N-methyl-d-aspartate Preconditioning Research Articles

Functional interplay between adenosine A2A receptor and NMDA preconditioning in fear memory and glutamate uptake in the mice hippocampus

N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.

Adenosine and NMDA Receptors Modulate Neuroprotection-Induced NMDA Preconditioning in Mice.

The severity score of quinolinic acid (QA)-induced seizures was investigated after N-methyl-D-aspartate (NMDA) preconditioning associated with adenosine receptors. Also, the levels of adenosine A1 and A2A receptors and subunits of NMDA receptors in the hippocampi of mice were determined to define components of the resistance mechanism. Adult CF-1 mice were treated intraperitoneally with saline or NMDA (75mg/kg), and some mice were treated intracerebroventricularly (i.c.v.) with 0.1pmol of adenosine receptor antagonists 8-cyclopentyltheophylline (CPT; receptor A1) or ZM241385 (receptor A2A) 0, 1, or 6h after NMDA administration. These adenosine receptor antagonists were administered to block NMDA's protective effect. Seizures and their severity scores were evaluated during convulsions induced by QA (36.8nmol) that was administered i.c.v. 24h after NMDA. The cell viability and content of subunits of the NMDA receptors were analyzed 24h after QA administration. NMDA preconditioning reduced the maximal severity 6 displayed in QA-administered mice, inducing protection in 47.6% of mice after QA-induced seizures. CPT increased the latency of seizures when administered 0 or 6h, and ZM241385 generated the same effect when administered 6h after NMDA administration. The GluN1 content was lower in the hippocampi of the QA mice and the NMDA-preconditioned animals without seizures. GluN2A content was unaltered in all groups. The results demonstrated the components of resistance evoked by NMDA, in which adenosine receptors participate in a time-dependent mode. Similarly, the reduction on GluN1 expression in the hippocampus may contribute to this effect during the preconditioning period.

Role of Phosphatidylinositol-3 Kinase Pathway in NMDA Preconditioning: Different Mechanisms for Seizures and Hippocampal Neuronal Degeneration Induced by Quinolinic Acid

N-methyl D-aspartate (NMDA) preconditioning is evoked by the administration of a subtoxic dose of NMDA and is protective against neuronal excitotoxicity. This effect may involve a diversity of targets and cell signaling cascades associated to neuroprotection. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases (MAPKs) such as extracellular regulated protein kinase 1/2 (ERK1/2) and p38MAPK pathways play a major role in neuroprotective mechanisms. However, their involvement in NMDA preconditioning was not yet fully investigated. The present study aimed to evaluate the effect of NMDA preconditioning on PI3K/Akt, ERK1/2, and p38MAPK pathways in the hippocampus of mice and characterize the involvement of PI3K on NMDA preconditioning-evoked prevention of seizures and hippocampal cell damage induced by quinolinic acid (QA). Thus, mice received wortmannin (a PI3K inhibitor) and 15min later a subconvulsant dose of NMDA (preconditioning) or saline. After 24h of this treatment, an intracerebroventricular QA infusion was administered. Phosphorylation levels and total content of Akt, glycogen synthase protein kinase-3β (GSK-3β), ERK1/2, and p38MAPK were not altered after 24h of NMDA preconditioning with or without wortmmanin pretreatment. Moreover, after QA administration, behavioral seizures, hippocampal neuronal degeneration, and Akt activation were evaluated. Inhibition of PI3K pathway was effective in abolishing the protective effect of NMDA preconditioning against QA-induced seizures, but did not modify neuronal protection promoted by preconditioning as evaluated by Fluoro-Jade B staining. The study confirms that PI3K participates in the mechanism of protection induced by NMDA preconditioning against QA-induced seizures. Conversely, NMDA preconditioning-evoked protection against neuronal degeneration is not altered by PI3K signaling pathway inhibition. These results point to differential mechanisms regarding protection against a behavioral and cellular manifestation of neural damage.

Neuroprotection induced by NMDA preconditioning as a strategy to understand brain tolerance mechanism.

Excitotoxicity refers to toxicity caused by abnormal concentrations of glutamate in the synaptic cleft that may lead to neuronal death. Since its description, the phenomenon of glutamatergic excitotoxicity has been implicated in the physiopathology of a wide range of neurological and psychiatric disorders, from acute brain damage such as traumatic brain injury, ischemia as well as chronic conditions like epilepsy, depression and neurodegenerative pathologies such as Huntington's, Parkinson's and Alzheimer's diseases. Excessive stimulation of glutamatergic receptors, mainly N-methyl-D-aspartate (NMDA) receptors (NMDAR), can have numerous adverse effects on the cell viability, including increased nitric oxide release (NO), activation of proteases, increased production of reactive oxygen (ROS) and nitrogen (RNS) species and massive influx of calcium ions (Ca2+), resulting in cell death. Thus, the use of strategies that modulate the excitotoxic cell damage represents a perspective for the treatment of diseases such as Parkinson's and Alzheimer's diseases, ischemia, traumatic brain injury (TBI) and seizures.

Adenosine A1 receptor activation modulates N-methyl-d-aspartate (NMDA) preconditioning phenotype in the brain

N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [3H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.

N-methyl-D-aspartate preconditioning prevents quinolinic acid-induced deregulation of glutamate and calcium homeostasis in mice hippocampus.

The search for new therapeutic strategies through modulation of glutamatergic transmission using effective neuroprotective agents is essential. Glutamatergic excitotoxicity is a major factor common to neurodegenerative diseases and in acute events such as cerebral ischemia, traumatic brain injury and epilepsy. We have previously demonstrated that N-methyl-D-aspartate (NMDA) preconditioning in mice showed 50% of protection against seizures and full protection against damage to neuronal tissue induced by quinolinic acid (QA). In this study, cellular and molecular mechanisms involved on NMDA preconditioning and neuroprotection were investigated in mice treated with NMDA 24h before QA insult. Calcium uptake and D-aspartate release from hippocampal slices obtained from mice treated with NMDA plus QA and not displaying seizures (protected mice) were similar to control (saline) or NMDA preconditioned mice. Increased calcium uptake and glutamate release is evidenced in unprotected (convulsed) mice as well as QA control, demonstrating that calcium and glutamate are involved in NMDA-induced preconditioning. Increased glutamate release evoked by QA was blocked by MK-801, whereas increased calcium uptake was abolished by voltage-dependent calcium channels inhibitors, but not MK-801. NMDA preconditioning is effective in normalizing the deregulation of glutamate transport and calcium homeostasis evoked by QA due to aberrant NMDA receptors activation that culminates in seizures and hippocampal cells damage.

Mitochondrial respiratory chain and creatine kinase activities following trauma brain injury in brain of mice preconditioned with N-methyl-d-aspartate

Traumatic brain injury (TBI) induces glutamatergic excitotoxicity through N-methyl-D-aspartate (NMDA) receptors, affecting the integrity of the mitochondrial membrane. Studies have pointed to mitochondria as the master organelle in the preconditioning-triggered endogenous neuroprotective response. The present study is aimed at understanding energy metabolism in the brains of mice after preconditioning with NMDA and TBI. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75mg/kg) and subjected to brain trauma. Mitochondrial respiratory chain and creatine kinase activities were assessed at 6 or 24h after trauma. The mice preconditioned and subjected to TBI exhibited augmented activities of complexes II and IV in the cerebral cortex and/or cerebellum. Creatine kinase activity was also augmented in the cerebral cortex after 24h. We suggest that even though NMDA preconditioning and TBI have similar effects on enzyme activities, each manage their response via opposite mechanisms because the protective effects of preconditioning are unambiguous. In conclusion, NMDA preconditioning induces protection via an increase of enzymes in the mitochondria.

Proteomic Analysis of the Mice Hippocampus After Preconditioning Induced by N-Methyl-d-Aspartate (NMDA)

Preconditioning induced by N-methyl-D-aspartate (NMDA) has been used as a therapeutic tool against later neuronal insults. NMDA preconditioning affords neuroprotection against convulsions and cellular damage induced by the NMDA receptor agonist, quinolinic acid (QA) with time-window dependence. This study aimed to evaluate the molecular alterations promoted by NMDA and to compare these alterations in different periods of time that are related to the presence or lack of neuroprotection. Putative mechanisms related to NMDA preconditioning were evaluated via a proteomic analysis by using a time-window study. After a subconvulsant and protective dose of NMDA administration mice, hippocampi were removed (1, 24 or 72 h) and total protein analyzed by 2DE gels and identified by MALDI-TOF. Differential protein expression among the time induction of NMDA preconditioning was observed. In the hippocampus of protected mice (24 h), four proteins: HSP70(B), aspartyl-tRNA synthetase, phosphatidylethanolamine binding protein and creatine kinase were found to be up-regulated. Two other proteins, HSP70(A) and V-type proton ATPase were found down-regulated. Proteomic analysis showed that the neuroprotection induced by NMDA preconditioning altered signaling pathways, cell energy maintenance and protein synthesis and processing. These events may occur in a sense to attenuate the excitotoxicity process during the activation of neuroprotection promoted by NMDA preconditioning.

NMDA preconditioning prevents object recognition memory impairment and increases brain viability in mice exposed to traumatic brain injury

Recent studies have focused on the role of N-methyl-d-aspartate (NMDA) in brain injury. The present study is aimed at verifying memory, anxiety/depression parameters, and cellular viability in the brain of mice preconditioned with NMDA and subjected to the model of mild traumatic brain injury. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75mg/kg) and subjected to brain trauma, and after 24h submitted to memory tasks and anxiety and depression-like behavioral tests. The memory tests were evaluated at 1.5h, 24h, and 7days after the training. In addition, the cellular viability was evaluated in the cerebral cortex and hippocampus 96h after the trauma. It was observed that the cellular viability was reduced in the hippocampus of the mice subjected to trauma and the preconditioning with NMDA was able to protect this damage. All mice learnt the task in the habituation test, but in the object recognition task the mice preconditioned with NMDA were protected against impairment induced by TBI in both short and long-term memory. On the other hand, in the step-down inhibitory avoidance test, only the mice treated with NMDA showed impairment of long-term memory (7days after training session). The evaluation of anxiety/depression behavior showed no changes after TBI. In conclusion, NMDA preconditioning induced impairment of the long-term memory; however, it was able to protect against the novel recognition memory impairment and increase the cellular survival in the hippocampus of mice exposed to traumatic brain injury.

Mild activation of poly(ADP‐ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7.

Molecular Alterations Associated with the NMDA Preconditioning-Induced Neuroprotective Mechanism Against Glutamate Cytotoxicity

Exposure of the brain to sub-lethal concentrations of glutamate activates, through stimulation of the glutamate N-methyl-D: -aspartate (NMDA) receptors, an endogenous brain protective mechanism (NMDA preconditioning) against glutamate cytotoxicity and various other injurious stimuli. Selective drug activation of this mechanism is considered to be a promising neuroprotective treatment against the devastating consequences of stroke and other traumatic brain insults. Although some properties of this mechanism have been characterized, many aspects concerning it are yet to be elucidated. In order to improve our understanding of the NMDA preconditioning mechanism, we have established an experimental in vitro model of primary rat neuronal cultures, in which NMDA preconditioning completely abolishes the glutamic acid insult-induced neuronal damage. Employing this model, we have monitored in the present study the level of activation or expression of several signal transducing proteins, assumed to be involved in the NMDA-activated protective mechanism, at various time points during the three successive periods of the model, preconditioning, insult, and reperfusion. The results demonstrated that the NMDA preconditioning-induced neuroprotective mechanism is associated with inactivation of p66ShcA, prevention of the insult-induced inactivation of Src, activation of AKT, inactivation followed by reactivation of FKHR-L1, and with increased expression of p52ShcA, EGFR, and MnSOD. The essential role of Src activity in the protective mechanism was further indicated by the demonstration that decreasing Src activation level by the Src inhibitor PP2 attenuated the NMDA preconditioning-induced protection. The alterations detailed above in the activation status or level of expression of the studied proteins are suggested to be part of the NMDA preconditioning-induced neuroprotective mechanism.

NMDA preconditioning protects against quinolinic acid-induced seizures via PKA, PI3K and MAPK/ERK signaling pathways

Preconditioning by N-methyl-d-aspartate (NMDA) may be promoted in vivo by the administration of a sub-convulsing dose of NMDA, with a neuroprotective effect against seizures and neuronal death induced by the infusion of quinolinic acid (QA) in mice. This study aimed to evaluate the participation of protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), Ca2+/calmodulin dependent protein kinase II (CaMKII) and phosphatidilinositol-3 kinase (PI3K) signaling pathways in this neuroprotection model. Adult Swiss male mice were preconditioned with NMDA 24h before the infusion of QA, and were treated with inhibitors of the aforementioned signaling pathways either 15min before the preconditioning or infusion of QA. Inhibition of the PKA and PI3K pathways abolished the protection evoked by NMDA, and inhibition of the MEK pathway significantly diminished this protection. Treatment with PKC and CaMKII inhibitors did not alter the protection rate. Inhibition of the MEK and PKC pathways resulted in an increased mortality rate when followed by the infusion of QA, or NMDA preconditioning and QA infusion, respectively. These results suggest that the PKA, PI3K and MEK pathways have a crucial role in the achievement of a neuroprotective state following preconditioning.

Preconditioning with NMDA protects against toxicity of 3-nitropropionic acid or glutamate in cultured cerebellar granule neurons

A brief sub-lethal ischaemic stimulus has been reported to protect against subsequent ischaemic damage in vivo, and in vitro following periods of hypoxia or oxygen-glucose deprivation (OGD). Preconditioning against neurotoxic stimuli has been linked to N-methyl-d-aspartate (NMDA) receptors, since receptor blockade prevents the protection afforded by OGD, and low doses of NMDA treatment are capable of preconditioning. The current study demonstrated that NMDA preconditioning also protects against 3-nitropropionic acid (3-NPA), a generator of both excitotoxic and oxidative damage, in addition to glutamate. Cerebellar granule neuronal (CGN) cultures prepared from 8-day neonatal Sprague-Dawley rats were maintained for 8 days prior to NMDA stimulation for 6h. At 9 days in vitro (DIV), preconditioned and control cultures were subjected to a toxic insult (1 microM-10 mM glutamate or 1 microM-10 mM 3-NPA). Neuronal viability was assessed by use of a fluorescein diacetate assay. Protection was effective with 100 microM NMDA preconditioning for 6 h against 1-100 microM glutamate, and also against 1-500 microM 3-NPA. The study demonstrates that NMDA preconditioning can be beneficial against excitotoxic treatments, even when these are potentially complicated by associated oxidative damage and metabolic compromise, as is the case for 3-NPA.

Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity

N-Methyl- d-aspartate (NMDA) preconditioning is evoked by subtoxic concentrations of NMDA (50 μM), which has been shown previously to lead to transient resistance to subsequent lethal dose of glutamate or NMDA in cultured neurons. The purpose of this study was to investigate the participation of adenosine A 1 and A 2A receptors on NMDA preconditioning against glutamate-induced cellular damage in cerebellar granule cells. NMDA preconditioning prevented the stimulatory effect induced by glutamate on AMP hydrolysis, but not on ADP hydrolysis. The neuroprotection evoked by NMDA preconditioning against glutamate-induced cellular damage was prevented by the presence of adenosine A 1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 100 nM), but not by the adenosine A 2A receptors antagonist, (4-(2[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a{1,3,5}triazian-5-yl-aminoethyl)phenol (ZM 241385, 50 nM). Interestingly, a long-term treatment with CPT or ZM 241385 alone protected cells against glutamate-induced neurotoxicity. Moreover, the functionality of adenosine A 1 receptor was not affected by NMDA preconditioning, but this treatment promoted adenosine A 2A receptor desensitization, measured by cAMP accumulation. Taken together, the results described herein suggest that the neuroprotection evoked by NMDA preconditioning against cellular damage elicited by glutamate occurs through mechanisms involving adenosine A 2A receptors desensitization co-operating with adenosine A 1 receptors activation in cerebellar granule cells.

NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice.

N-methyl D-aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)-induced seizures are used to average NMDA receptors-evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA-induced seizures and hippocampal damage in vivo. Mice were pretreated with nonconvulsant doses of NMDA for different times before i.c.v. QA infusion and observed for the occurrence of seizures. Hippocampal slices from mice were assayed to measure cellular viability. NMDA preconditioning presented 53% protection against QA-induced seizures, as well as QA-induced cellular death in the hippocampus. The NMDA receptor antagonist, MK-801, prevented the protection evoked by NMDA preconditioning. The adenosine A1 receptor antagonist, CPT, prevented the protection evoked by NMDA preconditioning against QA-induced seizures, but not against QA-induced hippocampal cellular damage. The adenosine A1 receptor agonist, CPA, did not mimic the NMDA preconditioning-evoked protective effects. These results suggest that in vivo preconditioning with subtoxic doses of NMDA protected mice against seizures and cellular hippocampal death elicited by QA, probably through mechanisms involving NMDA receptors operating with adenosine A1 receptors.

1313 Unilateral lesion of the hippocampal ca1 subfield due to transient forebrain ischemia plus unilateral hyperthermia, and its dynamics of cerebral blood flow and brain temperature

A brief sub-lethal ischaemic stimulus has been reported to protect against subsequent ischaemic damage in vivo, and in vitro following periods of hypoxia or oxygen–glucose deprivation (OGD). Preconditioning against neurotoxic stimuli has been linked to N-methyl-d-aspartate (NMDA) receptors, since receptor blockade prevents the protection afforded by OGD, and low doses of NMDA treatment are capable of preconditioning. The current study demonstrated that NMDA preconditioning also protects against 3-nitropropionic acid (3-NPA), a generator of both excitotoxic and oxidative damage, in addition to glutamate. Cerebellar granule neuronal (CGN) cultures prepared from 8-day neonatal Sprague–Dawley rats were maintained for 8 days prior to NMDA stimulation for 6 h. At 9 days in vitro (DIV), preconditioned and control cultures were subjected to a toxic insult (1 μM–10 mM glutamate or 1 μM–10 mM 3-NPA). Neuronal viability was assessed by use of a fluorescein diacetate assay. Protection was effective with 100 μM NMDA preconditioning for 6 h against 1–100 μM glutamate, and also against 1–500 μM 3-NPA. The study demonstrates that NMDA preconditioning can be beneficial against excitotoxic treatments, even when these are potentially complicated by associated oxidative damage and metabolic compromise, as is the case for 3-NPA.

1312 Effects of dopaminergic transplants on in vivo activity of tryptophan hydroxylase in the striatum of rats with unilateral 6-hydroxydopamine lesions

A brief sub-lethal ischaemic stimulus has been reported to protect against subsequent ischaemic damage in vivo, and in vitro following periods of hypoxia or oxygen–glucose deprivation (OGD). Preconditioning against neurotoxic stimuli has been linked to N-methyl-d-aspartate (NMDA) receptors, since receptor blockade prevents the protection afforded by OGD, and low doses of NMDA treatment are capable of preconditioning. The current study demonstrated that NMDA preconditioning also protects against 3-nitropropionic acid (3-NPA), a generator of both excitotoxic and oxidative damage, in addition to glutamate. Cerebellar granule neuronal (CGN) cultures prepared from 8-day neonatal Sprague–Dawley rats were maintained for 8 days prior to NMDA stimulation for 6 h. At 9 days in vitro (DIV), preconditioned and control cultures were subjected to a toxic insult (1 μM–10 mM glutamate or 1 μM–10 mM 3-NPA). Neuronal viability was assessed by use of a fluorescein diacetate assay. Protection was effective with 100 μM NMDA preconditioning for 6 h against 1–100 μM glutamate, and also against 1–500 μM 3-NPA. The study demonstrates that NMDA preconditioning can be beneficial against excitotoxic treatments, even when these are potentially complicated by associated oxidative damage and metabolic compromise, as is the case for 3-NPA.