NMDA receptor activation by residual glutamate in glutamine preparations: a cautionary note regarding weak NMDA receptor agonists

During an investigation of excitatory amino acids on cultured embryonic Xenopus neurons, we observed that commercial preparations of glutamine had weak agonist activity on NMDA-type glutamate receptors. Threshold responses were observed at 100 microM glutamine, and the dose-response relation did not show inflection or saturation at concentrations of up to 10 mM. However, NMDA receptor activation induced by glutamine probably represented activity of residual glutamate because: (a) recrystallization of glutamine reduced residual glutamate levels (measured by HPLC analysis) and NMDA receptor activation by comparable amounts; and (b) glutamate at concentrations close to those predicted to be present in glutamine preparations elicited currents of similar amplitudes. Our data indicate that residual glutamate at levels of less than 0.05% are sufficient to confound studies of weak NMDA receptor agonists.     

Phosphinothricin induces epileptic activity via nitric oxide production through NMDA receptor activation in adult mice

Phosphinothricin (PPT), the active component of a widely used herbicide, induces convulsions in rodents and humans. PPT shares structural analogy with glutamate, which could explain its powerful inhibitory effect on glutamine synthetase and its probable binding to glutamate receptors. To characterize the epileptogenic effect of PPT, electrographic and behavioural studies were carried out on PPT-treated adult mice. We investigated the role of N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) production in induction of seizures triggered by PPT, by using specific NMDA antagonist and nitric oxide synthase (NOS) inhibitor. The inhibitory effect of PPT on glutamine synthetase of mouse brain was assessed after in vitro and in vivo treatments. The results obtained show that PPT induces tonic-clonic seizures and generalized convulsions in mice. They suggest that these seizures are mediated through an NMDA receptor activation and NO production, without involvement of inhibition of glutamine synthetase.     

Stimulation of glutamine synthetase activity by excitatory amino acids in astrocyte cultures derived from aged mouse cerebral hemispheres may be associated with non-n-methyl-d-aspartate receptor activation

We have been using glial cells derived from aged mouse cerebral hemispheres (MACH) at several passages to study the responsiveness of astrocytes to microenvironmental signals in culture. In the present study, we examined the effects of excitatory amino acids on the activity of glutamine synthetase, a marker for astrocytes. MACH glia cell passages 25 to 29 were used. Culture groups were Dulbecco's modified Eagle's medium +10% fetal bovine serum (control); glutamate 100 μM; γ-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) 50 μM; kainic acid 10 μM; N-methyl-d-aspartate (NMDA) 10 μM. In all treated groups glutamine synthetase activity was significantly higher than in controls. We speculate that this increase represents an enhanced differentiation of immature astrocytes. In a second series, we examined the effects of glutamate receptor antagonists on glutamine synthetase activity as follows. MACH cultures were treated with glutamate 100 μM in combination with either L(+)-2-amino-3-phosphonopropionic acid (L-AP3; 50 μM); D(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 μM) or 6,7-dinitroquinoxaline-2,3-dione (DNQX; 50 μM). The increase in GS activity produced by glutamate was exhibited by the non-selective NMDA receptor antagonist, DNQX, but not by the metabotropic receptor antagonist, L-AP3 or a selective NMDA receptor antagonist, D-AP5. We also found that in cultures treated with glutamate, a number of astrocytes resembled “reactive astrocytes” morphologically. These astrocytes were absent in cultures treated with glutamate + DNQX. The findings provide supportive evidence that astrocytes from aged mouse cerebral hemispheres respond to excitatory amino acids and that this response is mediated by non-NMDA receptor activation.     

Enhancement of NMDA receptor-mediated neurotoxicity in the hippocampal slice by depolarization and ischemia

Evidence from animal stroke models suggests that the proximate cause of neuronal degeneration after ischemia is massive release of glutamate and activation of NMDA receptors. However, in the physiologic presence of oxygen and glucose in the rat hippocampal slice preparation, the neurotoxicity of glutamate, as measured by inhibition of protein synthesis, requires high concentrations and is not prevented by glutamate receptor antagonists. Thus, the NMDA receptor-mediated neurotoxic effects of extracellular glutamate accumulation during ischemia might depend on additional factors, such as neuronal depolarization. In the experiments reported here, slices were exposed to glutamate in a medium intended to mimic the ionic conditions found during ischemia, high potassium (128 mM) and low sodium (26 mM). This depolarizing medium itself inhibited protein synthesis in a manner which was partially mediated by NMDA receptor activation, since it was significantly reversed by the noncompetitive NMDA antagonist, MK-801. Furthermore, the effect of glutamate under depolarizing conditions was also significantly decreased by MK-801, suggesting that glutamate was acting at NMDA receptors. Thus, depolarization appears to enhance the sensitivity of neurons to toxic NMDA receptor activation by glutamate. Under conditions that mimic ischemia, hypoxia plus hypoglycemia, a similar protective effect of NMDA receptor antagonists was observed. Depolarization and ischemia both appeared to attenuate the neurotoxicity of non-NMDA receptor agonists. It appears that under conditions of normal glucose and oxygen, high concentrations of bath applied glutamate inhibit protein synthesis at sites other than the NMDA receptor. However, when the Na+ gradient is decreased, as occurs during ischemia, glutamate's NMDA effects predominate. These findings suggest that ionic shifts may play a central role in permitting NMDA receptor-mediated ischemic neuronal damage.     

Glutamate, NMDA and NMDA receptor antagonists: cardiovascular effects of intrathecal administration in the rat

Selected excitatory amino acids and antagonists were tested for their effects on arterial pressure and heart rate when administered intrathecally at the second (T2) or ninth (T9) thoracic spinal levels in urethane-anesthetized Sprague-Dawley rats with spontaneous or artificial respiration. Intrathecal administration of glutamate (1 mumol) and N-methyl-D-aspartic acid (NMDA; 2 nmol) at T9 increased arterial pressure and heart rate. The response began within 1 min, peaked at 2-3 min and persisted for 8-15 min. The maximum changes were 20-25 mm Hg for arterial pressure and 40-50 beats/min for heart rate. These responses were prevented by systemic administration of hexamethonium (10 mg/kg). Responses to administration of NMDA at the two spinal levels were essentially the same. Effects elicited by NMDA but not by glutamate were blocked by pretreatment with the NMDA receptor antagonists, D,L-2-amino-5-phosphonovaleric acid (APV; 10 nmol, intrathecal administration) and ketamine (7 mg/kg, i.v.). Intrathecal administration of APV (10, 50 and 200 nmol) at T2 produced dose-dependent decreases in arterial pressure without changing heart rate. The results support the hypothesis that NMDA receptors are involved in regulation of sympathetic output at the spinal level. They also indicate that in this preparation there is a tonic activation of NMDA receptors in sympathetic pathways to the vessels but not to the heart. Finally, the persistence of the response to glutamate in the presence of NMDA receptor antagonists suggests the involvement of non-NMDA receptors in spinal control of sympathetic output.     

NMDA receptor activation induces glutamate release through nitric oxide synthesis in guinea pig dentate gyrus

We tested the hypothesis that the release of glutamate following activation of N-methyl-d-aspartate (NMDA) receptors is mediated by nitric oxide (NO) production, using slices of the guinea pig hippocampus. The NMDA-induced glutamate release from slices of dentate gyrus or CA1, which was both concentration-dependent and Ca²⁺-dependent, was also Mg²⁺-sensitive and abolished by MK-801, a selective non-competitive NMDA receptor antagonist. In dentate gyrus, the NMDA-induced glutamate release was inhibited non-significantly by tetrodotoxin, whereas the NO synthase (NOS) inhibitor N^G-nitro-l-arginine (l-NNA) blocked the NMDA-induced release of glutamate in a concentration-dependent manner, but not a high K⁺-evoked release of glutamate. In addition, the l-NNA blockade of NMDA-induced release of glutamate was recovered by pretreatment with l-arginine, the normal substrate for NOS. These results suggest that activation of NMDA receptors in dentate gyrus, as well as subsequent Ca²⁺ fluxes, is required for the neuronal glutamate release mediated by NO production. On the other hand, the NMDA-evoked glutamate release from CA1 region was tetrodotoxin-sensitive and was not inhibited by l-NNA, thereby suggesting that activation of NMDA receptors in CA1 results in increased glutamate release in an NO-independent manner. Taken together, the NMDA receptor-mediated neuronal release of glutamate from the guinea pig dentate gyrus likely involves the recruitment of NOS activity.     

Parasynaptic NMDA receptor signaling couples neuronal glutamate transporter function to AMPA receptor synaptic distribution and stability

At synapses, two major processes occur concomitantly after the release of glutamate: activation of AMPA receptors (AMPARs) to conduct synaptic transmission and activation of excitatory amino acid transporters (EAATs) for transmitter removal. Although crosstalk between the receptors and EAATs is conceivable, whether and how the transporter activity affects AMPAR synaptic localization remain unknown. Using cultured hippocampal and cortical rat neurons, we show that inhibition of glutamate transporters leads to rapid reduction in AMPAR synaptic accumulation and total AMPAR abundance. EAAT inactivity also results in elevated internalization and reduced surface expression of AMPARs. The reduction in AMPAR amount is accompanied by receptor ubiquitination and can be blocked by suppression of proteasome activity, indicating the involvement of proteasome-mediated receptor degradation. Consistent with glutamate spillover, effect of EAAT inhibition on AMPAR distribution and stability is dependent on the activation of parasynaptically localized NR2B-containing NMDA receptors (NMDARs). Moreover, we show that neuronal glutamate transporters, especially those localized at the postsynaptic sites, are responsible for the observed effect during EAAT suppression. These results indicate a role for neuron-specific glutamate transporters in AMPAR synaptic localization and stability.     

BDNF regulates NMDA receptor activity in developing retinal ganglion cells

Brain-derived neurotrophic factor (BDNF) modulates glutamate receptors of the NMDA type in many areas of the brain. We assessed whether BDNF exerts an effect on NMDA receptor properties in retinal ganglion cells during early postnatal development. Electrophysiological responses to the glutamate agonist NMDA (500 microM-2 mM) in retinal slices of wildtype and BDNF deficient mice (bdnf-/-) were recorded using the whole-cell patch-clamp technique. Retinal ganglion cells of bdnf-/- mice displayed significantly smaller NMDA currents than those of age-matched wildtype mice. Remarkably, NMDA receptor activity was restored by incubating retinal slices of bdnf-/- mice in BDNF (50 ng/ml) for 1-3 h. We suggest that BDNF plays a role in the activation of functional NMDA receptors in early ganglion cell development.     

Manganese inhibits NMDA receptor channel function: implications to psychiatric and cognitive effects

Humans exposed to excess levels of manganese (Mn(2+)) express psychiatric problems and deficits in attention and learning and memory. However, there is a paucity of knowledge on molecular mechanisms by which Mn(2+) produces such effects. We now report that Mn(2+) is a potent inhibitor of [(3)H]-MK-801 binding to the NMDA receptor channel in rat neuronal membrane preparations. The inhibition of [(3)H]-MK-801 to the NMDA receptor channel by Mn(2+) was activity-dependent since Mn(2+) was a more potent inhibitor in the presence of the NMDA receptor co-agonists glutamate and glycine (K(i)=35.9+/-3.1 microM) than in their absence (K(i)=157.1+/-6.5 microM). We also show that Mn(2+) is a NMDA receptor channel blocker since its inhibition of [(3)H]-MK-801 binding to the NMDA receptor channel is competitive in nature. That is, Mn(2+) significantly increased the affinity constant (K(d)) with no significant effect on the maximal number of [(3)H]-MK-801 binding sites (B(max)). Under stimulating conditions, Mn(2+) was equipotent in inhibiting [(3)H]-MK-801 binding to NMDA receptors expressed in neuronal membrane preparations from different brain regions. However, under basal, non-stimulated conditions, Mn(2+) was more potent in inhibiting NMDA receptors in the cerebellum than other brain regions. We have previously shown that chronic Mn(2+) exposure in non-human primates increases Cu(2+), but not zinc or iron concentrations in the basal ganglia [Guilarte TR, Chen M-K, McGlothan JL, Verina T, Wong DF, Zhou Y, Alexander M, Rohde CA, Syversen T, Decamp E, Koser AJ, Fritz S, Gonczi H, Anderson DW, Schneider JS. Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates. Exp Neurol 2006a;202:381-90]. Therefore, we also tested the inhibitory effects of Cu(2+) on [(3)H]-MK-801 binding to the NMDA receptor channel. The data shows that Cu(2+) in the presence of glutamate and glycine is a more potent inhibitor of the NMDA receptor than Mn(2+). Our findings suggest that the inhibitory effect of Mn(2+) and/or Cu(2+) on the NMDA receptor may produce a deficit in glutamatergic transmission in the brain of individuals exposed to excess levels of Mn(2+) and produce neurological dysfunction.     

Activation of NMDA receptor of glutamate influences MMP-2 activity and proliferation of glioma cells

Glioblastoma multiforme (GBM) is the most common malignant glioma, which has high proliferative rate and an extremely invasive phenotype. Major limitations in the effective treatment of malignant gliomas are the proliferation and infiltration into the surrounding brain tissue. Although studies have shown that various stimuli promote glioma cell proliferation and invasion, the underlying mechanisms remain largely unknown. Glioma cells secrete significant amount of glutamate into surrounding tissue and intracellular signaling is thought to be initiated upon glutamate-induced modulation of the ion channels in GBM cells. The objective of the study was to investigate the effect of activation of NMDA (N-methyl-d-aspartate) receptors of glutamate on gelatinase subfamily MMPs and on proliferation of glioma cells. U251MG and U87MG cell lines were maintained in Dulbecco’s Modified Eagle’s Medium. Proliferation assay was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was investigated by gelatin zymography assay. We demonstrate that activated NMDA receptors (NMDAR) increased the activity of MMP-2 only in U251MG glioma cells at concentrations of 100 and 200 μM and increased the proliferation of both U87MG and U251MG glioma cells at concentrations of 50, 100, 150 and 200 μM. Inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly inhibited the effect of activation of NMDAR on MMP-2 activity and on proliferation. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells.     

Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, l-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.     

Ionotropic glutamate receptor modulation preferentially affects NMDA receptor expression in rat hippocampus

Electrophysiological data suggest that alterations in the function of one glutamate receptor subtype may affect the function of other subtypes. Further, previous studies have demonstrated that NMDA receptor antagonists affect NMDA and kainate receptor expression in rat hippocampus. In order to address the mutual regulation of NMDA, AMPA, and kainate receptor expression in rat hippocampus, we conducted two experiments examining the effects of NMDA and non-NMDA glutamate receptor modulators on NMDA, AMPA, and kainate receptor expression using in situ hybridization and receptor autoradiography. NMDA receptor expression was preferentially affected by systemic treatments, as all drugs significantly altered [(3)H]MK-801 binding, and several drugs increased [(3)H]ifenprodil binding. GYKI52466 and aniracetam treatments resulted in changes in both [(3)H]ifenprodil binding and NR2B mRNA levels, consistent with the association of this subunit and binding site in vitro. There were more modest effects on AMPA and kainate receptor expression, even by direct antagonists. Together, these data suggest that ionotropic glutamate receptors interact at the level of expression. These data also suggest that drug regimens targeting one ionotropic glutamate receptor subtype may indirectly affect other subtypes, potentially producing unwanted side effects.     

Evidence for a striatal NMDA receptor modulation of nigral glutamate release. A dual probe microdialysis study in the awake freely moving rat

Dual probe microdialysis was employed to characterize dialysate glutamate levels from the substantia nigra pars reticulata of awake freely moving rats, and to test its sensitivity to alterations in striatal neurotransmission including striatal N-methyl-d -aspartic acid (NMDA) receptor stimulation and blockade. Intranigral perfusion with low (0.1 mm ) Ca²⁺ medium (60 min) did not affect nigral glutamate levels, whereas intranigral perfusion with tetrodotoxin (10 μm , 60 min) increased nigral glutamate levels. Perfusion of KCl (100 mm , 10 min) in the dorsolateral striatum transiently stimulated nigral glutamate levels (maximal increase + 60%), whereas intrastriatal perfusion (60 min) with low Ca²⁺ medium and tetrodotoxin gradually increased nigral glutamate levels. Intrastriatal perfusion with NMDA (0.1–100 μm , 10 min) dose-dependently stimulated glutamate levels in the substantia nigra pars reticulata. The NMDA (1 μm )-induced increase in nigral glutamate release was transient and maximal (+60% within 20 min), whereas that for NMDA (10 μm ) had a slow onset but was long lasting (+35% after 60 min). Lower (0.1 μm ) and higher (100 μm ) NMDA concentrations were ineffective. The effect of intrastriatal NMDA (1 μm ) was prevented by coperfusion with MK-801 (1 μm ). Intrastriatal MK-801 (10 μm ) alone gradually increased glutamate levels up to +50% after 60 min of perfusion. The present results suggest that glutamate levels in the substantia nigra pars reticulata are sensitive to changes in neuronal transmission in the dorsolateral striatum, and that striatal NMDA receptors regulate nigral glutamate release in both a tonic and phasic fashion.     

Mechanism of NMDA receptor contribution to axon-to-glia signaling in the crayfish medial giant nerve fiber

Electrical stimulation of crayfish giant axons at high frequency activates group II metabotropic and NMDA glutamate receptors on adjacent glial cells via release of N-acetylaspartylglutamate and glutamate formed upon its hydrolysis. This produces a transient depolarization followed by a prolonged hyperpolarization of glial cells that involves nicotinic acetylcholine receptor activation. The hyperpolarization is nearly completely blocked by antagonists of metabotropic glutamate receptors but only slightly reduced by inhibition of NMDA receptors. We report that the NMDA-induced hyperpolarization of glial cells is reduced by decreased calcium in the solution bathing the giant nerve fiber, while removal of sodium ions or block of voltage-dependent calcium channels completely prevents the glial response to NMDA. Inhibition of nicotinic acetylcholine receptors or removal of extracellular Cl(-) converts the glial response from a hyperpolarization to a depolarization that is sensitive to NMDA receptor antagonist. We propose that NMDA receptor activation by glutamate, formed from extracellular N-acetylaspartylglutamate during nerve stimulation, contributes to glial hyperpolarization by increasing intracellular Ca(2+) via opening of voltage-sensitive Ca(2+) channels. Based on our previous work, we propose further that the added Ca(2+) supplements that produced by N-acetylaspartylglutamate and glutamate acting on group II metabotropic glutamate receptors to cause an increased release of acetylcholine and a larger hyperpolarization.     

Decreased N-methyl-D-aspartic acid (NMDA) receptor levels are associated with mercury exposure in wild and captive mink

Mercury (Hg) impairs glutamate homeostasis but little is known about its effects on the N-methyl-d-aspartic acid (NMDA) receptor. Here, we investigated NMDA receptor levels, as determined by [(3)H]-MK801 binding, in both wild and captive mink (Mustela vison) that experienced different levels of methylmercury (MeHg) exposure. Competitive in vitro binding experiments showed that inorganic Hg (HgCl(2); IC(50)=1.5-20.7 microM), but not MeHg (MeHgCl; IC(50)>320 microM), inhibited binding to the NMDA receptor in several brain regions of mink. In a survey of trapped wild mink, NMDA receptor levels in the brain were negatively correlated (p<0.005) with concentrations of total Hg (R=-0.618) and MeHg (R=-0.714). These findings were supported by a laboratory feeding study in which captive mink were exposed to dietary MeHg (0-2 ppm) for 89 days. Concentration-dependent decreases in NMDA receptor levels were found in the basal ganglia, cerebellum, brain stem and occipital cortex. These findings are of physiological and ecological concern because they demonstrate that Hg, at dietary concentrations as low as 0.1 ppm, can significantly reduce NMDA receptor levels.     

Subunit selective decrease of AMPA and metabotropic glutamate receptor mRNA expression in rat brain by systemic administration of the NMDA receptor blocker MK-801

Glutamate mediates its effects in mammals through both ionotropic and metabotropic receptors. Antagonists of ionotropic N-methyl-d-aspartate (NMDA) glutamate receptors elicit neuroprotective and neurotropic effects that have been attributed to Ca²⁺ block through the membrane ion channel. Nonetheless, molecular and biochemical effects of NMDA receptor antagonism on other glutamate receptor subunits remain poorly understood. We investigated the effects of acute administration of the noncompetitive NMDA receptor antagonist MK-801 on the mRNA expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate receptor (mGluR) subunits to determine the contribution of different glutamate receptors in response to blockade of NMDA receptor channels. In situ hybridization to rat brain sections revealed that AMPA receptor subunits GluR3 and GluR4, and mGluR3 were modestly but significantly decreased ∼10–20%, 8 h following 5 mg/kg MK-801 administration. A time course and dose response study revealed that the effect on mGluR3 was reversed by 24 h and occurred significantly at a dose range from 1 to 5 mg/kg. These results indicate that selected AMPA and mGluR subunit mRNAs respond at the RNA level to the blockade of NMDA receptors.     

NMDA receptor mediates dopamine release in the striatum of unanesthetized rats as measured by brain microdialysis.

We have studied the characteristics associated with the activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor on the release of dopamine (DA) in the striatum of awake rats as measured by brain microdialysis technique. NMDA dose-dependently stimulated the striatal DA release in Mg(2+)-free Ringer's solution. The stimulation was significant at 90 microM and the maximum observed effect was at the highest concentration tested (800 microM). The selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP5; 300 microM), blocked the stimulatory effect of NMDA. The NMDA-induced release of DA was reduced by 1.2 mM Mg2+ and totally blocked by 2.5 mM of the cation. Glycine (200 microM) potentiated the response evoked by 300 microM NMDA while 7-chloro-kynurenate (100 microM), an antagonist of the glycine site, reduced markedly this response. Neither atropine (100 microM) nor tetrodotoxin (TTX) (5 microM) prevented the stimulatory effect of NMDA. These results suggest that glutamate released from corticostriatal terminals presynaptically stimulates the release of DA via an NMDA receptor.     

Desensitization of the NMDA receptor complex by glycinergic ligands in cerebellar granule cell cultures

Glutamate neurotoxicity was examined in cultured cerebellar granule neurons following both prolonged (20–24 h) and brief (45 min) exposure to compounds acting at strychnine-insensitive glycine receptors. Glutamate neurotoxicity was reduced in a concentration-dependent fashion by brief exposure to the glycine partial agonists 1-aminocyclopropanecar☐ylic acid (ACPC) and(±)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) and the competitive antagonist, 7-chlorokynurenic acid (7-CK) with a rank order efficacy: 7-CK > HA-966 > ACPC. Neitherd-cycloserine (d-CS) nor glycine affected neurotoxicity produced by maximum glutamate concentrations, while glycine but notd-CS augmented the effects of submaximum glutamate concentrations. Prolonged exposure of cultures to either full (glycine) or partial agonists (ACPC,d-CS, HA-966) abolished the neuroprotective effects of ACPC and significantly diminished the neuroprotective effects of HA-966. In contrast, the neuroprotective effects of 7-CK were only marginally reduced by prolonged exposure to glycinergic ligands, while the neuroprotection afforded by compounds acting at other loci on the NMDA receptor complex (e.g. 2-amino-5-phosphonopentanoate (APV) and dizocilpine (MK-801)) were unaltered. These effects may represent homologous desensitization of the NMDA receptor complex at its strychnine-insensitive glycine receptor induced by prolonged exposure to glycinergic agonists and partial agonists. Nonetheless, levels of the NMDA receptor subunit ζ1 mRNA were unaffected by prolonged exposure to ACPC, indicating the apparent desensitization could involve a post-translational modification of the NMDA receptor complex.     

The NMDA receptor gating machine: lessons from single channels.

The slow component of the excitatory postsynaptic current in the central nervous system is generated by the activity of NMDA receptors. The activation properties of this class of glutamate receptor determine key features of the synaptic response and have important consequences for synaptic plasticity and cell physiology. NMDA receptor activation is complex and involves ligand binding, protein conformational changes, and channel blockade. Recently, two groups have proposed state models that encapsulate the essential features of NMDA receptor gating conformational changes. These models provide insight into the NMDA receptor as a molecular machine and should help us understand and manipulate NMDA receptor mediated synaptic function and pathologies.     

Testing NMDA receptor block as a therapeutic strategy for reducing ischaemic damage to CNS white matter

Damage to oligodendrocytes caused by glutamate release contributes to mental or physical handicap in periventricular leukomalacia, spinal cord injury, multiple sclerosis, and stroke, and has been attributed to activation of AMPA/kainate receptors. However, glutamate also activates unusual NMDA receptors in oligodendrocytes, which can generate an ion influx even at the resting potential in a physiological [Mg2+]. Here, we show that the clinically licensed NMDA receptor antagonist memantine blocks oligodendrocyte NMDA receptors at concentrations achieved therapeutically. Simulated ischaemia released glutamate which activated NMDA receptors, as well as AMPA/kainate receptors, on mature and precursor oligodendrocytes. Although blocking AMPA/kainate receptors alone during ischaemia had no effect, combining memantine with an AMPA/kainate receptor blocker, or applying the NMDA blocker MK-801 alone, improved recovery of the action potential in myelinated axons after the ischaemia. These data suggest NMDA receptor blockers as a potentially useful treatment for some white matter diseases and define conditions under which these blockers may be useful therapeutically. Our results highlight the importance of developing new antagonists selective for oligodendrocyte NMDA receptors based on their difference in subunit structure from most neuronal NMDA receptors.