1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.     

Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs.

Metabotropic glutamate (mGlu) receptors have been considered as potential targets for neuroprotective drugs, but the lack of specific drugs has limited the development of neuroprotective strategies in experimental models of acute or chronic central nervous system (CNS) disorders. The advent of potent and centrally available subtype-selective ligands has overcome this limitation, leading to an extensive investigation of the role of mGlu receptor subtypes in neurodegeneration during the last 2 years. Examples of these drugs are the noncompetitive mGlu1 receptor antagonists, CPCCOEt and BAY-36-7620; the noncompetitive mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)pyridine, SIB-1893, and SIB-1757; and the potent mGlu2/3 receptor agonists, LY354740 and LY379268. Pharmacologic blockade of mGlu1 or mGlu5 receptors or pharmacologic activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of N-methyl-d-aspartate (NMDA) receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes. A series of observations suggest a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer disease. MGlu2/3 receptor agonists inhibit glutamate release, but also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of CNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders. The advantage of all these drugs with respect to NMDA or AMPA receptor agonists derives from the evidence that mGlu receptors do not "mediate," but rather "modulate" excitatory synaptic transmission. Therefore, it can be expected that mGlu receptor ligands are devoid of the undesirable effects resulting from the inhibition of excitatory synaptic transmission, such as sedation or an impairment of learning and memory.     

In Vivo Potentiation of Glutamate-Mediated Neuronal Damage after Chronic Administration of the Glycolysis Inhibitor Iodoacetate

Neuronal damage associated with cerebral ischemia and hypoglycemia might be the consequence of the extracellular accumulation of excitatory amino acids. In previous studies we showed that elevation of glutamate and aspartate extracellular levels by inhibition of its uptake in vivo is not sufficient to induce neuronal damage unless mitochondrial energy metabolism is compromised. In the present study we show that chronic systemic administration of the glycolysis inhibitor iodoacetate (25 mg/kg) induces no damage to the brain per se but enhances neuronal vulnerability to glutamate-mediated neurotoxicity in the hippocampus. Tissue injury is well protected either by antagonizing NMDA glutamate receptors with MK-801 or by administration of pyruvate, a substrate of the tricarboxylic acid cycle. In contrast to systemic treatment, local infusions through a dialysis probe of 5 mM iodoacetate into the hippocampus induced acute lesions not sensitive to MK-801. Iodoacetate intrahippocampal perfusion induced substantial increases in the extracellular levels of glutamate (3.5-fold), taurine (8.8-fold), and particularly aspartate (35-fold). Neuronal damage under this conditions occurs very rapidly as revealed by the histological analysis of animals transcardially perfused immediately after iodoacetate perfusion. Aspartate might contribute to neuronal damage since intrahippocampal administration of this amino acid (600 nmol/μl) induces extensive lesions. The present study might suggest that impairment of glucose oxidation through the glycolytic pathway in vivo facilitates glutamate neurotoxicity. Additionally, the results indicate that pyruvate might prevent as efficiently as glutamate receptor antagonists glutamate-mediated neuronal damage associated with ischemia/hypoglycemia.     

Effects of adenosine and gamma-aminobutyric acid A receptor antagonists on N-methyl-D-aspartate induced neurotoxicity in the rat hippocampus

This study investigated the modulatory actions of adenosine and gamma-aminobutyric acid (GABA) on several aspects of N-methyl-D-aspartate (NMDA)-induced neurotoxicity, including neuronal loss, atrophy, necrosis, and calcium accumulation in the hippocampus. For this purpose, we combined unilateral intrahippocampal injections of NMDA (24 nmoles) with acute injections of the selective A1 adenosine receptor antagonist DPCPX (0.03 pmoles), the selective adenosine A2a receptor antagonist CSC (1.5 pmoles), a combination of these two antagonists, and injections of the selective GABA A receptor antagonist bicuculline (60 pmoles). Fifteen days after NMDA injection, neuronal loss with preservation of architecture was observed in stratum oriens, pyramidale, radiatum, lacunosum-moleculare, and stratum moleculare of Ammon's horn, and in radial and granular layers of the dentate gyrus. NMDA plus vehicle also produced a small degree of brain tissue necrosis (holes in the structure) in four of five brains. Acute injections of CSC, but not DPCPX or bicuculline, significantly increased the extent of neuronal loss produced by NMDA plus vehicle. CSC in combination with NMDA induced significantly more necrosis than NMDA plus vehicle. A significant degree of atrophy was observed in the hippocampus after treatment with NMDA plus vehicle, and bicuculline significantly increased the magnitude of this atrophy. NMDA-induced calcium deposits were detected within the radiatum and lacunosum-moleculare layers of the hippocampus and in the hilus of the dentate, but not in the stratum oriens, stratum pyramidale, or in the granular layer of the dentate gyrus. However, treatment with the different antagonists did not significantly modify the magnitude of the NMDA-induced calcium deposits. These results reveal a selective vulnerability of certain areas of the hippocampus to the accumulation of calcium deposits, and a selective interaction between adenosine receptors and NMDA-induced neurotoxicity in the hippocampus.     

Failure of glycine site NMDA receptor antagonists to protect againstl-2-chloropropionic acid-induced neurotoxicity highlights the uniqueness of cerebellar NMDA receptors

Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of excitatory amino acid-induced cell death mediated in part by the activation of NMDA receptors. However, the NMDA subunit stoichiometry, producing functional NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the NMDA receptors in forebrain regions. We have used thel-2-chloropropionic acid (l-CPA) (750 mg/kg) model of NMDA-medialed selective cerebellar granule cell necrosis in vivo to examine the role of the glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell necrosis. The abilities of various NMDA receptor antagonists were examined in vivo to determine the relative contribution of both glutamate and glycine sites involved in thel-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive NMDA receptor antagonist dizocilpine (MK-801) was compared with glutamate and glycine site NMDA antagonists. We have examined a number of markers for thel-CPA-induced granule cell necrosis. Thel-CPA-induced reduction in cerebellar aspartate and glutamate concentrations were used as markers of granule cell necrosis. We also measured the cerebellar water content and sodium concentrations as measures of thel-CPA-induced cerebellar edema that accompanies the granule cell necrosis. Finally the ability of the NMDA antagonists to attenuate thel-CPA-induced reductions in body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent glutamate antagonists, CPP and CGP40116 and dizocilpine prevented thel-CPA-induced locomotor dysfunction and granule cell necrosis as measured by their ability to preventl-CPA-induced reductions in aspartate and glutamate concentrations. CPR CGP40116 and dizocilpine also prevented the appearance of cerebellar edema followingl-CPA administration. In addition, dizocilpine, CPP and CGP40116 were able to partially prevent thel-CPA-induced loss in body weight over the 48 h experimental period. In contrast, none of the glycine partial agonists or antagonists, namely (±)HA-966,d-cycloserine, MDL-29951, DPCQ, MNQX or L-701 252 were able to prevent thel-CPA-induced loss in body weight,l-CPA-induced granule cell necrosis and behavioral disturbances when administered to rats. None of the NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for dizocilpine, CPP, CGP401 l6 and (±)HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrate that NMDA open channel blockade and glutamate antagonists can provide full neuroprotection against thel-CPA-induced granule cell necrosis. The failure of the glycine partial agonists and antagonists to provide any neuroprotection againstl-CPA-induced neurotoxicity in the cerebellum contrasts with their neuroprotective efficacy in other animal models of excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the glycine site plays a lesser role in modulating NMDA receptor function in the cerebellum and may explain why cells expressing NMDA receptors composed of NR1/NR2C subunits are particularly resistant to excitatory amino acid-induced neurotoxicity.     

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly. Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.     

Neuroprotective effects of MK-801, TCP, PCP and CPP against N-methyl-D-aspartate induced neurotoxicity in an in vivo perinatal rat model

Three non-competitive antagonists (MK-801, TCP, PCP) and one competitive antagonist (CPP) of N-methyl-D-aspartate (NMDA) receptors, were compared for their ability to antagonize neurotoxic actions of NMDA injected into the brains of 7-day-old rats. Unilateral intracerebral injection of NMDA (25 nmol/0.5 microliters) into the corpus striatum of pups consistently produced severe confluent neuronal necrosis in the striatum extending into the dorsal hippocampus and overlying neocortex. The distribution of damage corresponded to the topography of NMDA type glutamate receptors in the vulnerable regions. With this lesion in developing brain, the weight of the injected hemisphere 5 days later can be used as a quantitative measure of brain injury. Intraperitoneal administration of MK-801 (0.02-42.0 mumol/kg), TCP (3.5-54.0 mumol/kg), PCP (1.0-41.0 mumol/kg), and CPP (1.0-60.0 mumol/kg) 15 min after NMDA injection had prominent dose-dependent neuroprotective effects. MK-801 was 14 times more potent than other compounds tested and the 50% protective dose (PD50, that dose which reduced damage by 50% relative to untreated NMDA-injected controls) was 0.63 mumol/kg. Corresponding values for CPP, PCP, and TCP were 8.84, 10.85, and 24.05 mumol/kg respectively. The lowest dose of MK-801 that provided significant protection was 0.2 mumol/kg (0.04 mg/kg, 37.9 +/- 4.6% protection). Four mumol/kg (0.8 mg/kg) of MK-801 completely protected against NMDA-mediated damage. The study provides the first direct in vivo comparison of the neuroprotective abilities of these compounds. Systemic administrations of MK-801, TCP, PCP, and CPP all limit NMDA-induced neuronal injury in this model. The susceptibility of the immature brain to the neurotoxicity of NMDA provides a sensitive, reproducible, and quantitative in vivo system for comparing the effectiveness of drugs with protective actions against excitotoxic neuronal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

The removal of extracellular calcium: a novel mechanism underlying the recruitment of N-methyl-D-aspartate (NMDA) receptors in neurotoxicity

The involvement of NMDA-type glutamate receptor in neuronal injury established in experimental stroke and neurotrauma models has been recently challenged by failures in treatment of stroke/neurotrauma patients with NMDA receptor antagonists. NMDA receptor activity is known to be essential for mediating a multitude of physiological functions. However, how NMDA receptors are recruited to cause neuronal injury remains unclear. Here we report that the time period during which initial NMDA receptor up-regulation occurs is critical for the recruitment of NMDA receptors causing neuronal injury during extracellular calcium (Ca2+) reperfusion in cultured hippocampal neurons, and represents the key period for neuronal protection by NMDA receptor antagonists. Furthermore, we identified that via intracellular sodium (Na+), extracellular Ca2+ depletion induces the up-regulation of NMDA receptor gating. Taken together, our study provides direct experimental evidence suggesting that determination of when and how NMDA receptors are recruited to cause neurotoxicity is essential for guiding treatment via antagonism of NMDA receptor functions.     

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.     

MK-801 selectively protects mouse arcuate neurons in vivo against glutamate toxicity.

The receptor mediating glutamate toxicity in vivo has not yet been identified. The effects of NMDA antagonist MK-801 and the AMPA antagonist NBQX were studied on glutamate toxicity in the arcuate nucleus of newborn and adult mice. Morphometric methods were used to determine the effects of antagonists on glutamate toxicity. In the developing arcuate, MK-801 abolished glutamate neurotoxicity whereas NBQX has no effect. MK-801, but not NBQX, afforded significant but not complete protection in mature arcuate neurons. The residual toxicity could not be blocked by co-administration of NBQX which paradoxically partially inhibited the protective effect of MK-801. The results show that throughout development, glutamate neurotoxicity in the arcuate nucleus is predominantly mediated by NMDA receptors.     

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.     

Activation of NMDA receptors protects against glutamate neurotoxicity in the retina: evidence for the involvement of neurotrophins

Activation of glutamate receptors has been implicated in excitotoxicity. Here, we have investigated whether subtoxic concentrations of glutamate can modulate neuronal death in the developing retina. Explants of rat retinas were pre-incubated with glutamate, N-methyl-d-aspartate (NMDA), kainate, quisqualate or trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) for 18 h. Then, glutamate (6 mM) was added to the explants for an additional 6 h. Glutamate-induced degeneration was restricted to the emerging inner nuclear layer. Pre-incubation with glutamate, NMDA, or both, reduced glutamate-induced neuronal death and protected against neuronal death induced by irradiation (2 Gy). The NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid (d-APV; 30 microM) or 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate (MK-801; 30 microM), prevented glutamate-induced neuroprotection. To investigate whether this neuroprotection was mediated by neurotrophins, we incubated retinal explants with either brain-derived neurotrophic factor or neurotrophin-4. Both treatments resulted in partial protection against glutamate-induced neurotoxicity. Furthermore, NMDA mediated neuroprotection was totally reversed when a soluble form of the specific tyrosine kinase receptor B was simultaneously added to the explants. Our results suggest that activation of NMDA receptors may control neuronal death in the retina during development. This modulation seems to depend, at least in part, on the release of neurotrophins within the retina.     

An activity-dependent switch from facilitation to inhibition in the control of excitotoxicity by group I metabotropic glutamate receptors

Activation of group I metabotropic glutamate receptors (mGlu1 or -5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a "neuroprotective mode". The characteristics of the activity-dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1-45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype-selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity-dependent switch in regulating excitotoxic neuronal death and, therefore, the recent "history" of these receptors is critical for the response to agonists or antagonists.     

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.     

Neuroprotective and symptomatological action of memantine relevant for alzheimer’s disease — a unified glutamatergic hypothesis on the mechanism of action

The involvement of glutamate mediated neurotoxicity in the pathogenesis of Alzheimer's disease is finding increasingly more acceptance in the scientific community. Central to this hypothesis is the assumption that in particular glutamate receptors of the N-methyl-D-aspartate (NMDA) type are overactivated in a tonic rather than a phasic manner. Such continuous mild activation leads under chronic conditions to neuronal damage. Moreover, one should consider that impairment of plasticity (learning) may result not only from neuronal damage per se but also from continuous activation of NMDA receptors. To investigate this possibility we tested whether overactivation of NMDA receptors using either non-toxic doses/concentrations of a direct NMDA agonist or through an indirect approach--decrease in magnesium concentration--produces deficits in plasticity. In fact NMDA both in vivo (passive avoidance test) and in vitro (LTP in CA1 region) impaired learning and synaptic plasticity. Under these conditions memantine which is an uncompetitive NMDA receptor antagonist with features of "improved magnesium" (voltage dependence, affinity) attenuated the deficit. The more direct proof that memantine can act as a surrogate for magnesium was obtained in LTP experiments under low magnesium conditions. In this case as well, impaired LTP was restored in the presence of therapeutically relevant concentrations of memantine (1 microM). In vivo, doses leading to similar brain/serum levels produce neuroprotection in animal models relevant for neurodegeneration in Alzheimer's disease such as neurotoxicity produced by inflammation in the NBM or beta-amyloid injection to the hippocampus. Hence, we postulate that if in Alzheimer's disease overactivation of NMDA receptors occurs indeed, memantine would be expected to improve both symptoms (cognition) and slow down disease progression because it takes over the physiological function of magnesium.     

Blockade of both NMDA and non-NMDA receptors is required for optimal protection against ischemic neuronal degeneration in the in vivo adult mammalian retina.

Under ischemic conditions, the excitatory amino acids (EAA), glutamate and aspartate, accumulate in the extracellular compartment of brain and, by excessive stimulation of EAA receptors, trigger excitotoxic degeneration of CNS neurons. Since glutamate and aspartate exert excitotoxic activity through both of the generally recognized classes of EAA receptors [N-methyl-D-aspartate (NMDA) and non-NMDA], it follows that both receptor classes may play a role in ischemic neuronal degeneration. Although several laboratories have reported that NMDA receptor antagonists confer protection in vivo against ischemic neuronal degeneration, very little is known about the ability of non-NMDA antagonists to confer such protection, a major reason being that non-NMDA antagonists that penetrate blood-brain barriers have not been available. In the present study, we examined the ability of NMDA or non-NMDA antagonists, either individually or in combination, to prevent neuronal degeneration in vivo in the adult rat retina rendered ischemic by dye/photothrombotic occlusion of retinal blood vessels. In this model, delivery of drugs to the ischemic tissue is assured by intravitreal administration. Intravitreal administration of the NMDA antagonist, MK-801, reduced the severity of ischemic damage approximately 50% (a ceiling effect that could not be increased by administering higher doses). The predominantly non-NMDA antagonist, CNQX, when administered in the highest dose permitted by its solubility limitations, provided equivocal (statistically nonsignificant) protection, but the two drugs combined provided greater than 80% protection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by approximately 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 microM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3-MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 microM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.     

Positive allosteric modulation of metabotropic glutamate 5 (mGlu5) receptors reverses N-Methyl-D-aspartate antagonist-induced alteration of neuronal firing in prefrontal cortex.

BACKGROUND: Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptor hypofunction may be associated with schizophrenia. Activation of metabotropic glutamate 5 (mGlu5) receptors enhances NMDA receptor mediated currents in vitro, implying that allosteric modulation of mGlu5 receptors may have therapeutic efficacy for schizophrenia. The aim of this study was to determine if positive allosteric modulators of mGlu5 receptors are effective in reversing two cellular effects of NMDA receptor antagonists that are relevant to schizophrenia: increases in corticolimbic dopamine neurotransmission and disruption of neuronal activity in the prefrontal cortex (PFC). METHODS: In freely moving rats, we measured the effects of the positive modulator of mGlu5 receptor 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) alone or in combination with the NMDA antagonist MK801 on 1) spontaneous firing and bursting of medial PFC (mPFC) neurons, and 2) dopamine release as measured by microdialysis in the mPFC and nucleus accumbens (NAc). RESULTS: The predominant effect of CDPPB on mPFC neurons was excitatory, leading to an overall excitatory population response. Pretreatment with CDPPB prevented MK801-induced excessive firing and reduced spontaneous bursting. In contrast, CDPPB had no significant effect on basal dopamine release as compared with control rats and did not alter MK801-induced activation of dopamine release in the mPFC and NAc. CONCLUSIONS: These results show that positive modulation of mGlu5 receptors reverses the effects of noncompetitive NMDA antagonists on cortical neuronal firing without affecting dopamine neurotransmission. Thus, these compounds may be effective in ameliorating PFC mediated behavioral abnormalities that results from NMDA receptor hypofunction.     

Differential neuronal fates in the CA1 hippocampus after hypoxia in newborn and 7-day-old rats: effects of pre-treatment with MK-801

The brain displays an age-dependent sensitivity to ischemic insults. However, the consequences of oxygen deprivation per se in the developing brain remain unclear, and the role of glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is controversial. To gain a better understanding of the mechanisms involved in the cerebral response to severe hypoxia, cell damage was temporally monitored in the CA1 hippocampus of rat pups transiently exposed to in vivo hypoxia (100% N2) at either 24 h or 7 days of age. Also, the influence of a pre-treatment with the NMDA receptor antagonist MK-801 (5 mg/kg, i.p.) was examined. At both ages, morphometric analyses and cell counts showed hypoxia-induced significant neuronal loss (30-35%) in the pyramidal layer, with injury appearing more rapidly in rats exposed at 7 days. Morphological alterations of 4,6-diamidino-2-phenylindole (DAPI)-labeled nuclei, DNA fragmentation patterns on agarose gels, as well as expression profiles of the apoptosis-related regulatory proteins Bax and Bcl-2 showed that apoptosis was prevalent in younger animals, whereas only necrosis was detected in hippocampi of rats treated at 7 days. Moreover, pre-treatment with MK-801 was ineffective in protecting hippocampal neurons from hypoxic injury in newborn rats, but significantly reduced necrosis in older subjects. These data confirm that hypoxia alone may trigger neuronal death in vivo, and the type of cell death is strongly influenced by the degree of brain maturity. Finally, NMDA receptors are not involved in the apoptotic consequences of hypoxia in the newborn rat brain, but they were found to mediate necrosis at 7 days of age.     

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: pharmacological characterization

L-Glutamate neurotoxicity at the N-methyl-D-aspartate (NMDA) receptor was characterized in cultured cerebellar granule cells. When deprived of glucose for 40 min, these cells were killed by 20-60 microM L-glutamate. However, the neurons were resistant to glutamate at concentrations as high as 5 mM when glucose and Mg2+ were present throughout. Both competitive and non-competitive NMDA receptor antagonists completely blocked neurotoxicity due to glutamate and other NMDA receptor agonists. CPP [+/-)-3-(2-carboxypiperazin-4-yl)-prophyl-1-phosphonic acid) was the most effective competitive antagonist with full protection at 100 microM while MK-801 [+/-)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]-cyclohepten-5,10-imin e) was the most effective non-competitive antagonist with full protection at 20 nM. Other antagonists with higher selectivity for other subtypes of glutamate receptors were ineffective. We conclude that glutamate toxicity in energy-deprived cerebellar granule cells is mediated by NMDA receptors. Results are discussed in terms of an hypothesis offering an explanation for the transition of glutamate from neurotransmitter to neurotoxin which emphasizes the responsiveness of the receptor to agonists rather than focusing on the presence of high concentrations of agonist.