Interactions between excitotoxins and the Na/K-ATPase inhibitor ouabain in causing neuronal lesions in the rat hippocampus

A possible indirect role of glutamate in causing the neuronal death found after intracerebral administration of a low dose of ouabain (0.1 nmol) has been evaluated. This dose of ouabain produces a more extensive neuronal lesion than those caused by glutamate receptor agonists (kainate at an equimolar dose, or NMDA (N-methyl-d-aspartate) at a 50-fold higher dose). The selective glutamate receptor antagonists, dizocilpine (MK-801) and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), in doses which blocked the direct toxicity of glutamate receptor agonists acting on either the NMDA and non-NMDA classes of glutamate receptor, failed to provide more than a minor protection against ouabain-induced peuronal death in the rat dorsal hippocampus. In contrast, the non-selective glutamate receptor antagonist, kynurenate (100 nmol) reduced the damage by around 70%. The difference in neuroprotection found between the glutamate receptor antagonists suggests that kynurenate may protect by a non-glutamatergic mechanism. Co-administration of ouabain and glutamate receptor agonists (kainate, NMDA or glutamate) resulted in additive rather than synergistic damage to hippocampal neurons. The results suggest that in vivo, ouabain and excitotoxins probably cause neuronal death by independent mechanisms.     

Glutamate-Mediated Excitotoxic Death of Cultured Striatal Neurons Is Mediated by Non-NMDA Receptors

Considerable interest has focused on the role of glutamate-mediated excitotoxicity in neurodegenerative disorders of the basal ganglia. The in vitro data on the receptor mechanisms involved in this process, however, have been inconclusive. Some studies have indicated that excitotoxins acting at NMDA receptors kill striatal neurons and others have indicated that NMDA receptor-mediated excitotoxic death of striatal neurons is minimal in the absence of cortex. In the present study, we used a pharmacological approach to carefully reexamine this issue in 2-week-old cultures of striatal neurons dissociated from E17 rat embryos. The sensitivity of these neurons to glutamate agonists and antagonists was determined by monitoring cell loss in identified regions of the growth dishes. We found that glutamate killed striatal neurons with an EC₅₀ of 100 μM. This loss was not mediated by NMDA receptors, since it was not reduced by the NMDA receptor antagonist APV (0.1-1.0 mM). Consistent with this result, up to 50 mM NMDA receptor-specific excitotoxin quinolinic acid (QA) did not affect neuronal survival. Depolarizing the QA-exposed neurons with 35 mM potassium chloride to enhance NMDA receptor activation by QA also did not produce neuron loss. The metabotropic glutamate receptor antagonist AP3 (500 μM) also had no effect on the striatal neuron loss produced by 100 μM glutamate. In contrast, the non-NMDA antagonist GYKI 52466 (100 μM) did block the excitotoxic effect of glutamate (100 μM). Specific AMPA and KA receptor agonists and the non-NMDA antagonist GYKI 52466 revealed that the non-NMDA receptor-mediated excitotoxic effect of glutamate was mediated by KA receptors. These results suggest that cultured striatal neurons are directly vulnerable to non-NMDA glutamate agonists, but not to NMDA and metabotropic glutamate agonists. Thus, non-NMDA receptors may play a greater role in the excitotoxic death of striatal neurons in disease and experimental animal models than previously realized.     

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.     

Dissociation of Effects of Glutamate Receptor Antagonists on Excitotoxic and Hypoxic Neuronal Cell Death in a Novel Rat Cortical Culture System

A novel in vitro cell culture model has been developed to investigate the mechanisms of delayed neuronal cell death following exposure to excitatory amino acids and hypoxia. Medium change damages cortical cells possibly leading to preselection of the neuronal population. This model allowed compounds to be administered in the absence of a medium change. In this system, the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, attenuated the neurotoxic effects of overnight exposure to glutamate and NMDA completely, and partially protected neurones exposed to α-amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA). The non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, did not attenuate the effects of glutamate or NMDA but blocked the excitotoxic effects of AMPA completely. These results suggest partial involvement of NMDA receptor activation in AMPA-induced toxicity. By contrast, hypoxia-induced neuronal degeneration in this model was attenuated by either NMDA or non-NMDA antagonism, which confirms previous reports that the mechanisms of hypoxic and excitotoxic neurodegeneration in these in vitro models are not identical. A number of other compounds, which have been reported previously as neuroprotective in vitro and in vivo, including the calcium channel antagonists, SB 201823, flunarizine, and nifedipine, and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester, L-NAME, demonstrated no significant neuroprotective effects in this in vitro system. In common with other in vitro models that include a change of medium, these data suggest that this system does not have predictive validity for the identification of novel neuroprotective agents in vivo.     

Lactate induced excitotoxicity in hippocampal slice cultures

During the initial minutes of cerebral ischemia, lactic acid accumulates and acidifies brain pH to 6.0-6.7. Glutamate is also released during ischemia that activates glutamate receptors and induces excitotoxicity. While glutamate excitotoxicity is well established to induce ischemic injury, a role of lactic acidosis in ischemic brain damage is poorly understood. This study analyzes acidosis neurotoxicity in hippocampal slice cultures in the presence or absence of lactate. At pH 6.7, neuronal loss was similar whether or not lactate was present. At pH 6.4, neuronal loss was significantly greater in the presence of lactate suggesting that lactate potentiates the acidosis toxicity. At pH 6.4 in the presence of lactate, NMDA or non-NMDA receptor antagonists reduced neuronal loss, while in the absence of lactate, NMDA or non-NMDA receptor antagonists had little effect. [3H]-Glutamate uptake was inhibited by acidic pH, and the amount of inhibition was significantly greater in the presence of lactate. These findings suggest that lactate plays a role in acidosis neurotoxicity by inducing excitotoxicity. Lactic acidosis and excitotoxicity have been previously thought to be independent events during ischemia. This study suggests that during ischemia, lactic acidosis contributes to excitotoxic neuronal loss.     

Glutamate in the inferior colliculus plays a critical role in audiogenic seizure initiation.

Alterations of excitant amino acid (EAA) action are implicated in seizure susceptibility in the genetically epilepsy-prone rat (GEPR). The inferior colliculus (IC) is critical for audiogenic seizure (AGS) initiation in the GEPR. The present study observed that bilateral microinjection into the IC of L-canaline, a glutamate synthesis inhibitor, decreased AGS severity in the GEPR and also decreased potassium-evoked release of glutamate from IC slices. Bilateral microinjection of NMDA receptor antagonists, 2-amino-7-phosphonoheptanoate (AP7) or 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) into IC blocked AGS, and an antagonist at non-NMDA EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), also blocked AGS. NMDA receptor antagonists were 5-200 times more effective than CNQX. Microinjection of a non-competitive NMDA receptor antagonist, dizocilpine (MK-801), into IC had little effect except with very high doses. Microinjection of CPP or AP7 into the IC blocked AGS at considerably lower doses as compared to pontine reticular formation (PRF). However, MK-801 attenuated AGS when microinjected into PRF at doses that were ineffective in IC. Systemically administered CPP blocked AGS and significantly reduced IC neuronal firing in the behaving GEPR, suggesting an important action of systemically administered NMDA receptor antagonists on brainstem auditory nuclei critical to AGS. The present results support a critical role for glutamate acting, in part, through NMDA receptors in IC in initiation of AGS.     

Excitatory amino acid pathways in brain-stimulation reward

A range of agonists and antagonists active at different glutamate/aspartate (Glu/Asp) receptor subtypes were injected into rat ventral tegmental (VTA) sites downstream from self-stimulation electrodes in the medial forebrain bundle. Control injections were made into the contralateral tegmentum. Variable-interval (VI 10 s) self-stimulation was not significantly affected by a specific antagonist of N-methyl-D-aspartate (NMDA)-type receptors (D,L-2-amino-5-phosphonovaleric acid (2-AP5), 10 and 50 nmol). Broad-spectrum excitatory amino acid (EAA) antagonists viz cis-2,3-piperidine dicarboxylate (cPDA) (10 and 50 nmol), gamma-D-glutamylaminomethyl sulphonic acid (GAMS) (10 nmol) and p-chlorobenzoyl-2,3-piperazine dicarboxylic acid (pCB PzDA) (2.0 and 10 nmol), active at kainate, quisqualate, as well as NMDA receptors, all produced significant depression of responding when injected into the ipsilateral, but not the contralateral, tegmentum. Compounds inhibiting Glu/Asp reuptake had variable effects: strong depression with dihydrokainic acid (7.5 nmol), or no significant effect (L-threo-3-hydroxyaspartic acid, 2.0 and 10 nmol). The receptor agonist, NMDA (10 nmol), depressed responding regardless of injection side; kainic and responding regardless of injection side; kainic and quisqualic acid elicited myoclonic and other non-specific responses in preliminary tests, and were not examined further; enhanced responding was not seen. The side-specific blockade of responding by non-NMDA antagonists indicates the existence of non-NMDA EAA terminals in the VTA, signalling the receipt of hypothalamic brain-stimulation reward. Caudally directed EAA projections terminating on A10 dopamine cell bodies may account for depression of self-stimulation by EAA antagonists.     

Receptor mechanisms and circuitry underlying NMDA antagonist neurotoxicity.

NMDA glutamate receptor antagonists are used in clinical anesthesia, and are being developed as therapeutic agents for preventing neurodegeneration in stroke, epilepsy, and brain trauma. However, the ability of these agents to produce neurotoxicity in adult rats and psychosis in adult humans compromises their clinical usefulness. In addition, an NMDA receptor hypofunction (NRHypo) state might play a role in neurodegenerative and psychotic disorders, like Alzheimer's disease and schizophrenia. Thus, understanding the mechanism underlying NRHypo-induced neurotoxicity and psychosis could have significant clinically relevant benefits. NRHypo neurotoxicity can be prevented by several classes of agents (e.g. antimuscarinics, non-NMDA glutamate antagonists, and alpha(2) adrenergic agonists) suggesting that the mechanism of neurotoxicity is complex. In the present study a series of experiments was undertaken to more definitively define the receptors and complex neural circuitry underlying NRHypo neurotoxicity. Injection of either the muscarinic antagonist scopolamine or the non-NMDA antagonist NBQX directly into the cortex prevented NRHypo neurotoxicity. Clonidine, an alpha(2) adrenergic agonist, protected against the neurotoxicity when injected into the basal forebrain. The combined injection of muscarinic and non-NMDA Glu agonists reproduced the neurotoxic reaction. Based on these and other results, we conclude that the mechanism is indirect, and involves a complex network disturbance, whereby blockade of NMDA receptors on inhibitory neurons in multiple subcortical brain regions, disinhibits glutamatergic and cholinergic projections to the cerebral cortex. Simultaneous excitotoxic stimulation of muscarinic (m(3)) and glutamate (AMPA/kainate) receptors on cerebrocortical neurons appears to be the proximal mechanism by which the neurotoxic and psychotomimetic effects of NRHypo are mediated.     

Mechanisms underlying the neuropathological consequences of epileptic activity in the rat hippocampus in vitro

Blockade of γ-aminobutyric acid (GABA)ergic synaptic transmission in mature hippocampal slice cultures for a period of 3 days with convulsants was shown previously to induce chronic epileptiform activity and to mimic many of the degenerative changes observed in the hippocampi of epileptic humans. The cellular mechanisms underlying the induction of this degeneration were examined in the present study by comparing the effects of GABA blockers with the effects produced by the K⁺ channel blocker tetraethylammonium (2 mM). Both types of convulsant caused a comparable decrease in the number of Nissl-stained pyramidal cells in areas CA1 and CA3. No significant cell loss was induced by tetraethylammonium when epileptiform discharge was reduced by simultaneous exposure of cultures to tetrodotoxin (0.5 μM) or to the anticonvulsants pentobarbital (50 μM) or tiagabine (50 μM). We conclude that this degeneration was mediated by convulsant-induced epileptiform discharge itself. The hypothesis that N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity underlies cell death in this model was tested by applying convulsants together with specific antagonists of glutamate receptors. Whereas coapplication of antagonists of both non-NMDA and NMDA receptors strongly reduced the degeneration induced by the convulsants, application of either class of antagonist alone did not. Application of exogenous NMDA produced potent cell death, and this degeneration was blocked by the NMDA receptor antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801). Convulsants also induced a loss of dendritic spines that could be partially prevented by NMDA or non-NMDA receptor antagonists. We conclude that NMDA receptor activation is not solely responsible for the neuronal pathology resulting as a consequence of epileptiform discharge. © 1996 Wiley-Liss, Inc.     

Antagonism of non-NMDA receptors augments the neuroprotective effect of NMDA receptor blockade in cortical cultures subjected to prolonged deprivation of oxygen and glucose.

A 30-60 min period of oxygen and glucose deprivation induced widespread degeneration of cultured murine neocortical neurons. Neuronal degeneration could be blocked by adding the selective NMDA antagonist MK-801 to the bathing medium; however, if the deprivation period was prolonged to 90-105 min, the neuroprotective effect of MK-801 was overcome. The non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at 1-100 microM concentrations also failed to protect neurons against this prolonged insult, but the combination of CNQX with either MK-801 or D-APV produced marked neuroprotection. This synergistic action of CNQX was not due to enhanced blockade of NMDA receptors, as it was not mimicked by combining MK-801 with D-APV or 7-chlorokynurenate. These observations support the idea that combined NMDA and non-NMDA receptor blockade may have value in ameliorating the neuronal loss associated with prolonged ischemic insults in vivo.     

Selective blockade of non-NMDA receptors does not block rapidly triggered glutamate-induced neuronal death

The quinoxalinedione, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), has been introduced as a relatively selective antagonist of non-N-methyl-D-aspartate (non-NMDA) glutamate receptors. We studied the ability of CNQX to block excitatory amino acid-induced neurotoxicity in murine cortical cell cultures. 100 microM CNQX blocked the acute neuronal swelling induced by 500 microM kainate, but it also attenuated the swelling and degeneration induced by 500 microM NMDA. Addition of 1 mM glycine to the CNQX eliminated antagonism of NMDA toxicity, while preserving antagonism of the neuronal degeneration induced by kainate or AMPA. This selective non-NMDA antagonist combination of CNQX plus glycine substantially attenuated the acute neuronal swelling induced by brief exposure to 500 microM glutamate, but had little effect on subsequent late degeneration, supporting the conclusion that rapidly triggered glutamate-induced cortical neuronal death is predominantly mediated by NMDA receptors.     

Excitatory amino acids modulate epileptogenesis in the brain stem

Activation of cholinergic mechanisms in the pontine reticular formation by local microinjections of carbachol was shown to induce pontine electrographic seizures and clonic convulsions. In this study we found that glutamate microinjections into the pons induced similar electrographic seizures and clonic convulsions. Microinjections into the PRF of glutamate in subconvulsive doses prior to carbachol potentiated the epileptogenic effect of carbachol. The duration of the seizure activity increased and the convulsions became more severe. The NMDA receptor antagonist MK-801 and the non-NMDA receptor antagonists DNQX significantly reduced the potentiating effect of glutamate. These results indicate a possible role of EAA receptors in the generation of epilepsy in the pons. They also suggest the importance of studying the role of synergistic interactions between EAA mechanisms and cholinergic mechanisms in the various pontine functions.     

Excitatory amino-acids, a new class of neurotransmitters. Pharmacology and functional properties]

The pharmacology of excitatory amino acids (EAA) like glutamate or aspartate, has defined three main types of receptors: NMDA, quisqualate (now named AMPA) and ka?nate receptors, associated to cationic channels. The NMDA receptor, the best characterized, is a macromolecular complex with multiple specific sites: the agonist binding site (glutamate, aspartate, NMDA); the glycine site and polyamine site mediating allosteric regulations; the site located inside the channel for activity-dependent antagonists (phencyclidine, MK-801). This channel, permeable to calcium, is blocked by magnesium in a voltage-dependent manner. The structural complexity of the NMDA receptor suggests the existence of subtle regulations, but also offers many targets for pharmacological drugs. The calcium influx induced by NMDA receptor stimulation may account for the diversity of its functional properties. First, NMDA receptors modulate neuronal plasticity during the development and even long after. Indeed, NMDA receptor can induce long term potentiation (LTP; an experimental model of synaptic facilitation) and are involved in learning and memory. On the other hand, when over-stimulated, they induce neurotoxicity. The death of the cell occurs after several hours, during which NMDA antagonists can prevent irreversible damages. EAA systems are distributed in the whole brain, interacting with numerous other neurotransmitters, but particularly concentrated in the cortico-striatal and cortico-cortical fibers and in the hippocampus. Several neuro-psychiatric disorders could be related to a glutamatergic dysfunction: acute neuronal lesions (stroke, viral disease like AIDS) and epilepsy; but also chronic neurodegenerative disorders (Alzheimer's dementia, Huntington and Parkinson diseases). A glutamatergic hypothesis of schizophrenia arose from the phencyclidine model of psychosis, arguing for an imbalance between glutamate and dopamine. The therapeutic perspectives of glutamatergic substances in these diseases will be discussed.     

DNQX blockade of amphetamine behavioral sensitization

The role of the N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid (EAA) receptors in the mechanism of behavioral sensitization to amphetamine-induced sterotypy was investigated in mice. The results confirm previous observations that NMDA antagonists can block the induction of the phenomenon but not the expression; in contrast, DNQX, a non-NMDA receptor antagonist, can block both the induction and the expression of the sensitization. The differential effects of the two classes of antagonists suggest that the induction and the expression are the result of different mechanisms, both of which involve the EAA system. The DNQX results differ from those of haloperidol, which can also block both the induction and expression, because haloperidol can completely block the amphetamine-induced responses in naive and in sensitized animals; whereas DNQX is without effect on the amphetamine activity in naive animals and, in the sensitized animal, can block only that portion of the response that is derived from the sensitization phenomenon. The effects of the EAA antagonists support the hypothesis that the enhanced responsiveness in the sensitized animals is derived from the activation of EAA receptors, which, in turn, increases the release of dopamine in the striatum. Finally, the involvement of the non-NMDA receptors in the expression of the behavioral sensitization further substantiates the postulate that the amphetamine-induced sensitization is a behavioral manifestation of long-term potentiation (LTP).     

Neuroprotection against excitotoxicity by N-alkylglycines in rat hippocampal neurons

Excessive activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype is considered a relevant initial step underlying different neurodegenerative diseases. Recently, with the approval of memantine to treat Alzheimer dementia, NMDA receptors have regained clinical interest. Accordingly, the development and validation of NMDA receptor antagonists is being reconsidered. We recently identified a family of trialkylglycines that act as channel blockers of the NMDA receptor. Their neuroprotective activity against excitotoxic insults remains elusive. To address this issue, we first characterized the contribution of glutamate receptor sub-types to hippocampal death in culture as a function of days in culture in vitro (DIV). Whereas at 7 DIV neither NMDA nor glutamate produced a significant neuronal death, at 14 and 21 DIV, NMDA produced the death of 40% of the neurons exposed to this receptor agonist that was fully protected by MK-801. Similar results were obtained for l-glutamate at 14 DIV. In contrast, when neurons at 21 DIV were used, glutamate killed 51.1±4.9% of the neuronal population. This neuronal death was only partially prevented by MK-801, and fully abrogated by a combination of MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glucose deprivation injured 37.1±9.2% of the neurons through a mechanism sensitive to MK-801. The family of recently identified N-alkylglycines tested protected neurons against NMDA and glucose-deprivation toxicity, but not against glutamate toxicity. Noteworthy, N-alkylglicines with a moderate protection against NMDA-induced toxicity strongly protected from β-amyloid toxicity. Collectively, these findings imply both NMDA and non-NMDA receptors in excitotoxicity of hippocampal neurons, and suggest that blockade of NMDA receptors alone may not suffice to efficiently abrogate neurodegeneration.     

Platelet-derived growth factor-BB pretreatment attenuates excitotoxic death in cultured hippocampal neurons

Neuronal excitotoxic death results from excess stimulation by elevated levels of extracellular glutamate acting on N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. While excitotoxicity is typically attenuated by using glutamate receptor antagonists, we report here that neuronal deaths induced directly by brief exposures to glutamate or NMDA were both attenuated by preincubation with platelet-derived growth factor-BB (PDGF-BB). The neuroprotection was concentration and time dependent; preincubation for at least 24 h with a minimum of 10 ng/mL of PDGF-BB was required for maximal neuroprotective effect. The NMDA receptor antagonist MK-801 also afforded partial protection, and when MK-801 was used with PDGF-BB, neuronal survival was comparable to that of untreated controls. When protection of inhibitory and excitatory neurons by PDGF treatment was compared, the excitatory neurons appeared to be selectively protected. The present results demonstrate that PDGF pretreatment can protect neurons from direct glutamate-induced excitotoxicity in vitro and suggests that PDGF might possibly function as a neuroprotective agent in potential therapeutic applications.     

Pharmacology of N-methyl-D-aspartate-induced brain injury in an in vivo perinatal rat model

Intrastriatal injection of the glutamate analogue N-methyl-D-aspartate (NMDA, 25 nmol) in postnatal day (PND) 7 rats provides a rapid, sensitive, and reproducible assay in which potential neuroprotective strategies against excitotoxic neuronal injury can be examined in vivo. Brain injury is quantified 5 days postinjection by comparison of the weights of the injected and contralateral cerebral hemispheres. Intraperitoneal injections (15 minutes post-NMDA) of competitive and noncompetitive NMDA receptor antagonists attenuated the severity of NMDA-induced brain injury. The rank order of neuroprotective potency of these antagonists was CGS-19755 greater than DOIPG greater than dextromethorphan greater than HA-966. Of these compounds only the competitive antagonist CGS-19755 provided complete neuroprotection. NMDA-mediated brain injury was also reduced by the specific sigma receptor ligands +PPP and haloperidol (35% reduction). In contrast, drugs that reduce presynaptic neurotransmitter release (adenosine) or enhance neuronal inhibition (baclofen) were not effective against NMDA toxicity. Although all five of the anticonvulsants tested limited NMDA-induced seizure activity, only carbamazepine reduced NMDA-mediated brain injury (36% reduction). These findings extend earlier observations that NMDA receptor antagonists can limit NMDA-induced toxicity in vivo and suggest that sigma receptors contribute to the pathophysiology of NMDA-mediated brain injury in vivo. Furthermore, NMDA-induced seizures and brain injury appear dissociable in this in vivo model. The results illustrate important practical limitations of neuroprotection in vivo vs. in vitro.     

Müller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina

We characterized morphological effects of the endogenous excitotoxin, glutamate in ex vivo retinal segments prepared from 30-day-old rats. Initial changes induced by glutamate consisted of reversible, sodium-dependent Müller cell swelling. This glial swelling was mimicked by glutamate transport substrates but not by ionotropic glutamate receptor agonists. Only very high concentrations of exogenous glutamate (3,000 microM) produced excitotoxic neuronal damage. The neuronal damage was accompanied by severe glial swelling and was blocked by an antagonist of non-N-methyl-D-aspartate (NMDA) receptors but not by an NMDA receptor antagonist. Because glutamate uptake can be influenced by changes in cellular energy levels, we studied the effects of oxidative and glycolytic energy depletion on glutamate-mediated Müller cell swelling. Oxygen deprivation produced little morphological change and did not alter either glutamate-mediated Müller cell swelling or glutamate-induced excitotoxicity. In contrast, inhibition of glycolysis by iodoacetate produced severe neuronal damage without Müller cell swelling. In the presence of iodoacetate, exogenous glutamate failed to cause glial swelling. The neuronal damage produced by iodoacetate was inhibited by pyruvate, a substrate that sustains oxidative energy pathways. In the presence of iodoacetate plus pyruvate, glutamate failed to cause Müller cell swelling but became neurotoxic at low concentrations through activation of non-NMDA receptors. These results indicate that glycolytic energy metabolism plays a critical role in sustaining ionic balances required for Müller cell glutamate uptake and glial uptake helps to prevent glutamate-mediated excitotoxicity.     

Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.     

Simultaneous blockade of non-NMDA ionotropic receptors and NMDA receptor-associated ionophore partially protects hippocampal slices from protein synthesis impairment due to simulated ischemia

A large body of evidence exists to demonstrate that excitatory amino acids (EAA) and their receptors are involved in the pathophysiological mechanisms linking several acute brain insults, such as cerebral ischemia, to neuronal degeneration and death. Accordingly, the use of EAA receptor antagonists can be beneficial in attenuating or preventing the neuronal irreversible damage subsequent to various neuropathological syndromes. We have investigated the effect of 15 min of simulated ischemic conditions, i. e., oxygen/glucose deprivation, on hippocampal slices preparation measuring, as neurotoxicity indexes, both the amino acids efflux in the incubation medium, detected by HPLC, and the inhibition of protein synthesis, evaluated as ³H-Leucine incorporation into proteins. Accumulation of neurotransmitter amino acids was measured in the medium during the “ischemic” period. Glutamate increased 30-fold over the basal level while aspartate was sevenfold and GABA 12-fold higher than in normal conditions. After a reoxygenation period of 30 min, the rate of protein synthesis of hippocampal slices subjected to “ischemia” was reduced to 35–50% of controls. The non-competitive NMDA antagonist MK-801 (100 μM) and the competitive NMDA antagonist CGP 39551 (100–250 μM) as well as the non-NMDA receptor antagonist NBQX (100 μM) and AP3 (300 μM) were unable to counteract the metabolic impairment when they were present alone in the incubation fluid during simulated “ischemia.” An incomplete, but highly significant (p < 0.001), protection from protein synthesis impairment was achieved in the presence of an equimolar concentration (100 μM) of MK-801 and NBQX. A similar protective effect could be reproduced using 100 μM NBQX in concomitance with a high Mg⁺⁺ (20 μM) voltage-dependent block of the NMDA receptor-associated channel but not exposing the slices to a NBQX (100 μM) and CGP 39551 (100–250 μM) mixture. The recovery of protein synthesis in the presence of the MK-801/NBQX effective combination was not paralleled by a detectable decrease in the amount of amino acids released in the incubation medium during the “ischemic” period. Taken together, the present data allow new insights into neurotoxicity-mediating mechanisms, suggesting that multiple additive processes are involved and that antagonists acting at different sites on excitatory amino acid receptor subtype can show different neuroprotective potency. © 1995 Wiley-Liss, Inc.