N-methyl-D-aspartate Receptor-mediated Neurotoxicity Research Articles

E2F1 in neurons is cleaved by calpain in an NMDA receptor-dependent manner in a model of HIV-induced neurotoxicity.

The transcription factor E2F1 activates gene targets required for G1 -S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson's Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in N-methyl-d-aspartate (NMDA) receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. In addition, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in post-mitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HIV-associated neurocognitive disorders pathogenesis as well as several other diseases of the CNS. After crossing the blood-brain barrier, HIV-infected monocytes differentiate into macrophages and release excitotoxins and inflammatory factors including glutamate into the brain parenchyma (1). These factors stimulate neuronal N-Methyl-d-aspartate (NMDA) receptors (2), causing calcium influx (3) and subsequent activation of the cysteine protease calpain (4). Activated calpain cleaves multiple substrates including E2F1, producing a stabilized protein fragment with truncations at the N- and C-terminus (5). Calpain-cleaved E2F1 may contribute to calpain-mediated neuronal damage observed in NMDA receptor-mediated neurotoxicity (6).

Cellular origin and regulation of D- and L-serine in in vitro and in vivo models of cerebral ischemia.

D-Serine is known to be essential for the activation of the N-methyl-D-aspartate (NMDA) receptor in the excitation of glutamatergic neurons, which have critical roles in long-term potentiation and memory formation. D-Serine is also thought to be involved in NMDA receptor-mediated neurotoxicity. The deletion of serine racemase (SRR), which synthesizes D-serine from L-serine, was recently reported to improve ischemic damage in mouse middle cerebral artery occlusion model. However, the cell type in which this phenomenon originates and the regulatory mechanism for D-/L-serine remain elusive. The D-/L-serine content in ischemic brain increased until 20 hours after recanalization and then leveled off gradually. The results of in vitro experiments using cultured cells suggested that D-serine is derived from neurons, while L-serine seems to be released from astroglia. Immunohistochemistry studies of brain tissue after cerebral ischemia showed that SRR is expressed in neurons, and 3-phosphoglycerate dehydrogenase (3-PGDH), which synthesizes L-serine from 3-phosphoglycerate, is located in astrocytes, supporting the results of the in vitro experiments. A western blot analysis showed that neither SRR nor 3-PGDH was upregulated after cerebral ischemia. Therefore, the increase in D-/L-serine was not related to an increase in SRR or 3-PGDH, but to an increase in the substrates of SRR and 3-PGDH.

Toward Safer Thrombolytic Agents in Stroke: Molecular Requirements for NMDA Receptor-Mediated Neurotoxicity

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (K(D)=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.

Angiotensin II attenuates NMDA receptor-mediated neuronal cell death and prevents the associated reduction in Bcl-2 expression

While angiotensin II (Ang II) plays a major role in the regulation of blood pressure, fluid homeostasis and neuroendocrine function, recent studies have also implicated the peptide hormone in cell growth, differentiation and apoptosis. In support of this, we have previously demonstrated that Ang II attenuates N-methyl- d-aspartate (NMDA) receptor signaling [Molec. Brain Res. 48 (1997) 197]. To further examine the modulatory role of Ang II on NMDA receptor function, we investigated the effect of angiotensin receptor (AT) activation on NMDA-mediated cell death and the accompanying decrease in Bcl-2 expression. The viability of differentiated N1E-115 and NG108-15 neuronal cell lines was reduced following exposure to NMDA in a dose-dependent manner. MTT analysis (mitochondrial integrity) revealed a decrease in cell survival of 49.4±12.3% in NG108 cells and 79.9±6.8% in N1E cells following treatment with 10 mM NMDA for 20 h. Cytotoxicity in N1E cells was inhibited by the noncompetitive NMDA receptor antagonist, MK-801. Further, NMDA receptor-mediated cell death in NG108 cells was attenuated by treatment with Ang II. The Ang II effect was inhibited by both AT 1 and AT 2 receptor antagonists, losartan and PD123319, respectively, suggesting that both receptor subtypes may play a role in the survival effect of Ang II. Since it has been shown that activation of NMDA receptors alters the expression of Bcl-2 family proteins, Western blot analysis was performed in N1E cells to determine whether Ang II alters the NMDA-induced changes in Bcl-2 expression. A concentration-dependent decrease of intracellular Bcl-2 protein levels was observed following treatment with NMDA, and this reduction was inhibited by MK801. Addition of Ang II suppressed the NMDA receptor-mediated reduction in Bcl-2. The Ang II effect on NMDA-mediated changes in Bcl-2 levels was blocked by PD123319, but was not significantly changed by losartan, suggesting AT 2 receptor specificity. Taken together, these results suggest that Ang II attenuates NMDA receptor-mediated neurotoxicity and that this effect may be due, in part, to an alteration in Bcl-2 expression.

Chronic treatment with glutaric acid induces partial tolerance to excitotoxicity in neuronal cultures from chick embryo telencephalons.

Glutaryl-CoA dehydrogenase deficiency (GDD) is characterized biochemically by an accumulation of glutaric (GA) and 3-hydroxyglutaric (3-OH-GA) acids and clinically by the development of acute striatal degeneration. 3-OH-GA was recently shown to induce neuronal damage via N-methyl-D-aspartate (NMDA) receptors. The pathogenetic role of GA, however, remains unclear. We demonstrate that GA exerts a dual action in cultured chick embryo neurons. Short-term incubation with millimolar concentrations of GA induces a weak neuronal damage, adding to 3-OH-GA neurotoxicity. In contrast, chronic treatment with subtoxic, micromolar concentrations of GA results in partial tolerance to 3-OH-GA- and NMDA-induced cell damage. A downregulation of NMDA receptors, in particular of the NR2B subunit, is critically involved in this GA-induced effect, resulting in a reduced Ca(2+) increase and generation of reactive oxygen species after acute exposure to NMDA or 3-OH-GA. Furthermore, GA decreases Na(+)/K(+)-ATPase activity, which is prevented by glutathione, suggesting a modulation of NMDA receptor function via resting membrane potential and Na(+)-dependent glutamate transport. In contrast, GA does not inhibit mitochondrial respiratory chain and beta-oxidation of fatty acids, virtually excluding an activation of NMDA receptors secondary to ATP depletion. These results strongly suggest that GA modulates the NMDA receptor-mediated neurotoxicity of 3-OH-GA, providing an explanatory basis for the non-linear relationship between organic acid concentrations and disease progression in GDD patients. Furthermore, GA-induced downregulation of NMDA receptors might be involved in the delayed cerebral maturation of GDD patients, resulting in frontotemporal atrophy and a reduced opercularization, which are common neuroradiological findings in GDD patients.

Chapter 3 Ibogaine as a glutamate antagonist: Relevance to its putative antiaddictive properties

The chapter presents a discussion on ibogaine as a glutamate antagonist. The chapter presents an overview data demonstrating that pharmacologically relevant concentrations of ibogaine produce a blockade of N methyl-D-aspartate (NMDA) receptors and relates the relevance of these findings to its antiaddictive properties. If the neurochemical and electrophysiological studies with ibogaine are pharmacologically meaningful, then like other NMDA antagonists, ibogaine should protect against NMDA receptor-mediated neurotoxicity. The chapter presents in vitro data that provide compelling evidence on ibogaine acting as an NMDA antagonist. Glycine is a coagonist at NMDA receptors. Because of the presence of specific transporters that appear colocalized with NMDA receptors, it is unlikely that these strychnine-insensitive glycine sites are saturated under physiological conditions. Like other NMDA antagonists, memantine (a lowaffinity, uncompetitive NMDA antagonist) blocks the expression of morphine withdrawal in mice.

Sustained exposure to a glycine receptor partial agonist differentially alters NMDA receptor agonist and antagonist potencies in cultured spinal cord neurons

Sustained (20 h) exposure to the glycine partial agonist 1-aminocyclopropanecarboxylic acid (ACPC) significantly reduced N-methyl- d-aspartate (NMDA)-induced neurotoxicity in cultured spinal cord neurons when the NMDA (25 and 100 μM) was added to the cultures 30 min after removal of the ACPC (1 mM). In contrast, ACPC preexposure failed to protect against kainate-induced neuronal injury. The magnitude of neuronal protection against NMDA (100 μM) was further enhanced if the neurons pretreated with ACPC were reexposed to this drug during the NMDA challenge. In addition, the potencies of both the competitive NMDA antagonist AP5 and the noncompetitive antagonist dizocilpine to block NMDA toxicity were significantly increased following ACPC preexposure, while the potency of the competitive glycine receptor antagonist 7-chlorokynurenate (7-CK) was unchanged. Analysis of Northern blots suggest that ACPC-induced changes in NMDA receptor function were not associated with alterations in the levels of the mRNAs encoding the NMDAR-1, -2A, -2B, or -2C subunits. These results indicate that sustained exposure to ACPC modifies NMDA receptors in a manner that diminishes NMDA receptor-mediated neurotoxicity while selectively enhancing the potencies of several NMDA receptor antagonists. These effects do not appear to be related to changes in expression of specific NMDA receptor subunits, and may instead involve a post-translational modification of one or more subunit proteins.

Aging and the effects of MK-801 on anoxic damage in rat hippocampal slices

We examined whether age-related differences in N-methyl- d-aspartate (NMDA) receptor-mediated neurotoxicity contribute to the increased vulnerability of the aged brain to anoxic damage. In both adult and aged hippocampal slices, NMDA receptor blockade with MK-801 did not affect the onset of anoxic depolarization. MK-801 improved the postanoxic recovery of synaptic transmission by the same percentage in both age groups. Thus, the faster onset of anoxic depolarization and diminished postanoxic recovery of synaptic transmission seen in aged hippocampal slices cannot be attributed to age-related differences in NMDA receptor-mediated neurotoxicity.

Roles of neuroactive amino acids in ammonia neurotoxicity.

Many neurologic disorders are related to congenital or acquired hyperammonemia (HA). Advanced symptoms of HA range from seizures in acute stages to stupor and coma in more chronic conditions, manifesting variable imbalance between the inhibitory and excitatory neurotransmission. Evidence obtained with the use of experimental HA models suggests that acute neurotoxic effects of ammonia are mediated by overactivation of ionotropic glutamate (GLU) receptors, mainly the N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree the KA/AMPA receptors. NMDA receptor-mediated neurotoxicity may be potentiated by impaired control of their function by metabotropic GLU receptors, which are inactivated by ammonia. Prolonged overactivation of the NMDA receptors upon extended ammonia exposure causes their downregulation. The GLU receptor changes may be related to their excessive exposure to extrasynaptic GLU. Ammonia promotes GLU accumulation in the extrasynaptic space by enhancing its release from neurons, and/or by decreasing its reuptake to the nerve endings and astrocytes, where the effect results from inactivation (downregulation) of the astrocytic glutamate transporter GLT1. Excitotoxic effects of ammonia are augmented by increased synthesis of nitric oxide (NO), which is associated with NMDA receptor activation and/or increased synaptic transport of arginine (ARG). A shift toward neural inhibition is promoted by positive modulation of the gamma-aminobutyric acid (GABA)ergic tone resulting from excessive accumulation in the brain of endogenous central benzodiazepine receptor agonists, and from upregulation of astrocytic peripheral benzodiazepine receptors leading to elevated levels of prognenelone-derived neurosteroids, which positively modulate the GABA(A) receptor complex. Inhibitory neurotransmission may also be favored by enhanced release from astrocytes of an inhibitory amino acid, taurine.

EFFECT OF HYPERVENTILATION ON THE SPERMINE AND MAGNESIUM SITES OF THE N-METHYL-D-ASPARTATE RECEPTOR IN THE BRAINS OF NEWBORN PIGLETS. †272

The present study tests the hypothesis that cerebral ischemia induced by severe hypocapnia results in tissue hypoxia and modifies the N-methyl-D-aspartate (NMDA) receptor/ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in a control(normoxic) group (n=4) and hypocapnic (brain tissue hypoxic) group (n=4) of anesthetized and ventilated newborn piglets. Hypocapnia was induced by hyperventilation (PaCO2 = 9-11 mmHg) for 1 hr. Brain tissue hypoxia was confirmed biochemically in the hypocapnic (ischemic) group by an 80% decrease in [phosphocreatinine]. 3[H]MK-801 binding studies, an index of NMDA receptor activation, was performed at 32°C for 3 hours in an assay medium containing 20mM HEPES (pH 7.0), 75μg protein, glycine (100μM) and glutamate (100μM). Nonspecific binding was measured in the presence of 10μM unlabeled MK-801. Spermine-dependent activation was determined by adding increasing [spermine] from 2.5 to 50μM. Mg++ dependent binding was assayed separately in the presence of [Mg2+] from 2.5 to 100 μM. Maximal spermine-dependent activation in the control and hypocapnic groups was 19.8 ± 4.3% (at 5μM) and 47.6 ± 23.7% (at 6.7μM) above baseline, respectively. Maximal 3[H]MK-801 binding in the control and hypocapnic groups occurred at a [Mg2+] of 25μM and 5.8μM, respectively (p < 0.001). The data show that hypocapnic-induced tissue hypoxia results in increased spermine-dependent maximal activation and increased sensitivity to Mg++, indicating modification of the polyamine and Mg++ sites of the NMDA receptor. We conclude that hypocapnia-induced modification of the spermine and magnesium sites leads to increased activation of the NMDA receptor, resulting in increased NMDA receptor-mediated neurotoxicity in the newborn brain. (NIH-HD20337, MOD-6FY940135, UCP-R50693).

NMDA receptor-mediated excitoprotection of cultured cerebellar granule neurons fails to alter glutamate-induced expression of c- fos and c- jun mRNAs

Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N- methyl- d-aspartate (NMDA) induces a state of excitoprotection when measured by subsequent exposure to toxic concentrations of glutamate. This NMDA-induced excitoprotective state is prevented by inhibitors of new RNA and protein synthesis. Since the neurotrophic and excitoprotective effects of NMDA in cerebellar granule neurons may involve changes in the expression of the immediate early genes c- fos and c- jun, we measured c- fos and c- jun mRNAs in cerebellar granule neurons after exposure to either toxic concentrations of glutamate or excitoprotective (subtoxic) concentrations of NMDA. Exposure of cerebellar granule neurons to toxic concentrations of glutamate induced a dramatic increase in c- fos and c- jun mRNAs which was not associated with a corresponding increase in c- fos and c- jun proteins as measured immunocytochemically. However, the increase in c- fos and c- jun mRNAs induced by toxic concentrations of glutamate was not altered by preexposing cerebellar granule neurons to NMDA, suggesting that increased expression of c- fos and c- jun mRNAs is not sufficient for glutamate toxicity of these neurons. Preexposure of cerebellar granule neurons to NMDA for 24 h, which induced a maximal excitoprotective state, resulted in a transient increase in c- fos, and to a lesser degree c- jun, mRNAs similar to that induced by toxic concentrations of glutamate. The induction of c- fos, but not that of c- jun, mRNA both by excitoprotective concentrations of NMDA and by neurotoxic concentrations of glutamate was blocked by the non-competitive NMDA receptor antagonist, MK-801. These data show that c- fos and c- jun may play a role in NMDA receptor-mediated excitoprotection as well as in NMDA receptor-mediated neurotoxicity. NMDA receptor-mediated excitoprotection is likely to interfere with the intracellular cascade of glutamate toxicity by acting on an element downstream of c- fos and c- jun-dependent transcriptional regulation.

The non-psychotropic cannabinoid (+)-(3 S,4 S)-7-hydroxy- Δ6-tetrahydrocannabinol 1,1-dimethylheptyl (HU-211) attenuates [formula omitted] receptor-mediated neurotoxicity in primary cultures of rat forebrain

The non-psychotropic cannabinoid (+)-(3 S,4 S)-7-hydroxy- Δ 6-tetrahydrocannabinol 1,1-dimethylheptyl (HU-211), a stereoselective inhibitor of the N- methyl- d-aspartate (NMDA) receptor, protects primary cultures of rat forebrain against NMDA receptor-mediated neurotoxicity. Cell mortality produced by exposure for 10 min to NMDA or glutamate was reduced to approximately 18 or 27%, respectively, by application of 50 μM HU-211 for 10–15 min during or after exposure of cultures to excitatory amino acid. This effect of HU-211 was dependent on its concentration (EC 50 = 8.7 ± 4 μM). HU-211 also reduced the toxicity induced by brief exposure (10 min) to kainate or quisqualate, though less effectively. HU-211 may therefore prove useful as a non-psychoactive drug that protects against neurotoxicity mediated by the NMDA receptor.

Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl- d-aspartate neurotoxicity

We examined the role of nitric oxide in N-methyl- d-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures. In rat and mouse cultures, the NO synthase inhibitor, N G-Nitro- l-arginine, blocked cGMP formation but not neuronal cell death following a 5–10 min exposure to 300–500 μM NMDA. N G-Monomethyl- l-arginine was also unable to prevent neuronal death. In contrast, the non-competitive NMDA receptor antagonist, dextrophan, prevented both cGMP formation and cell death. While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required.

Ethanol inhibits NMDA receptor-mediated excitotoxicity in rat primary neuronal cultures.

Excessive or prolonged stimulation of N-methyl-D-aspartate (NMDA) receptors appears to play an important role in many neurodegenerative processes in brain through a process known as excitotoxicity. This study examined the effects of ethanol on NMDA receptor-mediated excitotoxicity in primary neuronal cultures obtained from embryonic rat whole brain. Neurotoxicity was quantitated by measuring the amount of lactate dehydrogenase released into the media during a 20-hr time period following NMDA washout. Exposure of 12- to 14-day-old cultures to NMDA in Mg(2+)-free HEPES buffer (pH 7.4) for a 25-min period resulted in a concentration-dependent toxicity (EC50 = 54 microM). Time-course experiments showed that exposure to NMDA for as little as 5 min was excitotoxic and reached a plateau after a 20-min exposure period. Preincubation of the cultures with ethanol (25 to 200 mM) resulted in a concentration-dependent inhibition of NMDA-mediated toxicity with approximately 38% inhibition produced by 25 mM ethanol and essentially complete inhibition at 200 mM ethanol (IC50 = 60 mM). Increasing the glycine concentration to 100 microM did not potentiate NMDA neurotoxicity or antagonize the neuroprotective effect of ethanol. NMDA-Mediated excitotoxicity was reduced by approximately 50% by the glycine antagonist 7-chlorokynurenate (50 microM). Ethanol (50 mM) reduced NMDA neurotoxicity similar to 7-chlorokynurenate, and the two together produced greater inhibition than either alone. These results show that intoxicating concentrations of ethanol can potently inhibit NMDA receptor-mediated excitotoxicity and may have important implications in terms of ethanols interactions with brain trauma, ischemia, and other neuropathologies associated with NMDA receptor-mediated neurotoxicity.

Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity

Excessive activation of NMDA receptors is thought to mediate the calcium-dependent neurotoxicity associated with hypoxic-ischemic brain injury, trauma, epilepsy, and several neurodegenerative diseases. For this reason, various NMDA antagonists have been investigated for their therapeutic potential in these diseases, but heretofore none have proven to be both effective and safe. In the present study, memantine, an adamantane derivative similar to the antiviral drug amantadine, is shown to block the channels activated by NMDA receptor stimulation. From whole-cell and single-channel recording experiments, the mechanism of action of memantine is deduced to be open-channel block, similar to MK-801; however, unlike MK-801, memantine is well tolerated clinically. Compared to MK-801, memantine's safety may be related to its faster kinetics of action with rapid blocking and unblocking rates at low micromolar concentrations. Furthermore, at these levels memantine is an uncompetitive antagonist and should theoretically allow near-normal physiological NMDA activity throughout the brain even in the face of pathologically high focal concentrations of glutamate. These pharmacological properties confer upon memantine a therapeutic advantage against NMDA receptor-mediated neurotoxicity with few side effects compared with other organic NMDA open-channel blockers. Moreover, memantine is increasingly effective against escalating levels of glutamate, such as those observed during a stroke. Low micromolar concentrations of memantine, levels known to be tolerated by patients receiving the drug for the treatment of Parkinson's disease, prevent NMDA receptor-mediated neurotoxicity in cultures of rat cortical and retinal ganglion cell neurons; memantine also appears to be both safe and effective in a rat stroke model. These results suggest that memantine has considerable therapeutic potential for the myriad of clinical entities associated with NMDA receptor-mediated neurotoxicity.

Effects of transforming growth factor-beta on murine astrocyte glutamine synthetase activity. Implications in neuronal injury.

Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In the present in vitro study, we investigated the effects of cytokines on astrocyte glutamine synthetase (GS) activity and subsequently on N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity. Proinflammatory cytokines IL-1 alpha, IL-1 beta, and IL-6 at a concentration of 20 ng/ml did not affect GS activity; however, tumor necrosis factor-alpha inhibited this activity by 20% in mixed neuronal/astrocyte cultures. Treatment for 24 h with transforming growth factor (TGF)-beta 1 or -beta 2 inhibited up to 60% GS activity. TGF-beta 2 also inhibited GS in enriched astrocyte cultures with an ED50 of 10 pg/ml. Antibodies specific to TGF-beta 2 blocked this effect. Treatment of astrocytes with TGF-beta 2 (250 pg/ml) resulted in markedly dilated rough endoplasmic reticulum. Since astrocyte GS may play a protective role in NMDA receptor-mediated neurotoxicity, we treated mixed neuronal/astrocyte cultures with TGF-beta 2 (250 pg/ml) and found a threefold potentiation of NMDA receptor-mediated neurotoxicity. These data suggest that TGF-beta impairs astrocyte GS function and enhances neurotoxicity, thus providing insight into understanding one mechanism of cytokine-mediated central nervous system disease.

Reduction of posttraumatic transneuronal "early gene" activation and dendritic atrophy by the N-methyl-D-aspartate receptor antagonist MK-801.

The removal of a major hippocampal afferent system, the glutamatergic fibers from the entorhinal cortex, results in transneuronal changes in postsynaptic inhibitory neurons using gamma-aminobutyric acid (GABA) as a neurotransmitter. This study shows that these transneuronal alterations are reduced by the selective N-methyl-D-aspartate (NMDA) receptor antagonist (+)-MK-801. Thus, systemic injection of (+)-MK-801 prior to and after entorhinal lesion abolishes the retraction of distal dendrites from the termination zones of degenerating entorhinal fibers and reduces the swelling of their distal segments. Also, entorhinal lesion results in the appearance of c-fos protein-like immunoreactivity in hippocampal neurons and glial cells, which again is blocked by (+)-MK-801 administration. These data suggest that NMDA receptor-mediated neurotoxicity due to postlesional glutamate elevation results in early gene expression and in transneuronal dendritic changes. Similar processes may play a role in Alzheimer's disease, since there is a severe degeneration of the glutamatergic entorhino-hippocampal projection in this neurodegenerative disorder.

Difluoromethyl ornithine protects against the neurotoxic effects of intrastriatally administered N-methyl-D-aspartate in vivo

The neurotoxic effects of intrastriatally administered N-methyl-D-aspartate (NMDA) (250 nmol), as measured by reductions in striatal choline acetyl transferase activity and by increased binding of the glial marker [ 3H]PK 11195 10 days later, were reduced by coinfusion of the irreversible ornithine decarboxylase inhibitor difluoroniethylornithine (250 nmol) in the rat. The data suggest a crucial role for the polyamines in NMDA receptor-mediated neurotoxicity.

Calcium channel antagonists attenuate NMDA receptor-mediated neurotoxicity of retinal ganglion cells in culture

Dihydropyridine calcium channel antagonists block a prolonged or ‘L-type’ component of voltage-dependent Ca 2+ current in patch-clamp recordings of postnatal rat retinal ganglion cells. In the present study on these neurons, calcium channel antagonists were found at 500–1000 nM concentrations to attenuate the early rise in [Ca 2+] i and the subsequent toxic effects of exogenous glutamate, N-methyl- d-aspartate (NMDA), or an endogenous glutamate-related compound present in the retinal cultures. Previous data have shown that the neurotoxicity engendered by these agents can also be prevented by selective NMDA antagonists. The present observation raise the possibility, at least in this preparation, that activation of both voltage-dependent calcium channels and NMDA receptor-operated channels contribute to the injurious effects triggered by molecules binding to the NMDA receptor.

GABAergic neocortical neurons are resistant to NMDA receptor-mediated injury.

N-methyl-D-aspartate (NMDA) receptors are thought to mediate much of the central neuronal loss produced by certain neurologic insults, including hypoxia-ischemia, hypoglycemia, and trauma. Therefore, the specific vulnerability of GABAergic inhibitory neurons to NMDA receptor-mediated toxicity might be an important determinant of the potential for epileptogenesis following these insults. We have examined the fate of GABAergic cortical neurons in mouse cell cultured neuronal population) were identified either by immunoreactivity with antisera to GABA or by autoradiography following high-affinity uptake of 3H-GABA. Cultures exposed for 5 min to 20 to 750 microM NMDA showed NMDA concentration-dependent, widespread neuronal loss. However, GABAergic neurons were relatively spared, and thus represented an enhanced fraction of neuronal survivors. These observations suggest that GABAergic cortical neurons may possess some intrinsic resistance to NMDA receptor-mediated neurotoxicity, a property which might convey an anticonvulsant "inhibitory safety factor" to neocortex against certain forms of injury.