Efficacy of competitive vs noncompetitive blockade of the NMDA channel following traumatic brain injury

N-methyl-d-aspartate (NMDA) receptor antagonists have been demonstrated widely to be neuroprotective in cerebral ischemia, hypoxia, and traumatic brain injury. However, although noncompetitive NMDA antagonists have typically proven efficacious under all of these conditions, competitive antagonists have not been shown to be beneficial following moderate traumatic brain injury. The present study has used phosphorus magnetic resonance spectroscopy ([³¹P]MRS) to examine the effects of the competitive antagonist cis-4-(phosphonomethyl) piperidine-2-carboxylic acid (CGS-19755) and the noncompetitive antagonist dextromethorphan on biochemical outcome following fluid percussion-induced traumatic brain injury in rats. Five minutes prior to induction of moderate (2.8±0.2 atm) fluid percussion brain injury, animals received either CGS-19755 (10 mg/kg iv), dextromethorphan (10 mg/kg iv), or equal volume saline vehicle. [³¹P]MRS spectra were then acquired for 4 h post-trauma and intracellular pH, free magnesium concentration, cytosolic phosphorylation potential, and oxidative capacity determined. Both CGS-19755-treated animals and saline treated controls demonstrated significant and sustained declines in intracellular free magnesium concentration and bioenergetic status following trauma. In contrast, administration of dextromethorphan significantly attenuated free magnesium decline and improved bioenergetic state during the post-traumatic monitoring period. These results suggest that the neuroprotective actions of NMDA antagonists following traumatic brain injury are associated with attenuation of free magnesium decline and that such actions seem to be preferentially mediated by noncompetitive blockers.     

The action of CGS-19755 on the redox enhancement of NMDA toxicity in rat cortical neurons in vitro.

The effects of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) were studied in cultures of rat cerebral cortex under normal and altered redox conditions. CGS-19755 was effective in preventing delayed neuronal death produced by an acute exposure to either glutamate (500 microM) or NMDA (200 microM), but was ineffective in protecting neurons against the toxicity induced by a prolonged exposure to kainate (500 microM). We observed that the reducing agent dithiothreitol (DTT, 500 microM), could dramatically enhance toxicity and electrophysiological responses produced by 50 microM NMDA. CGS-19755 (100 microM) could effectively block both of these effects of DTT. Any toxicity produced by DTT alone was also antagonized by CGS-19755. In contrast, oxidized DTT did not enhance NMDA toxicity nor was it toxic when added alone. These results indicate that CGS-19755 is an effective and specific neuroprotectant acting at the NMDA receptor in vitro, and that the enhancement in NMDA toxicity induced by DTT is mediated by an increase in activity at this receptor complex.     

Glutamate receptor antagonists modulate heat shock protein response in focal brain ischemia

Neurons and glia reacting to ischemic injury exhibit delayed expression of heat shock proteins (HSPs). We tested the hypothesis that glutamate receptor antagonists alter neuronal and glial activation during focal cerebral ischemia, as shown by spatio-temporal changes in HSP immunoreactivity. Rats underwent focal ischemia by permanent occlusion of the middle cerebral artery. All animals were pre-treated with NBQX (30 mg kg-1), a competitive antagonist of the AMPA/kainate receptor, or CGS-19755 (10 mg kg-1), a competitive NMDA receptor antagonist, and euthanatized after 6 or 24 h of ischemia to demonstrate regional immunoreactivity of HSP-72 or 32 in brain. Neurons immunolabeled for HSP-72 appeared in the penumbral region adjacent to the infarct at 24 h and increased in number and distribution after pretreatment with NBQX or CGS-19755. Immunolabeling for HSP-32 revealed that pre-treatment with CGS-19755 caused ramified glia to infiltrate the ischemic cortex at 6 h, a pattern that was not seen in ischemic controls until 24 h. Blockade of the NMDA or AMPA/kainate receptor modulates cellular stress responses in both neurons and glia within the developing infarct. We conclude that early, rather than delayed, expression of HSP-32 is a sensitive indicator of glial activation induced specifically by CGS-19755.     

CGS-19755 and MK-801 selectively prevent rat striatal cholinergic and gabaergic neuronal degeneration induced by N-methyl-D-aspartate and ibotenate in vivo

Summary The in vivo efficacies and potencies of various excitatory amino acid agonists in inducing cholinergic neuronal degeneration were compared following unilateral injections into the rat striatum. Kainic acid (KA), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), ibotenic acid (IBO), and N-methyl-D-aspartic acid (NMDA) all produced dose-related decreases in choline acetyltransferase (ChAT) activity. The relative order of potency was KA>AMPA>IBO>NMDA. Quisqualic acid (QUIS) was about as potent as NMDA, but the maximal decrease in ChAT activity was less (36%). N-acetylaspartyl-L-glutamate (NAAG) did not significantly decrease ChAT activity when up to 1,000 nmoles was injected. Approximate equitoxic doses of agonists were then used to examine the ability of i.p. administered CGS-19755 and MK-801 to prevent in vivo excitatory amino acid-induced cholinergic and GABAergic neuronal degeneration. NMDA-induced decreases in ChAT and glutamic acid decarboxylase (GAD) activities were prevented by CGS-19755 (10–40 mg/kg) and MK-801 (1–10 mg/kg). CGS-19755 (40 mg/kg) and MK-801 (10 mg/kg) did not prevent loss of ChAT or GAD induced by KA or AMPA, but did prevent the degenerative effects of IBO. This study shows that CGS-19755 and MK-801, two NMDA receptor antagonists that act by different mechanisms, are completely selective following systemic administration. Moreover, the in vivo excitotoxic effects of IBO are mediated at NMDA receptor sites that are blocked by these compounds.     

Glutamate receptor antagonists modulate heat shock protein response in focal brain ischemia

Abstract

Neurons and glia reacting to ischemic injury exhibit delayed expression of heat shock proteins (HSPs). We tested the hypothesis that glutamate receptor antagonists alter neuronal and glial activation during focal cerebral ischemia, as shown by spatio–temporal changes in HSP immunoreactivity. Rats underwent focal ischemia by permanent occlusion of the middle cerebral artery. All animals were pre-treated with NBQX (30 mg kg^-1), a competitive antagonist of the AMPA/kainate receptor, or CGS-19755 (10 mg kg^-1), a competitive NMDA receptor antagonist, and euthanatized after 6 or 24 h of ischemia to demonstrate regional immunoreactivity of HSP-72 or 32 in brain. Neurons immunolabeled for HSP-72 appeared in the penumbral region adjacent to the infarct at 24 h and increased in number and distribution after pretreatment with NBQX or CGS-19755. Immunolabeling for HSP-32 revealed that pre-treatment with CGS19755 caused ramified glia to infiltrate the ischemic cortex at 6 h, a pattern that was not seen in ischemic controls until 24 h. Blockade of the NMDA or AMPA/kainate receptor modulates cellular stress responses in both neurons and glia within the developing infarct. We conclude that early, rather than delayed, expression of HSP-32 is a sensitive indicator of glial activation induced specifically by CGS-19755.     

Quantitative assessment of neuroprotection against NMDA-induced brain injury

In immature rodent brain, unilateral intrastriatal injections of selected excitatory amino acid (EAA) receptor agonists, such as N-methyl-D-aspartate (NMDA), produce prominent ipsilateral forebrain lesions. In Postnatal Day (PND) 7 rats that receive a right intrastriatal injection of NMDA (25 nmol) and are sacrificed 5 days later, there is a considerable and consistent reduction in the weight of the injected cerebral hemisphere relative to that of the contralateral side (-28.5 +/- 1.9%, n = 6). In animals treated with specific NMDA receptor antagonists, the severity of NMDA-induced damage is markedly reduced. We have previously reported that the efficacy of potential neuroprotective drugs in limiting NMDA-induced lesions can be assessed quantitatively by comparison of hemisphere weights after a unilateral NMDA injection. In this study, we compared three quantitative methods to evaluate the severity of NMDA-induced brain injury and the degree of neuroprotection provided by NMDA receptor antagonists. We characterized the severity of brain injury resulting from intrastriatal injections of 1-50 nmol NMDA in PND 7 rats sacrificed on PND 12 by (i) comparison of cerebral hemisphere weights; (ii) assay of the activity of the cholinergic neuronal marker, choline acetyltransferase (ChAT) activity; and (iii) measurement of regional brain cross-sectional areas. The severity of the resulting brain injury as assessed by comparison of hemisphere weights increased linearly with the amount of NMDA injected into the striatum up to 25 nmol NMDA. The magnitude of injury was highly correlated with the degree of reduction in ChAT activity (r2 = 0.97).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective interaction effects of NMDA and AMPA receptor antagonists in an in vitro model of cerebral ischemia

An in vitro model of ischemia was developed and characterized using the acute rat hippocampal slice preparation. Neuroprotective concentrations of several competitive and noncompetitive glutamate subtype-selective antagonists (CGS-19755, MK-801, YM90K and GYKI-52466) were initially determined in anoxia-enhanced agonist-induced excitotoxicity experiments. Concentrations which proved to be effective in these studies were subsequently tested for their effectiveness against an ischemic episode. Ischemia was defined as a 30-min exposure to aglycemic media ending in 5 min of concurrent anoxia, a protocol which was arrived at by empirically determining the effect of various hypoglycemic and anoxic insults on the ability of hippocampal slices to retain their electrophysiological viability. Exposure to such an ischemic episode resulted in a loss of viability by most slices, an effect which was strongly dependent on extracellular calcium. AMPA antagonists applied alone produced no neuroprotective effect in the present model of in vitro ischemia, while NMDA antagonists applied alone had a modest neuroprotective effect. In contrast, the coapplication of 10 μM MK-801 and 300 μM GYKI-52466, noncompetitive NMDA and AMPA receptor antagonists, respectively, resulted in almost complete neuroprotection. This protection was comparable to that obtained by withholding extracellular calcium, indicating that the toxic effects of glutamate receptor overstimulation can be accounted for solely by calcium influx. The effect of this combination treatment on the survival rate of hippocampal slices was synergistic, that is greater than the sum of the effects of the individual compounds. The results indicate that neuroprotection against acute ischemic insults may require a combination therapy approach.     

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)     

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)     

Lack of neuroprotective effect of some sigma ligands in a model of focal cerebral ischemia in the mouse.

The potential neuroprotective effect of seven sigma ligands has been evaluated in a mouse model of focal cerebral ischemia (induced by coagulation of the left middle cerebral artery) and compared to that of known N-methyl-D-aspartate (NMDA) antagonists. When given after the induction of cerebral ischemia, the NMDA receptor antagonists dizocilpine and CGS 19755 and the mixed NMDA antagonist/sigma ligand eliprodil (SL 82.0715) afforded very substantial protection against cortical infarction (92, 44 and 72%, at the doses of 1, 10 and 10 mg/kg, i.p., for dizocilpine, CGS 19755 and eliprodil, respectively). In contrast, none of the sigma ligands investigated--DTG, DMTG, GBR 12909, (+)- and (-)-3-PPP (up to 10 mg/kg), BMY 14802 (up to 30 mg/kg), except haloperidol at a high dose (3 mg/kg)--had neuroprotective effects.     

Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.     

The effects of a competitive NMDA receptor antagonist (CGS-19755) on cerebral blood flow and pH in focal ischemia.

We report the effects of intravenous infusion of CGS-19755, a potent competitive N-methyl-D-aspartate (NMDA) antagonist, on local cerebral pH (LCpH) and local CBF (LCBF) in rats with occluded left middle cerebral and common carotid arteries. LCpH and LCBF were determined simultaneously by a double-label autoradiographic technique 4 h after vascular occlusion in three groups: no treatment, carrier infused, and a group receiving CGS-19755 at 10 mg/kg bolus immediately after occlusion followed by infusion at 5 mg kg-1 h-1 for 4 h. Compared with rats receiving carrier, several cortical structures on the side of occlusions showed significantly higher CBF in rats receiving CGS-19755. This drug also corrected the pH in several left cortical structures to values significantly higher than in the rats receiving carrier. The correction in LCpH was not limited to those regions showing significant elevations in LCBF. In the nonoccluded hemisphere, CGS-19755 significantly increased the hemispheric mean blood flow from 122 +/- 17 to 221 +/- 64 ml 100 g-1 min-1 (mean +/- SD of all structures, p less than 0.01) without any changes in LCpH. Cortical but not basal ganglia infarct volume was significantly smaller in rats receiving CGS-19755 than in the carrier-treated group. These results suggest that, at least partially, the neuroprotective effect of CGS-19755 in ischemia may be related to changes in CBF and pH in addition to its antagonist effect on the NMDA receptor.     

NMDA receptor antagonists influence early development of GABAergic interneurons in the mammalian striatum

Neurotransmitters influence a wide variety of developmental processes. We hypothesize that N-methyl-d-aspartate (NMDA) glutamate receptors influence proliferation of populations of forebrain neurons. As our model, we use a subclass of GABAergic striatal interneurons that express the calcium binding protein parvalbumin (PV). To separate proliferative and post-proliferative effects of NMDA receptor antagonists on PV neurons, we first determined the birth-date of rat striatum PV neurons at the coronal level selected for analysis. Dividing striatal progenitor cells were marked by intraperitoneal injections of 5′-bromodeoxyuridine (BrdU) given to timed pregnant rats at selected time points between embryonic days (E) 12–22. Double immunohistochochemistry for BrdU and PV was used in adult progeny to determine the time course of neurogenesis of striatal PV neurons. The results of the neurogenetic analysis were then used for rational timing of treatment with competitive (CGS19755) and non-competitive (MK-801) NMDA receptor antagonists. In comparison to pair-fed and vehicle-injected controls, gestational rats given CGS-19755 and MK-801 during the proliferative phase (E15–E18) showed a marked reduction of striatal PV neuron cell density as adults. In contrast, animals given NMDA antagonists during the post-proliferative period (E18–E21) showed no significant reduction in PV neuron cell density compared to pair-fed controls. These results suggest that glutamate influences cell proliferation of a population of striatal neurons by an NMDA-mediated mechanism, providing evidence for a novel role for excitatory amino acids in early forebrain development.     

Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in vitro and improvesoOutcome after brain trauma

The effects of selective blockade of group I metabotropic glutamate receptor subtype 1 (mGluR1) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. The selective mGluR1 antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), and (S)-(+)-alpha-amino-4-carboxy-2-methylbezeneacetic acid (LY367385) provided significant neuroprotection in rat cortical neuronal cultures subjected to mechanical injury, in both pretreatment or posttreatment paradigms. Administration of the antagonists also attenuated glutamate-induced neuronal cell death in the cultures. Coapplication of these antagonists with the N-methyl-d-aspartate (NMDA) receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had additive neuroprotective effects in glutamate injured cultures. Intracerebroventricular administration of AIDA to rats markedly improved recovery from motor dysfunction after lateral fluid percussion induced traumatic brain injury (TBI). Treatment with mGluR1 antagonists also significantly reduced lesion volumes in rats after TBI, as evaluated by MRI. It appears that these compounds mediate their neuroprotective effect through an mGluR1 antagonist action, as demonstrated by inhibition of agonist induced phosphoinositide hydrolysis in our in vitro system. Moreover, AIDA, CPCCOEt, and LY367385, at concentrations shown to be neuroprotective, had no significant effects on the steady state NMDA evoked whole cell current. Taken together, these data suggest that modulation of mGluR1 activity may have substantial therapeutic potential in brain injury.     

Recombinant plasma-type platelet-activating factor acetylhydrolase attenuates NMDA-induced hippocampal neuronal apoptosis

The bioactive lipid platelet-activating factor (PAF) accumulates in brain during injury, seizures and ischemia and may, in addition, be significant in AIDS dementia and in other neurodegenerative diseases. We have used plasma-type recombinant PAF acetylhydrolase (rPAF-AH) to test the hypothesis that PAF accumulation is involved in early events leading to neuronal apoptosis during excitotoxic neuronal injury. Neuronal cultures were labeled with FITC-12-dUTP (TUNEL technique) and propidium iodide, digitized using fluorescence microscopy and a chilled 3CCD color camera, and analyzed with 2D graphics analysis software. N-methyl-D-aspartate (NMDA) (50 μM, 2 hr) induced a 2.5-fold increase in apoptosis of hippocampal neurons compared with controls when analyzed 24 hr after NMDA treatment. Hippocampal neurons receiving rPAF-AH (20 μg/ml) before, during, and after NMDA treatment demonstrated a concentration-dependent neuroprotective effect which resulted in 47% and 30% neuroprotection against 50 and 100 μM NMDA, respectively. The noncompetitive NMDA receptor antagonist MK-801(300 nM) completely inhibited apoptosis caused by NMDA. The neuroprotective effect of rPAF-AH against NMDA-induced apoptosis was confirmed using as additional criteria, histone release, electron microscopy, and DNA laddering. Neuroprotection elicited by rPAF-AH demonstrates that PAF is an injury mediator in NMDA-induced neuronal apoptosis and that the recombinant protein is potentially useful as a therapeutic approach. J. Neurosci. Res. 53:677–684, 1998. © 1998 Wiley-Liss, Inc.     

Phencyclidine-induced discriminative stimulus is mediated via phencyclidine binding sites on the N-methyl-D-aspartate receptor-ion channel complex, not via sigma(1) receptors

The effects of several N-methyl-D-aspartate (NMDA) receptor- and sigma receptor-related compounds on the discriminative stimulus effects of phencyclidine (PCP) were examined in rats trained to discriminate PCP (1.5 mg/kg, i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. PCP produced a dose-dependent increase in PCP-appropriate responding. A non-competitive NMDA receptor antagonist, dizocilpine (0.2 mg/kg, i.p.) and a putative sigma(1) receptor agonist, (+)-SKF-10047 (10 mg/kg, i.p.) fully substituted for PCP in every rat tested. Neither a competitive NMDA receptor antagonist, CGS-19755 (0.1-3 mg/kg, i.p.), sigma(1) receptor agonist, (+)-pentazocine (10-30 mg/kg, i.p.) nor dextromethorphan (10-20 mg/kg, i.p.) produced PCP-like discriminative stimulus effects. The discriminative stimulus effects of PCP (1.5 mg/kg, i.p.), dizocilpine (0.2 mg/kg, i.p.) and (+)-SKF-10047 (10 mg/kg, i.p.) were significantly attenuated by CGS-19755 (1 mg/kg, i.p.), but not by sigma(1) receptor antagonist BMY-14802 (10 mg/kg, i.p.) and NE-100 (5 mg/kg, i.p.). These results suggest that the discriminative stimulus effects of PCP are predominantly mediated via PCP binding sites on the NMDA receptor-ion channel complex, not via sigma(1) receptors. In addition, the PCP-like discriminative stimulus effects of (+)-SKF-10047 were demonstrated to be mediated via PCP binding sites.     

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.     

Interaction of the putative essential nutrient pyrroloquinoline quinone with the N-methyl-D-aspartate receptor redox modulatory site.

The putative essential nutrient pyrroloquinoline quinone (PQQ) can efficiently mediate reduction and oxidation reactions in a variety of systems. Therefore, we investigated whether this compound could alter the function of the NMDA receptor via a recently described redox modulatory site. In rat cortical neurons in vitro, 50 microM PQQ could reverse the enhancement of 30 microM NMDA-induced whole-cell ionic currents produced by the reducing agent dithiothreitol (DTT; 2-4 mM). PQQ also depressed native responses in a DTT-reversible fashion. In addition, 50-200 microM PQQ produced a significant degree of neuroprotection in an acute model of NMDA-mediated neurotoxicity in astrocyte-rich cultures of rat cerebral cortex. Under certain conditions, PQQ can lead to the formation of oxygen-derived free radicals, and we have previously observed that these reactive species can oxidize the NMDA receptor. Nevertheless, the enzymatic free radical scavengers superoxide dismutase and catalase (10 micrograms/ml each) did not abolish the actions of PQQ. This observation held true even in astrocyte-poor cortical cultures, where neuronal processes are directly exposed to the extracellular milieu. Therefore, under in vitro conditions in which PQQ is presented without an exogenous electron donor, it appears as if the entire neuroprotective effect of PQQ is attributable to a direct oxidation of the NMDA receptor redox site. These results suggest the possibility of a novel role for PQQ, PQQ-like substances, and quinone-containing proteins in the brain, and may represent a novel therapeutic approach for the amelioration of NMDA receptor-mediated neurotoxic injury.     

The kappa opioid-related anticonvulsants U-50488H and U-54494A attenuate N-methyl-D-aspartate induced brain injury in the neonatal rat.

The neuroprotective effects of the kappa opioid-related anticonvulsants U-50488H and U-54494A were tested in a model of N-methyl-D-aspartate (NMDA)-induced brain injury in the neonatal rat. Seven-day-old rat pups were injected intracerebrally with 7.5 nmol NMDA. Five days later, the ensuing unilateral hemisphere weight reduction was measured and used to assess the severity of insult. Control animals (n = 85) exhibited a 21.7 +/- 0.5% hemisphere weight reduction. Animals treated with U-54494A in split doses before and after NMDA administration showed significant neuroprotection at 10, 15, and 20 mg/kg, with the maximum effect observed at 15 mg/kg (33.8% protection). Animals treated with U-50488H on a similar dosing schedule showed significant neuroprotection at all doses tested, with peak protection observed at 30 mg/kg (51.8% protection). Both compounds exhibited a neuroprotective effect when hemisphere cross-sectional area and hippocampal histology were assessed. Treatment with U-54494A after NMDA administration also afforded neuroprotection at various doses, as measured by hemisphere weight disparity, with peak protection occurring at a dose of 20 mg/kg (32.4% protection). These data show that both U-50488H and U-54494A afford neuroprotection against NMDA-induced neuronal injury in the neonatal rat brain.     

Neuroprotectant effects of LY 274614, a structurally novel systemically active competitive NMDA receptor antagonist

Summary Antagonists for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may have therapeutic potential as neuroprotectant agents in conditions of neuronal degeneration that include brain ischemia, Huntington's chorea, and Alzheimer's disease. Here we have investigated the pharmacological actions of LY 274614, a structurally novel competitive NMDA receptor antagonist, for pharmacological selectivity and neuroprotectant effects following systemic administration. LY 274614 potently displaced NMDA receptor ([³H]CGS19755) binding (IC₅₀=58.8±10.3 nM), but had no appreciable affinity at [³H]AMPA or [³H]kainate receptor sites at up to 10,000 nM. NMDA-induced convulsions in neonatal rats or NMDA-induced lethality in mice are potently and selectively antagonized by i.p. or p.o. LY 274614. Oral doses showed a delayed but prolonged duration of effect. In adult rats, the neuro-degenerative effects (loss of choline acetyltransferase activity) following the intrastriatal infusions of NMDA or quinolinate, but not kainate, were prevented by LY 274614 (2.5 to 20mg/kg i.p.). LY 274614 is an effective neuroprotectant agent against NMDA receptor-induced toxicity when administered systemically and is a promising therapeutic agent for conditions where glutamate plays a role in the pathology of neuronal degeneration.