Potent Analgesia Induced in Rats by Combined Action at PCP and Polyamine Recognition Sites of the NMDA Receptor Complex

The present study was performed to examine the analgesic effects of the intrathecal administration of agents acting at various sites in the N -methyl- d -aspartic acid (NMDA) receptor complex on the nociceptive responses to s.c. formalin injection in rats. Both the competitive NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) and the non-competitive NMDA antagonist dizocilpine maleate (MK-801) produced dose-dependent analgesic effects in the late, but not the early, phase of the formalin test. The polyamine antagonist ifenprodil, and the strychnine-insensitive glycine antagonists DCQX and 7-chlorokynurenic acid, failed to produce any analgesic effects in either the early or the late phase of the formalin test. The analgesic effects of APV were enhanced slightly by combined administration with a non-analgesic dose of glycine, and the analgesic effects of MK-801 were dramatically potentiated by combined adminstration of a non-analgesic dose of the polyamine spermine. The results indicate that much more potent analgesia can be produced in the formalin test by a combination of open channel blockers (such as MK-801) with agonists acting at the polyamine site, than by a single treatment with antagonists to either glycine allosteric or polyamine sites within the NMDA receptor complex.     

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.     

N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis." Copyright 1999 Academic Press.     

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.     

Developmental differences in antagonism of NMDA toxicity by the polyamine site antagonist ifenprodil.

Antagonists of 4 distinct regulatory sites on the N-methyl-D-aspartate (NMDA) receptor were tested for their ability to attenuate NMDA-mediated acute excitotoxicity in isolated chick retina of various embryonic ages between days 11 and 19 in ovo. Acute excitotoxicity was monitored by histology and by release of endogenous gamma-aminobutyric acid (GABA) into the medium during 30 min of incubation with 50 microM NMDA. The uncompetitive PCP channel site antagonist, MK-801, the competitive antagonist, CGS 19755, and the strychnine-insensitive glycine site antagonist, 7-chlorokynurenate, completely blocked NMDA-induced cell swelling and increased GABA release at all ages tested. Potencies versus NMDA were MK-801 greater than CGS 19755 greater than 7-chlorokynurenate with IC50S of 0.02, 0.62, and 15 microM, respectively. NMDA antagonism by the polyamine site antagonist, ifenprodil, differed from other classes of antagonists in several respects. At the earlier embryonic ages tested (E12-13) ifenprodil provided differential protection; completely blocking somal and neuritic swelling in most but not all inner nuclear layer neurons and inner plexiform processes. In dose-response studies, ifenprodil attenuated the NMDA-induced increase in medium GABA at all ages tested with an Imax of 10 microM. Ifenprodil, however, showed a decreased ability to completely protect some NMDA-sensitive neurons. This was reflected both histologically and by GABA release. Maximal attenuation of NMDA evoked GABA release was 83, 80, 62 and 50% at days E12, 13, 15 and 19, respectively. Histologically, differential protection was seen at E12 and 13, in limited areas at E15, and was no longer present at E19.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of three NMDA receptor antagonists in the treatment of prolonged status epilepticus

Three different classes of NMDA receptor antagonists were compared for their effectiveness in terminating prolonged status epilepticus (SE), induced by continuous hippocampal stimulation. Animals were treated after 150 min of SE by intraperitoneal administration of increasing doses of 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), MK-801 (dizocilpine), ifenprodil, or saline. EEG recordings were used to determine seizure termination. The first experiment (n = 57 animals) determined the most effective anticonvulsant dose of each agent by determining its ability to terminate SE within the next 300 min. Five control rats treated with normal saline after 150 min of SE continued to exhibit continuous seizures for the next 300 min. All drugs were administered after 150 min of SE. CPP terminated seizures with an ED(50) of 6.4 mg/kg; the maximal effective dose was 15 mg/kg. MK-801 has an ED(50) of 1.4 mg/kg; the maximal effective dose was 2 mg/kg. Ifenprodil was maximally effective at 30 mg/kg. However, an ED(50) could not be calculated. In a subsequent experiment, the NMDA antagonists were compared for their ability to terminate prolonged SE within 60 min of their administration at the most effective dose. MK-801 (2.0 mg/kg) terminated SE in 6 of 10 animals within 60 min, CPP (15 mg/kg) terminated it in 1 of 9 animals; ifenprodil (30 mg/kg) did not terminate it in any of 9 animals treated. In the 300 min following administration, CPP (6/9) and MK-801 (6/10) were equally efficacious in terminating SE but ifenprodil (2/7) was less effective (P = 0.065, chi-square test). The results indicate that the non-competitive NMDA receptor antagonist MK-801 was superior to the competitive antagonist CPP and the pH-sensitive site antagonist ifenprodil, in terminating prolonged experimental SE.     

The N-methyl-d-aspartate (NMDA) receptor glycine site antagonist ACEA 1021 does not produce pathological changes in rat brain

ACEA 1021 is a potent, selective N-methyl-

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-aspartate (NMDA) receptor glycine site antagonist under clinical evaluation as a neuroprotectant for stroke and head trauma. The potential of ACEA 1021 to produce morphologic changes in cerebrocortical neurons of the rat was assessed since it is known that noncompetitive (e.g., MK-801) and competitive (e.g., CGS 19755) NMDA receptor antagonists produce neuronal vacuolization and necrosis in the rat posterior cingulate/retrosplenial cortex. Male and female adult rats were treated intravenously with either vehicle (Tris) or 10 mg/kg or 50 mg/kg ACEA 1021. MK-801 (5 mg/kg, s.c.) served as positive control. Whereas MK-801 produced characteristic neuronal vacuolization and necrosis in the posterior cingulate/retrosplenial cortex, neither dose of ACEA 1021 had any effect on neuronal morphology. The absence of neuropathological changes in rats supports the further clinical evaluation of ACEA 1021 for stroke and head trauma, and suggests that glycine site antagonists may be devoid of neurotoxic potential.     

Radioligand binding studies reveal agmatine is a more selective antagonist for a polyamine-site on the NMDA receptor than arcaine or ifenprodil

Ifenprodil, arcaine and agmatine have all been reported to inhibit the NMDA receptor by actions at polyamine-sites, however the specific sites with which these compounds interact is unknown. Here we used radioligand binding of [3H]MK-801 to a membrane preparation from rat cerebral cortex to investigate the interactions of these compounds with the NMDA receptor complex. In the absence of exogenous polyamines, agmatine reduced [3H]MK-801 binding only at concentrations over 500 micro M, as opposed to the putative polyamine-site antagonists arcaine and ifenprodil which directly reduce ligand binding at much lower concentrations (5 micro M) in the absence of polyamines. In our studies, all three compounds significantly reduced spermidine-potentiated [3H]MK-801 binding, however agmatine was the only compound effective at concentrations below those that produced direct inhibition of [3H]MK-801 binding. Under these conditions, agmatine had a K(i)=14.8 micro M for spermidine-potentiated [3H]MK-801 binding and displayed characteristics of a competitive antagonist. Agmatine, as well as ifenprodil and arcaine, also displaced [3H]spermidine from rat cortical membranes at concentrations similar to those that were effective at reducing spermidine-potentiated [3H]MK-801 binding. In conclusion, these data suggest that agmatine reduces the potentiating effects of polyamines by competitive antagonism at a specific site on the NMDA receptor complex, and that these actions of agmatine differ from those of ifenprodil and arcaine.     

Kinetic mechanisms of glycine requirement for N-methyl-D-aspartate channel activation.

Glycine potentiates N-methyl-D-aspartate (NMDA) receptor-mediated responses via its interaction with a strychnine-insensitive glycine recognition site. We have previously shown that the potent glycine receptor antagonist 7-chlorokynurenic acid (7Cl-KYN) dose-dependently inhibits [3H]MK-801 binding to the PCP receptor and that this effect is reversed by glycine. [3H]MK-801 binding to the PCP receptor within the NMDA receptor-gated ion channel is a measure of channel activation. Association of PCP receptor ligands is biexponential with the fast component of binding serving as a marker of activated NMDA channels. In the present study we utilize 7Cl-KYN as a probe of the kinetic mechanism of the glycine effect upon NMDA receptor functioning. In the presence of L-glutamate, incubation with 7Cl-KYN completely abolished the fast component of [3H]MK-801 association in 4 out of 5 experiments. In the fifth experiment where the fast component was detected, it accounted for less than half of that seen in the presence of L-glutamate alone. 7Cl-KYN-induced inhibition of the fast component of [3H]MK-801 association was reversed by the addition of glycine. Since the fast component represents ligand binding to the PCP receptor via the open NMDA channel, selective reduction of this component by 7Cl-KYN indicates that glycine receptor antagonists reduce the probability of channel opening, and also that the selective reduction in the component of [3H]MK-801 binding that manifests fast kinetics can serve as a marker for glycine antagonists.     

In vitro and in vivo antagonistic activities of SM-31900 for the NMDA receptor glycine-binding site

The purpose of this study was to clarify the in vitro pharmacological profile and the in vivo activity of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900). SM-31900 inhibited the binding of [3H]glycine and [3H]5,7-dichlorokynurenic acid, radioligands for the N-methyl-D-aspartate (NMDA) receptor glycine-binding site, to rat brain membranes in a competitive manner, with K(i) values of 11+/-2 and 1.0+/-0.1 nM, respectively, and completely prevented the binding of [3H]dizocilpine (MK-801), a radioligand for the NMDA receptor channel site. In cultures of rat cortical neurons, SM-31900 markedly prevented the neuronal cell death induced by transient exposure to glutamate, in a concentration-dependent manner. Its neuroprotective potency was much stronger than those of other glycine-binding site antagonists (4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline (L-689,560), 5,7-dichlorokynurenic acid, and 7-chlorokynurenic acid). Furthermore, SM-31900 showed anticonvulsant activity when administered systemically, unlike other antagonists. These data indicate that SM-31900 is a systemically active antagonist with high affinity for the NMDA receptor glycine-binding site.     

Inhibition of N-methyl-D-aspartate evoked sodium flux by MK-801

The inhibition of N-methyl-D-aspartate (NMDA) stimulated 22Na+ efflux from rat hippocampal slices was studied using competitive and non-competitive receptor antagonists. There was a good correlation between the abilities of the competitive antagonists to block NMDA evoked 22Na+ efflux and their potencies as inhibitors of L-[3H]glutamate binding. The recently reported novel NMDA receptor antagonist, (+)-5-methyl-16,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) was shown to non-competitively inhibit NMDA stimulated 22Na+ efflux with an IC50 value of 0.4 microM. Relatively high (10 microM) concentrations of MK-801 had no effects on quisqualic acid, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), or kainic acid stimulated efflux. However, MK-801 was able to block 22Na+ efflux induced by ibotenic acid and L-homocysteic acid, amino acids that act as NMDA receptor agonists. MK-801, (-)-MK-801, and non-competitive NMDA receptor antagonists of the arylcyclohexylamine and dioxolane classes inhibited NMDA stimulated 22Na+ efflux with potencies that reflected their abilities to compete for [3H]MK-801 binding sites in rat cortical membranes. These results indicate the utility of the 22Na+ efflux assay in studying the properties of NMDA receptors and confirm the nature and selectivity of the inhibition of NMDA receptor linked ion channel activation by MK-801.     

Reducing conditions significantly attenuate the neuroprotective efficacy of competitive, but not other NMDA receptor antagonists in vitro

Inappropriate activation of NMDA receptors during a period of cerebral ischaemia is a crucial event in the pathway leading to neuronal degeneration. However, significant research has failed to deliver a clinically active NMDA receptor antagonist, and competitive NMDA antagonists are ineffective in many experimental models of ischaemia. The NMDA receptor itself has a number of modulatory sites which may affect receptor function under ischaemic conditions. Using rat organotypic hippocampal slice cultures we have investigated whether the redox modulatory site affects the neuroprotective efficacy of NMDA receptor antagonists against excitotoxicity and experimental ischaemia (OGD). NMDA toxicity was significantly enhanced in cultures pretreated with a reducing agent. The noncompetitive antagonist MK-801 and a glycine-site blocker were equally neuroprotective in both normal and reduced conditions, but there was a significant rightward shift in the dose-response curves of the competitive antagonists APV and CPP and the uncompetitive antagonist memantine. OGD produced neuronal damage predominantly in the CA1 region, which was prevented by MK-801 and memantine, but not by APV or CPP. Inclusion of an oxidizing agent during the period of OGD had no effect alone, but significantly enhanced the neuroprotective potency of the competitive antagonists. These data clearly demonstrate that chemical reduction of the redox modulatory site of the NMDA receptor decreases the ability of competitive antagonists to block NMDA receptor-mediated neuronal damage, and that the reducing conditions which occur during simulated ischaemia are sufficient to produce a similar effect. This may have important implications for the design of future neuroprotective agents.     

Rodent data and general hypothesis: antipsychotic action exerted through 5-HT2A receptor antagonism is dependent on increased serotonergic tone

Summary. The locomotor stimulation induced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) in mice was regarded as a model of at least some aspects of schizophrenia. The serotonin synthesis inhibitor dl-p-chlorophenylalanine (PCPA) was used to evaluate the involvement of endogenous serotonin in (a) the induction of MK-801-induced hyperlocomotion in NMRI mice, and (b) the inhibition of MK-801-induced hyperlocomotion by each of five monoaminergic antagonists (M100907, clozapine, olanzapine, raclopride, SCH23390). Further, brain monoaminergic biochemistry was characterised in rats and mice after various drug treatments. PCPA pretreatment did not significantly reduce MK-801-induced hyperlocomotion in any of the experiments performed; however in a meta-analysis of six experiments, the locomotion displayed by MK-801-treated animals was diminished 17% by PCPA pretreatment. The selective 5-HT2A receptor antagonist M100907 exerted a dose-dependent inhibition of MK-801-induced hyperlocomotion. This effect was abolished in mice pretreated with PCPA, but could be restored in a dose-dependent manner by restitution of endogenous 5-HT by means of 5-hydroxytryptophan (5-HTP). On the other hand, the inhibition of MK-801-induced hyperlocomotion exerted by the selective dopamine D-2 receptor antagonist raclopride or the dopamine D-1 receptor antagonist SCH23390 was unaffected by PCPA pretreatment. The antipsychotics clozapine and olanzapine displayed a split profile. Hence, the inhibitory effect on MK-801-induced hyperlocomotion exerted by low doses of these compounds was diminished after PCPA pretreatment, while inhibition exerted by higher doses was unaffected by PCPA. These results suggest that (1) MK-801-induced hyperlocomotion is accompanied by an activation of, but is not fully dependent upon, brain serotonergic systems. (2) In the hypoglutamatergic state induced by MK-801, endogenous serotonin exerts a stimulatory effect on locomotion through an action at 5-HT2A receptors, an effect that is almost completely counterbalanced by a concomitant inhibitory impact on locomotion, mediated through stimulation of serotonin receptors other than 5-HT2A receptors. M100907, by blocking 5-HT2A receptors, unveils the inhibitory effect exerted on locomotion by these other serotonin receptors. (3) Dopamine D-2 receptor antagonistic properties of antipsychotic compounds, when they come into play, override 5-HT2A receptor antagonism. Possible implications for the treatment of schizophrenia with 5-HT2A receptor antagonists are discussed. It is hypothesized that treatment response to such agents is dependent on increased serotonergic tone. Accepted February 9, 1998; received December 16, 1997     

Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801

There is experimental evidence indicating that the non-competitive NMDA receptor antagonist MK-801 impairs cognition and produces a series of schizophrenia-like symptoms in rodents (hypermotility, stereotypies, and ataxia). The present study was designed to investigate the efficacy of the nitric oxide (NO) donor molsidomine in counteracting these MK-801-induced behavioral effects in the rat. In a first study, post-training administration of molsidomine (at 4 but not 2 mg/kg) successfully antagonized MK-801-induced performance deficits in a recognition memory test. In a subsequent study, molsidomine (2 and 4 mg/kg) was shown to be unable to reverse MK-801-induced hypermotility but attenuated stereotypies (continuous movement whole cage, body sway, and head weaving) produced by MK-801. Moreover, at 4 mg/kg this NO donor counteracted MK-801-induced ataxia. Our findings indicate that molsidomine attenuates behavioral effects related to the hypofunction of the NMDA receptor suggesting that NO might be involved in the psychotomimetic effects of non-competitive NMDA receptor antagonists.     

A slow intravenous infusion of N-methyl-DL-aspartate as a seizure model in the mouse.

A seizure model involving slow i.v. infusion of the excitatory amino acid N-methyl-DL-aspartate (NMDLA) in the mouse is described. It allows determination of the threshold doses of NMDLA required to elicit clonic and tonic seizures in individual mice. The NMDA receptor antagonists MK-801, CPP, ifenprodil and 7-chlorokynurenic acid (7-CLK), and diazepam dose-dependently increased the dose of NMDLA required to elicit a tonic seizure. CPP, 7-CLK and diazepam also increased the dose of NMDLA inducing clonic seizures. In contrast, ifenprodil at doses which antagonised tonic seizures had no effect on clonic seizures. The glycine and polyamine modulatory site agonists, D-serine and spermidine respectively, dose-dependently reduced the dose of NMDLA required to induce clonic and tonic seizures. The NMDLA infusion model appears to be more sensitive than the classical bolus injection test and can detect both anticonvulsant and proconvulsant actions mediated by the NMDA receptor complex.     

Anti-convulsant and adverse effects of the glycineB receptor ligands, D-cycloserine and L-701,324: comparison with competitive and non-competitive N-methyl-D-aspartate receptor antagonists

In this study, the anticonvulsant and adverse effects of compounds that belong to four different categories of systemically available N-methyl-d-aspartate (NMDA) receptor ligands were compared, namely the competitive antagonist CGP 40116, the noncompetitive antagonist MK-801 (dizocilpine), the glycine_B receptor antagonist L-701,324, and the glycine_B receptor high-efficacy partial agonist d-cycloserine. The maximal electroshock seizures (MES), which are widely used to detect drug efficacy against generalized tonic–clonic seizures in humans, were produced by transcorneal electrical stimulation. Abolition of tonic hind-limb extension was taken as the end-point. The drug-induced motor and long-term memory deficits were quantified by using the inverted screen test and the step-through passive-avoidance test, respectively. All tested compounds exhibited significant anticonvulsant effect. The rank order of potency for the respective compounds was: MK-801 = CGP 40116 > L-701,324 ? d-cycloserine. All of these compounds induced motor impairment at doses close to those found to be anticonvulsant, however, hyperlocomotion and stereotyped behavior occurred only with MK-801. The highest protective indices [PI = TD₅₀ (inverted screen)/ED₅₀ (MES)] were calculated for CGP 40116 and d-cycloserine (2.4 and 2.2, respectively). When tested for memory impairment at one-half the MES ED₅₀, again only MK-801 induced significant memory disruption in the passive avoidance test. In conclusion, these results suggest that glycine_B receptor high-efficacy partial agonists and competitive NMDA receptor antagonists may be advantageous to noncompetitive NMDA antagonists and glycine_B receptor antagonists as potential antiepileptic drugs.     

Studies on neuronal apoptoisis in primary forebrain cultures: Neuroprotective/anti-apoptotic action of NR2B NMDA antagonists

While the role of apoptosis in neuronal injury is continually being re-defined, approaches to intervene in the progression of apoptotic injury have been documented to provide neuroprotection against a variety of insults. The present studies were undertaken to systematically study the effects of certain neuroprotective agents against neuronal apoptosis mediated by staurosporine (ST). ST (0.01-5 micro M) produced a dose-related apoptotic injury (as characterized by cellular morphology, 'Comet' assay analysis [single cell gel electrophoresis] and caspase-3 activation) in primary cultures of forebrain neurons. ST significantly increased caspase-3 activity. The NMDA receptor subtype non-selective antagonist dizocilpine [(+) MK-801; 0.1-50 micro M] and a novel sodium channel blocker RS100642 (1.0-250 micro M) had no significant effects against ST-induced neurotoxicity. Conversely, NR2B-selective NMDA receptor antagonists CGX-1007 (0.01-50 micro M) and ifenprodil (0.01-50 micro M) provided dose-dependent neuroprotection against ST-induced neurotoxicity (as measured by neuronal viability and comet assay analysis). CGX-1007 had no significant effect on ST-induced caspase-3 activity; however, ifenprodil did block activation of caspase-3. These studies demonstrate that NR2B NMDA receptor antagonists are anti-apoptotic and may mediate their action via mechanism(s) that are dependent or independent of caspase-3 activation.     

Effects of chronic administration of MK-801 upon local cerebral glucose utilisation and ligand binding to the NMDA receptor complex

Although clinical use of N-methyl-D-aspartate (NMDA) receptor antagonists will involve prolonged drug administration, knowledge of the functional consequences of chronic NMDA receptor blockade is limited. Local cerebral glucose utilisation was measured in conscious rats in 74 discrete brain regions after chronic administration of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine (MK-801) (0.5 mg/kg i.p.). Chronic treatment with MK-801 caused small, significant changes in glucose use in 4 of the 74 brain areas; parietal cortex (-13%), frontal cortex (-10%), subthalamic nucleus (-14%) and nucleus accumbens (-17%). These focal alterations in glucose use were not associated with changes in ligand binding to various sites within the NMDA receptor complex (i.e. agonist recognition site, glycine site, ion channel site) which were assessed autoradiographically. The acute effects of MK-801 on glucose utilisation were significantly enhanced after chronic MK-801 in 7 brain regions (e.g. frontal and parietal cortices) and attenuated in 6 brain regions (e.g. nucleus accumbens, hippocampus, posterior cingulate cortex). Neither local enhancement nor attenuation of the acute response to MK-801 was due to alterations in ligand binding to sites within the NMDA receptor complex. The data clearly indicate that the functional consequences of NMDA blockade are altered after chronic MK-801 treatment in an anatomically organised, though complex manner. These adaptive functional changes after chronic MK-801 treatment cannot be attributed readily to alterations in the NMDA receptor complex in affected regions.     

Intrahippocampal -cycloserine improves MK-801-induced memory deficits: radial-arm maze performance in rats

In order to investigate whether strychnine-insensitive glycine sites coupled with hippocampal NMDA (N-methyl- -aspartate) receptors are involved in spatial memory in rats, we examined the effects of intrahippocampal treatment of -cycloserine (DCS), a glycine-site agonist, on spatial-memory deficits which were produced by an NMDA antagonist MK-801 (dizocilpine) on the radial-arm maze task. After the acquisition of this task, the radial-maze performance was tested under the combined treatments of intraperitoneal MK-801 or saline (SAL) and intrahippocampal DCS or SAL. The results showed that MK-801 impaired the performance, and that DCS improved the MK-801-induced performance impairment. These results suggest that glycine sites are involved in spatial memory through their modulatory action on hippocampal NMDA receptors.