occurring dipeptide and promising antioxidant, as a neuroprotective agent. The addition of 2 mM carnosine prevented the decrease in cell viability caused by a 2-hour incubation with 50 μM NMDA, while it showed no effect on the viability of the cell culture in the control. Based on the results, we consider the further study of carnosine, its complexes, and analogues as neuroprotectors in cerebral ischemia promising.     

P2 purinoceptor blocker suramin antagonises NMDA receptors and protects against excitatory behaviour caused by NMDA receptor agonist (RS)-(tetrazol-5-yl)-glycine in rats

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P₂ receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P_2X subclass of P₂ receptors are ligand-gated Ca²⁺-selective channels and, since the increased influx of Ca²⁺ into neurons has been linked to neurotoxicity, simultaneous inhibition of P_2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [³H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [³H]kainate binding in vitro. In vivo, intracerebroventricular (ICV) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by ICV administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression. J. Neurosci. Res. 49:627–638, 1997. © 1997 Wiley-Liss, Inc.     

Neuroprotective potential of ionotropic glutamate receptor antagonists

From the therapeutic point of view, the real challenge is not only to improve the symptoms, but to interfere with the pathomechanism of the disease. That is why a considerable interest has recently been devoted to developing glutamate receptors antagonists (mainly of the NMDA type) for acute and chronic neurodegeneration. Developing such a treatment that slows down the progression of the disease is extremely time and costs consuming. At present there is consensus that competitive NMDA receptor antagonists will not find therapeutic applications, in contrast to agents acting at the glycine_B site, or channel blockers. Recently, at least seven glycine_B antagonists (e.g. ACEA 1021, GV-150526, GV-196771A, ZD-9379, MRZ 2/576) and over 10 NMDA channel blockers (e.g. Remacemide, ARL-15896AR, HU-211, ADCI, CNS-5161, Neramexane-MRZ 2/579) have been under development, most of them as neuroprotective agents for acute (stroke, trauma) or chronic insult (e.g. Huntington’s or Alzheimer’s disease). Several substances selective for NR2B NMDA receptor subtypes such as: eliprodil, CP-101606 and Ro-25-6981 have been claimed to have a good neuroprotective profile. This presentation is an attempt to critically review preclinical and scarce clinical experience in the development of new NMDA receptor antagonists as neuroprotective agents according to the following scheme: rational, preclinical findings in animal models and finally clinical experience if available. The general impression is that NMDA receptor antagonists may find use in chronic type of neurodegeneration while AMPA antagonists seems to show better promise in acute insult.     

Direct suppression by odorants of ionotropic glutamate receptors in newt retinal neurons

Summary. Odorants are known to suppress voltage-gated channels not only in olfactory receptor cells but also in neurons of outside of the olfactory system. Here we found that odorants suppress glutamate-gated channels in newt retinal neurons using the Ca²⁺ imaging technique. Bath application of 100 μM glutamate rose [Ca²⁺]_i under application of the voltage-gated Ca²⁺ channel blocker. Thus, [Ca²⁺]_i rises in the neurons were most likely attributable to Ca²⁺ influx via Ca²⁺-permeable glutamate-gated channels rather than voltage-gated Ca²⁺ channels. A similar increase of [Ca²⁺]_i was observed by application of 100 μM NMDA and 50 μM kainate, suggesting that both NMDA and AMPA/kainate receptors were expressed in newt retinal neurons. Application of odorants, 1 mM amyl acetate and acetophenone, reversibly reduced [Ca²⁺]_i increased by glutamate, NMDA and kainate. This suggests that odorants can suppress not only voltage-gated channels but also ligand-gated channels such as NMDA and AMPA/kainate receptors. Received January 9, 2002; accepted February 25, 2002 Published online June 28, 2002 Acknowledgements We thank H. Kurajyo, A. Mamiya, and Y. Hikita for their technical assistance. This work was supported by Japan Society of the Promotion of Science (No. 12780620 to F.K.), the SKYLARK Food Science Institute, the Fujisawa Foundation, Narishige Neuroscience Research Foundation, the Research Foundation for Pharmaceutical Sciences, Daiko Foundation, and the Naito Foundation. Authors' address: Dr. M. Ohkuma, Department of Physiology, School of Medicine, Fujita Health University, 1-98 Dengakugakubo, Kutsukakechou, Toyoake, Aichi, 470-1192, Japan, e-mail: m-ohkuma@fujita-hu.ac.jp     

Regulation of Intracellular Ca2+ in Response to Muscarinic and Glutamate Receptor Agonists During the Differentiation of NTERA2 Human Embryonal Carcinoma Cells into Neurons

Single cell microfluorimetry was used to study intracellular calcium ion signals ([Ca²⁺]_i) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, [Ca²⁺]_i) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in [Ca²⁺]_i) was dependent upon both Ca²⁺ influx and Ca²⁺ mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, [Ca²⁺]_i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in [Ca²⁺li were governed by both Ca²⁺ influx and Ca²⁺ mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca²⁺ mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M₁, M₃, M₄ and possibly M₅ receptor subtypes linked to changes in [Ca²⁺]_i), whilst only M₃ and M₅ are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in [Ca²⁺]_i), and changes in the expression of muscarinic ACh receptor subtypes.     

Changes in extracellular calcium concentration in the immature rat cerebral cortex during anoxia are not influenced by MK-801

The extracellular calcium concentration ([Ca²⁺]_ec) was recorded by calcium-sensitive microelectrodes in the parietal cortex of 9–11 day old rats during anoxia. During the first 10 min of anoxia, [Ca²⁺]_ec increased from 1.1 mM to 1.5 ± 0.23 mM, and thereafter it started to decrease reaching below basal level after around 13 min. The [Ca²⁺]_ec decrease was either slow and continuous, or biphased with a rapid initial decrease followed by a continuous slow decrease. After 60 min of anoxia, the [Ca²⁺]_ec had reached 0.2–0.3 mM. Changes in [Ca²⁺]_ec in animals treated with the NMDA receptor antagonist MK-801 (0.3 mg/kg i.p.) did not display any significant differences compared to controls. Thus, the strong neuroprotective effect of MK-801 in ischemic situations in the immature brain can not be explained by a prevention of calcium entry during anoxic depolarization.     

Asphyxia induced by umbilical cord occlusion alters glutamatergic and GABAergic synaptic transmission in neurons of the superior colliculus in fetal rats

Using optical recordings, we studied the effects of asphyxia on intracellular Cl⁻ and Ca²⁺ concentrations ([Cl⁻]i; [Ca²⁺]i) in the superior colliculus of fetal rats, which were connected via the umbilical cord to the dam. Acute asphyxia was induced by umbilical cord occlusion. The number of fetal superior colliculus neurons showing GABA-mediated increases in [Cl⁻]i (leading to hyperpolarization) following local synaptic electrical stimulation had decreased by 3 h post-asphyxiation, while the number showing GABA-mediated decreases in [Cl⁻]i (leading to depolarization) increased. [Ca²⁺]i rise, which occurred after acute asphyxiation, was antagonized by both non-NMDA and NMDA receptor antagonists. The increase in [Ca²⁺]i following focal superior colliculus stimulation was markedly attenuated at 3 h post-asphyxiation.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

Blockade of NMDA receptor-mediated mobilization of intracellular Ca prevents neurotoxicity

NMDA receptor activation leads to elevated Ca²⁺ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca²⁺ response in associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca²⁺ response to NMDA was due to mobilization of intracellular Ca²⁺ stores rather than merely to Ca²⁺ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca²⁺ stores. Henceforth, despite normal extracellular Ca²⁺, NMDA elicited only about half of its former Ca²⁺ response. Similarly, when dantrolene was used to block Ca²⁺ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca²⁺ responses. These results quite surprisingly indicate that at least half the Ca²⁺ response to NMDA is due to release of intracellular Ca²⁺, a process triggered by influx of extracellular Ca²⁺. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca²⁺ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.     

Decoding glutamate receptor activation by the Ca2+ sensor protein hippocalcin in rat hippocampal neurons

Hippocalcin is a Ca²⁺‐binding protein that belongs to a family of neuronal Ca²⁺sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin‐YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor‐dependent depolarization and following Ca²⁺influx via voltage‐operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin‐YFP translocation in a dendritic shaft at the application site due to direct Ca²⁺influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin‐YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back‐propagating action potentials. The translocation to spines required Ca²⁺influx via synaptic NMDA receptors in which Mg²⁺ block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1–2 μm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site‐ and time‐specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.     

Role of nonsynaptic GluN2B-containing NMDA receptors in excitotoxicity: Evidence that fluoxetine selectively inhibits these receptors and may have neuroprotective effects

In acute ischaemic brain injury and chronic neurodegeneration, the primary step leading to excitotoxicity and cell death is the excessive and/or prolonged activation of glutamate (Glu) receptors, followed by intracellular calcium (Ca²⁺) overload. These steps lead to several effects: a persistent depolarisation of neurons, mitochondrial dysfunction resulting in energy failure, an increased production of reactive oxygen species (ROS), an increase in the concentration of cytosolic Ca²⁺ [Ca²⁺]_i, increased mitochondrial Ca²⁺ uptake, and the activation of self-destructing enzymatic mechanisms. Antagonists for NMDA receptors (NMDARs) are expected to display neuroprotective effects, but no evidence to support this hypothesis has yet been reported. A number of clinical trials using NMDAR antagonists have failed to demonstrate neuroprotective effects, either by reducing brain injury or by preventing neurodegeneration. Recent advances in NMDAR research have provided an explanation for this phenomenon. Synaptic and extrasynaptic NMDARs are composed of different subunits (GluN2A and GluN2B) that demonstrate opposing effects. Synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs have different co-agonists: d-serine for synaptic NMDARs and glycine for extrasynaptic NMDARs. Both co-agonists are of glial origin.The mechanisms of cell destruction or cell survival in response to the activation of NMDAR receptors depend in part on [Ca²⁺]_i and the route of entry of this ion and more significantly on the subunit composition and localisation of the NMDARs. While synaptic NMDAR activation is involved in neuroprotection, the stimulation of extrasynaptic NMDARs, which are composed of GluN2B subunits, triggers cell destruction pathways and may play a key role in the neurodegeneration associated with Glu-induced excitotoxicity. In addition, it has been found that synaptic and extrasynaptic NMDA receptors have opposing effects in determining the fate of neurons. This result has led to the targeting of nonsynaptic GluN2B-containing NMDARs as promising candidates for drug research. Under hypoxic conditions, it is likely that the failure of synaptic glutamatergic transmission, the impairment of the GluN2A-activated neuroprotective cascade, and the persistent over-activation of extrasynaptic GluN2B-containing NMDARs lead to excitotoxicity. Fluoxetine, a drug widely used in clinical practice as an antidepressant, has been found to selectively block GluNR2B-containing NMDARs. Therefore, it seems to be a potential candidate for neuroprotection.