dizocilpine-maleate and ifenprodil

A growing body of evidence indicates that the mesolimbic dopaminergic (DAergic) pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.) play a critical role in the initiation of psychological dependence on morphine. As well as DAergic system, the involvement of non-DAergic neurotransmitter and neuromodulator systems in rewarding effects induced by morphine has been recently documented. We previously demonstrated that the morphine-induced rewarding effect was dramatically suppressed by co-treatment with NMDA receptor antagonists, such as dizocilpine (MK-801), ketamine and ifenprodil. Therefore, we propose here that inhibiting the N-methyl-D-aspartate (NMDA) receptor and its associated protein kinase in the N.Acc. is useful for the treatment for psychological dependence on morphine. The following review provides a summary of recent our findings regarding the role of NMDA receptor and its associated protein kinase in the development of psychological dependence on morphine.

Topics: Animals; Disks Large Homolog 4 Protein; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Humans; Intracellular Signaling Peptides and Proteins; Ketamine; Membrane Proteins; Mice; Morphine Dependence; Nucleus Accumbens; Piperidines; Protein Kinase C; Receptors, N-Methyl-D-Aspartate; Signal Transduction

We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms.. Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect.. Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect.. Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

Topics: Animals; Corpus Striatum; Dizocilpine Maleate; Dopamine; Dopamine Antagonists; Dopamine Plasma Membrane Transport Proteins; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Haloperidol; Hypoxia; Ligands; Nomifensine; Ouabain; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, sigma

The N-methyl-D-aspartate (NMDA) receptor has been recently identified as an important mediator of impulsive choice, as assessed in delay discounting. Although discounting is independently influenced by sensitivity to reinforcer magnitude and delayed reinforcement, few studies have examined how NMDA receptor ligands differentially affect these parameters.. The current study examined the effects of various NMDA receptor ligands on sensitivity to reinforcer magnitude and delayed reinforcement in a delay-discounting procedure.. Following behavioral training, rats received treatments of the following NMDA receptor ligands: the uncompetitive antagonists ketamine (0, 1.0, 5.0, or 10.0 mg/kg; i.p.), MK-801 (0, 0.003, 0.01, or 0.03 mg/kg; s.c.), and memantine (0, 2.5, 5.0, or 10.0 mg/kg; i.p.), the competitive antagonist CGS 19755 (0, 5.0, 10.0, or 20.0 mg/kg; s.c.), the non-competitive NR2B subunit-selective antagonist ifenprodil (0, 1.0, 3.0, or 10.0 mg/kg; i.p), and the partial agonist D-cycloserine (0, 3.25, 15.0, or 30.0 mg/kg; s.c.).. When an exponential model was used to describe discounting, CGS 19755 (5.0 mg/kg) increased impulsive choice without altering sensitivity to reinforcer magnitude. Conversely, ketamine (10.0 mg/kg), memantine (5.0 mg/kg), and ifenprodil (10.0 mg/kg) decreased sensitivity to reinforcer magnitude without altering impulsive choice. MK-801 and D-cycloserine did not alter delay-discounting performance, although two-way ANOVA analyses indicated D-cycloserine (15.0 mg/kg) decreased impulsive choice.. The behavioral changes observed in delay discounting following administration of NMDA receptor antagonists do not always reflect an alteration in impulsive choice. These results emphasize the utility in employing quantitative methods to assess drug effects in delay discounting.

Topics: Animals; Behavior, Animal; Choice Behavior; Cycloserine; Delay Discounting; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Impulsive Behavior; Ketamine; Ligands; Male; Memantine; Pipecolic Acids; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Reinforcement, Psychology

Drugs of abuse act as reinforcers because they influence learning and memory processes resulting in long-term memory of drug reward. We have previously shown that mice conditioned by fixed daily dose of cocaine (Fix-C) or daily escalating doses of cocaine (Esc-C) resulted in short- and long-term persistence of drug memory, respectively, suggesting different mechanisms in acquisition of cocaine memory. The present study was undertaken to investigate the differential contribution of N-methyl-D-aspartate receptor (NMDAR) subunits in the formation of Fix-C and Esc-C memory in C57BL/6J mice. Training by Esc-C resulted in marked elevation in hippocampal expression of Grin2b mRNA and NR2B protein levels compared with training by Fix-C. The NR2B-containing NMDAR antagonist ifenprodil had similar attenuating effects on acquisition and reconsolidation of Fix-C and Esc-C memory. However, the NMDAR antagonist MK-801 had differential effects: (1) higher doses of MK-801 were required for post-retrieval disruption of reconsolidation of Esc-C memory than Fix-C memory; and (2) pre-retrieval MK-801 inhibited extinction of Fix-C memory but it had no effect on Esc-C memory. In addition, blockade of NMDAR downstream signaling pathways also showed differential regulation of Fix-C and Esc-C memory. Inhibition of neuronal nitric oxide synthase attenuated acquisition and disrupted reconsolidation of Fix-C but not Esc-C memory. In contrast, the mitogen-activating extracellular kinase inhibitor SL327 attenuated reconsolidation of Esc-C but not Fix-C memory. These results suggest that NMDAR downstream signaling molecules associated with consolidation and reconsolidation of cocaine-associated memory may vary upon changes in the salience of cocaine reward during conditioning.

Topics: Aminoacetonitrile; Animals; Cocaine; Conditioning, Operant; Dizocilpine Maleate; Dopamine Uptake Inhibitors; Excitatory Amino Acid Antagonists; Male; Memory; Mice, Inbred C57BL; Nitric Oxide Synthase Type I; Piperidines; Receptors, N-Methyl-D-Aspartate; Reward

Epidemiological studies have shown that early life adverse events have long-term effects on the susceptibility to subsequent stress exposure in adolescence, but the precise mechanism is unclear. In the present study, mice on postnatal day 21-28 were randomly assigned to either a group or isolated cages for 8 weeks. The socially isolated (SI) mice exhibited a higher level of spontaneous locomotor activity, a longer duration of immobility in the forced swimming test (FST), significantly less prepulse inhibition (PPI) and an increase in aggressive (but not attack) behavior. However, acute stress markedly exacerbated the attack counts of the SI mice but did not affect the group housing (GH) mice. SI mice exhibited higher synaptosomal NR2A and NR2B levels in the hippocampus as compared to the GH mice. Whole-cell patch clamp recordings of CA1 neurons in hippocampal slices showed that the SI mice exhibited a higher input-output relationship of NMDAR-EPSCs as compared to the GH mice. Application of the NR2B -specific antagonist ifenprodil produced a greater attenuating effect on NMDAR-EPSCs in slices from the SI mice. NMDAR EPSCs recorded from the SI mice had a slower deactivation kinetic. MK-801, CPP and ifenprodil, the NMDA antagonists, reversed acute stress-induced exaggeration of aggressive and depressive behaviors. Furthermore, acute stress-induced exacerbation of attack behavior in the SI mice was abolished after the knockdown of NR2B expression. These results suggest that social isolation-induced increased expression of NMDA receptors in the hippocampus involves stress exacerbation of aggressive behaviors. Amelioration of aggressive behaviors by NMDA antagonists may open a new avenue for the treatment of psychopathologies that involve outbursts of emotional aggression in neglected children.

Topics: Affective Symptoms; Aggression; Animals; Animals, Newborn; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Exploratory Behavior; Gene Expression Regulation; Hippocampus; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Neurons; Piperidines; Prepulse Inhibition; Random Allocation; Receptors, N-Methyl-D-Aspartate; Reflex, Startle; Social Isolation; Swimming; Synaptosomes

The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR, the channel binding site labeled with [(3)H]MK-801 and the [(3)H]ifenprodil binding site. Most analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR inhibition by the majority of the compounds was reminiscent of other long-chain dicationic NR blockers. The potency of the parent compound as NR blocker was increased by modifying the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive bisbenzamidines to a recently described acidic interface between the N-terminal domains of GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain contributed by GluN1 as the most relevant target.

Topics: Animals; Binding Sites; Brain; Dizocilpine Maleate; Molecular Docking Simulation; Pentamidine; Piperazine; Piperazines; Piperidines; Protein Structure, Tertiary; Rats; Receptors, N-Methyl-D-Aspartate; Tritium

NMDA antagonists consistently produce social inhibition in adult animals, although effects of these manipulations on social behavior of adolescents are relatively unknown.. The aim of this study was to assess potential age differences in the socially inhibitory effects of the non-competitive NMDA antagonist, MK-801, as well as NR2 subunit selective effects, given the regional and developmental differences that exist for the NR2 subunit during ontogeny.. In separate experiments, adolescent and adult male Sprague-Dawley rats were treated acutely with MK-801 (0, 0.05, 0.1, 0.2 mg/kg, i.p.), the NR2A antagonist, PEAQX (2.5, 5, 10, 20 mg/kg, s.c.), or the NR2B antagonist, ifenprodil (1.5, 3, 6, 12 mg/kg, i.p.), 10 min prior to a social interaction test.. Adolescents required higher doses of MK-801 (0.1 and 0.2 mg/kg) to induce social suppression, whereas adults demonstrated reductions in social activity after all doses. Likewise, adolescents required higher doses of ifenprodil (6 and 12 mg/kg) to produce social inhibitory effects relative to adults (all doses). In contrast, adults were less sensitive to PEAQX than adolescents, with adults showing social inhibition after 20 mg/kg whereas adolescents showed this effect following 10 and 20 mg/kg. Although locomotor activity was generally reduced at both ages by all drugs tested, ANCOVAs using locomotor activity as a covariate revealed similar patterns of social inhibitory effects.. Adolescents are less sensitive than adults to the disruption of social behavior by NMDA and NR2B-selective receptor antagonism, but not by an NR2A antagonist-age differences that may be related to different subunit expression patterns during development.

Topics: Aging; Animals; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Inhibition, Psychological; Male; Motor Activity; Piperidines; Quinoxalines; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Social Behavior

Obesity is a worldwide epidemic that is increasing at an alarming rate. One of its causes is the increased availability and consumption of diets rich in fat. In the present study, we investigated the effects of short-term consumption of a high fat diet (HFD) on dietary preferences in Swiss CD1 mice and its relation in time to specific metabolic effects. Mice that were weaned 21days postpartum and fed a chow diet for one week were afterward subjected to a diet preference test for 5days, exposed to both a regular diet (RD) and HFD. We found that mice did not show any preferences. In a second experiment, two groups of mice that were weaned 21days postpartum and subjected to a chow diet for one week were fed either RD or HFD for 18days, and a diet preference test was performed for 5days. After this short-term consumption of HFD, mice preferred HFD, while mice subjected to RD did not show any preference. Importantly, no differences in blood glucose levels were found between the groups prior to and after the experiments. The results support our hypothesis that the preference for HFD is not a spontaneous behavior in CD1 mice, but it can be observed after short-term consumption; additionally, this preference develops before metabolic effects appear. Finally, this preference for HFD could not be observed when the mice were i.p. injected daily with low doses of the NMDA receptor antagonists, ketamine, ifenprodil or MK-801 during the HFD feeding period. These data suggest that acquisition of dietary preference for HFD is a NMDA receptor-dependent learning process.

Topics: Animals; Blood Glucose; Diet, High-Fat; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Food Preferences; Ketamine; Male; Mice; Mice, Inbred Strains; Piperidines; Receptors, N-Methyl-D-Aspartate

Adolescents display high levels of interactions with peers relative to other age groups, with these interactions further enhanced by ethanol under some circumstances. Understanding of the neural mechanisms underlying these high levels of social interactions is important given that alcohol use is initiated during adolescence and adolescents tend to report drinking for social reasons. Given that ethanol's effects are associated in part with functional antagonism of the NMDA receptor system, the current experiment explored the role of NMDA antagonists for facilitating adolescent social behavior. Adolescent male Sprague-Dawley rats were challenged acutely with either the non-competitive NMDA antagonist, MK-801 (0.01, 0.03mg/kg), the NR2A antagonist, PEAQX (1.25, 3.75mg/kg) or the NR2B antagonist, ifenprodil (0.75, 2.25mg/kg) 30min prior to a 10-min social interaction test. All compounds generally increased overall social activity (i.e., sum of social investigation, contact behavior, and play), with ifenprodil also significantly enhancing play and social contact behaviors. Although the frequencies of peer-directed social behaviors were typically greater following administration with these NMDA antagonists, social preference, indexed via the number of crossovers to the side with the partner relative to crossovers away, was significantly reduced in MK-801 and PEAQX-treated rats. None of these changes were associated with concomitant alterations in overall locomotor activity under these test circumstances. These data support the suggestion that the increases in social interactions observed in adolescents following acute ethanol may be driven in part by NMDA receptor antagonism - particularly of the NR2B subunit - given that ifenprodil stimulated social behavior in a manner similar to that produced by low doses of ethanol.

Topics: Animals; Behavior, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Male; N-Methylaspartate; Piperidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Social Facilitation

Hippocampal sclerosis, the main pathological sign of chronic temporal lobe epilepsy (TLE), is associated with oxidative injury, altered N-methyl d-aspartate receptor (NMDAR) stoichiometry, and loss of hippocampal neurons. However, the mechanisms that drive the chronic progression of TLE remain elusive. Our previous studies have shown that NADPH oxidase activation and ERK 1/2 phosphorylation are required for the up-regulation of the predominantly pre-synaptic NR2B subunit auto-receptor in both in vitro and in vivo pilocarpine (PILO) models of TLE. To provide further understanding of the cellular responses during the early-stages of hyper excitability, we investigated the role of oxidative damage and altered NR2B functions. In rat primary hippocampal cultures, we found that N-acetylcysteine (NAC) prevented PILO-mediated thiol oxidation, apoptosis, cell death and NR2B subunit over-expression. Interestingly, NAC did not block thiol oxidation when added to the neurons 6h after the PILO exposure, suggesting that disulfide formation could rapidly become an irreversible phenomenon. Moreover, NAC pre-treatment did not prevent PILO-induced NR2A subunit over-expression, a critical event in hippocampal sclerosis. Pre-treatment with the highly specific NR2B subunit inhibitor, ifenprodil, partially decreased PILO-mediated thiol oxidation and was not effective in preventing apoptosis and cell death. However, if acutely administered 48h after PILO exposure, ifenprodil blocked glutamate-induced aberrant calcium influx, suggesting the crucial role of NR2B over-expression in triggering neuronal hyper-excitability. Furthermore, ifenprodil treatment was able to prevent NR2A subunit over-expression by means of ERK1/2 phosphorylation. Our findings indicate oxidative stress and NR2B/NMDA signaling as promising therapeutic targets for co-treatments aimed to prevent chronic epilepsy following the seizure onset.

Topics: Acetylcysteine; Animals; Apoptosis; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Epilepsy, Temporal Lobe; Excitatory Amino Acid Antagonists; Hippocampus; Male; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Oxidation-Reduction; Oxidative Stress; Pilocarpine; Piperidines; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sulfhydryl Compounds

Neurocognitive disorders afflict approximately 20% of HIV-infected patients. HIV-1-infected cells in the brain shed viral proteins such as transactivator of transcription (Tat). Tat elicits cell death and synapse loss via processes initiated by NMDA receptor activation but mediated by separate downstream signalling pathways. Subunit selective NMDA receptor antagonists may differentially modulate survival relative to synaptic changes.. Tat-evoked cell death was quantified by measuring propidium iodide uptake into rat hippocampal neurons in culture. The effects of Tat on synaptic changes were measured using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein.. Dizocilpine, a non-competitive NMDA receptor antagonist, inhibited Tat-induced synapse loss, subsequent synapse recovery and Tat-induced cell death with comparable potencies. Memantine (10 µM) and ifenprodil (10 µM), which preferentially inhibit GluN2B-containing NMDA receptors, protected from Tat-induced cell death with no effect on synapse loss. Surprisingly, memantine and ifenprodil induced synapse recovery in the presence of Tat. In contrast, the GluN2A-prefering antagonist TCN201 prevented synapse loss and recovery with no effect on cell death.. Synapse loss is a protective mechanism that enables the cell to cope with excess excitatory input. Thus, memantine and ifenprodil are promising neuroprotective drugs because they spare synaptic changes and promote survival. These GluN2B-preferring drugs induced recovery from Tat-evoked synapse loss, suggesting that synaptic pharmacology changed during the neurotoxic process. NMDA receptor subtypes differentially participate in the adaptation and death induced by excitotoxic insult.

Topics: Animals; Cell Culture Techniques; Cell Survival; Culture Media; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Image Processing, Computer-Assisted; Immunohistochemistry; Memantine; Microscopy, Confocal; Neurons; Neuroprotective Agents; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Sulfonamides; Synapses; tat Gene Products, Human Immunodeficiency Virus; Transfection

Achievements made over the past few years have demonstrated the important role of the creatine and phosphocreatine system in the buffering and transport of high-energy phosphates into the brain; however, the non-energetic processes elicited by this guanidine compound in the hippocampus are still poorly understood. In the present study we disclosed that the incubation of rat hippocampal slices with creatine (10mM) for 30 min increased Na(+),K(+)-ATPase activity. In addition, intrahippocampal injection of creatine (5 nmol/site) also increased the above-mentioned activity. The incubation of hippocampal slices with N-methyl-d-aspartate (NMDA; MK-801, 10 μM) and NMDA Receptor 2B (NR2B; ifenprodil, 3 μM) antagonists but not with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate antagonist (DNQX, 10 μM) and nitric oxide synthase inhibitor (NOS; l-NAME, 100 μM), blunted the effect of creatine on Na(+),K(+)-ATPase activity. Furthermore, the calcineurin inhibitor (cyclosporine A, 200 nM) as well as the Protein Kinase C (PMA, 100 nM) and Protein Kinase A (8-Br-cAMP, 30 μM) activators attenuated the creatine-induced increase of Na(+),K(+)-ATPase activity. In addition, the incubation of hippocampal slices with creatine (10mM) for 30 min increased calcineurin activity. The results presented here suggest that creatine increases Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway, proposing an putative underlying non-energetic role of this guanidine compound. However, more studies are needed to assess the contribution of this putative alternative role in neurological diseases that present decreased Na(+),K(+)-ATPase activity.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Cell Membrane; Creatine; Cyclic AMP-Dependent Protein Kinases; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Isoenzymes; Male; N-Methylaspartate; Nerve Tissue Proteins; Nitric Oxide Synthase; Piperidines; Protein Kinase C; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Sodium-Potassium-Exchanging ATPase

Oxaliplatin is a platinum-based chemotherapy drug characterized by the development of acute and chronic peripheral neuropathies. The chronic neuropathy is a dose-limiting toxicity. We previously reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats. In the present study, we investigated the involvement of NR2B-containing N-methyl-D-aspartate (NMDA) receptors in oxaliplatin-induced mechanical allodynia in rats.. Repeated administration of oxaliplatin (4 mg/kg, i.p., twice a week) caused mechanical allodynia in the fourth week, which was reversed by intrathecal injection of MK-801 (10 nmol) and memantine (1 μmol), NMDA receptor antagonists. Similarly, selective NR2B antagonists Ro25-6981 (300 nmol, i.t.) and ifenprodil (50 mg/kg, p.o.) significantly attenuated the oxaliplatin-induced pain behavior. In addition, the expression of NR2B protein and mRNA in the rat spinal cord was increased by oxaliplatin on Day 25 (late phase) but not on Day 5 (early phase). Moreover, we examined the involvement of nitric oxide synthase (NOS) as a downstream target of NMDA receptor. L-NAME, a non-selective NOS inhibitor, and 7-nitroindazole, a neuronal NOS (nNOS) inhibitor, significantly suppressed the oxaliplatin-induced pain behavior. The intensity of NADPH diaphorase staining, a histochemical marker for NOS, in the superficial layer of spinal dorsal horn was obviously increased by oxaliplatin, and this increased intensity was reversed by intrathecal injection of Ro25-6981.. These results indicated that spinal NR2B-containing NMDA receptors are involved in the oxaliplatin-induced mechanical allodynia.

Topics: Animals; Dizocilpine Maleate; Enzyme Inhibitors; Gene Expression Regulation; Hyperalgesia; Indazoles; Male; Memantine; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organoplatinum Compounds; Oxaliplatin; Phenols; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Spinal Cord

Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimeŕs disease.

Topics: Amyloid beta-Peptides; Animals; Calcium; Cells, Cultured; Dizocilpine Maleate; Electrophysiological Phenomena; Female; Humans; Memantine; Neurons; Oocytes; Peptide Fragments; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Valine; Xenopus laevis

The aim of our study was to investigate the effects of ifenprodil and MK-801 on D,L-homocysteine thiolactone induced seizures in adult rats.Male Wistar rats were divided into following groups: 1. Saline-treated (C, n=10); 2. D,L-homocysteine thiolactone 8 mmol/kg, i.p. (H, n=7); 3. Ifenprodil 20 mg/kg i.p. (IF, n=8); 4. MK-801 0.5 mg/kg, i.p. (MK, n=8) and 5. Groups that received IF or MK 30 minutes prior to H (IFH, n=8 and MKH, n=8). Seizure behavior was assessed by incidence, latency, number and intensity of seizure episodes. Seizure severity was described by a descriptive scale with grades 0-4. Lethality in experimental group was recorded 90 min and 24 h upon D,L-homocysteine thiolactone administration.There were no behavioral signs of seizure activity in groups C, IF and MK.Pre-treatment with MK-801 (MKH) showed tendency to reduced incidence of convulsions, latency to the first seizure onset and the severity of seizure episodes, but statistical significance was not attained comparing to the H group. However, median number of seizure episodes was significantly decreased in MKH (p<0.05), comparing to the H group. On the other hand, ifenprodil (IFH) decreased the latency to the first seizure onset and increased the median number of seizure episodes (p<0.05). The majority of seizure episodes in IFH (72.1%, p<0.05) and MKH (73.1%, p<0.05) groups was grade 2 and significantly different comparing to the H (36.0%). Our findings suggest that MK-801 has a mild anticonvulsive effect on D,L-homocysteine thiolactone induced seizures in adult rats.

Topics: Animals; Dizocilpine Maleate; Homocysteine; Male; Piperidines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Seizures

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Topics: Animals; Astrocytes; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Guanylate Cyclase; Hyperalgesia; Indazoles; JNK Mitogen-Activated Protein Kinases; Male; Neuralgia; Neurons; Nitric Oxide Synthase Type I; Pain Measurement; Phenols; Phosphorylation; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Spinal Cord

We report a series of new stereoisomeric γ-amino alcohols comprising an N-methyl isoindoline moiety as ligands for the ifenprodil binding site of the NMDA receptor. Among the four series of stereoisomers, 8a-c, 9a-c, 10a-c, and 11a-c, synthesised, the highest potencies and NMDA-NR2B subtype selectivity was found for the methyl derivative 11a and the chloro derivative 11c, both possessing the [1S,1'S] configuration. However, additional moderate potency of 11a and 11c at the hERG channel with values of 2.6 ± 2.4% and 1.6 ± 2.0%, respectively, rendered them unsuitable for medical use.

Topics: Amino Alcohols; Binding Sites; Dizocilpine Maleate; Drug Design; Ether-A-Go-Go Potassium Channels; Excitatory Amino Acid Antagonists; Inhibitory Concentration 50; Ligands; Molecular Structure; Molecular Targeted Therapy; N-Methylaspartate; Neuroprotective Agents; Piperidines; Receptors, N-Methyl-D-Aspartate; Stereoisomerism

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.

Topics: Animals; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Mice; Patch-Clamp Techniques; Piperidines; Protein Subunits; Pyramidal Cells; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses

Although there is evidence of the involvement of N-methyl-D-aspartate receptors (NMDAR) in the action of lithium, its role in the antidepressant effects of lithium in a behavioural model remains unclear. In this study, we evaluated the effects of NMDAR antagonists on the antidepressant-like effects of lithium in the mouse forced swimming test. Lithium (30 and 100 mg/kg, i.p.) significantly (P < 0.01) reduced the immobility times of mice, whereas at lower doses (5 and 10 mg/kg) had no effect. NMDA antagonists ketamine (2 and 5 mg/kg, i.p.), MK-801 (0.1 and 0.25 mg/kg, i.p.) and ifenprodil (1 and 3 mg/kg, i.p.) significantly (P < 0.05) decreased the immobility time. Lower doses of ketamine (0.5 and 1 mg/kg), MK-801 (0.01 and 0.05 mg/kg) and ifenprodil (0.1 and 0.5 mg/kg) had no effect. Combined treatment of subeffective doses of lithium (10 mg/kg) and ketamine (1 mg/kg), MK-801 (0.05 mg/kg) or ifenprodil (0.5 mg/kg) robustly (P < 0.001) exerted an antidepressant-like effect. The noneffective dose of a NMDA agonist (NMDA, 75 mg/kg, i.p.) prevented the antidepressant-like effect of lithium (30 mg/kg). None of the drugs at subactive doses or in combination with lithium had significant effect on the locomotor activity in the open field test. We for the first time suggested a role for NMDAR signalling in the antidepressant-like effects of lithium, providing a new approach for treatment of depression.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Injections, Intraperitoneal; Ketamine; Lithium Chloride; Male; Mice; Models, Animal; Motor Activity; N-Methylaspartate; Piperidines; Receptors, N-Methyl-D-Aspartate; Swimming; Time Factors

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl-D-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [(125)I]MK-801 binding) and spermidine (100 microM)-potentiated [(125)I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1-10 microM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 microM) or ifenprodil (100 microM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [(125)I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 microM) exposure produced marked neurodegeneration (approximately 350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or DL-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [(125)I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

Topics: Animals; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Dentate Gyrus; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Hippocampus; In Vitro Techniques; Male; N-Methylaspartate; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Piperidines; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine; Valine

Histamine potentiates activation of native and recombinant N-methyl-d-aspartate receptors (NMDARs), but its mechanisms of action and physiological functions in the brain remain controversial. Using four different models, we have further investigated the histamine-induced potentiation of various NMDAR-mediated responses. In single cultured hippocampal neurons, histamine potentiated NMDA currents. It also potentiated the NMDA-induced increase in intracellular calcium in the absence, as well as with saturating concentrations, of exogenous d-serine, indicating both glycine-dependent and glycine-independent components of its effect. In rat hippocampal synaptosomes, histamine strongly potentiated NMDA-induced [(3)H]noradrenaline release. The profile of this response contained several signatures of the histamine-mediated effect at neuronal or recombinant NMDARs. It was NR2B-selective, being sensitive to micromolar concentrations of ifenprodil. It was reproduced by tele-methylhistamine, the metabolite of histamine in brain, and it was antagonized by impromidine, an antagonist/inverse agonist of histamine on NMDA currents. Up to now, histamine was generally considered to interact with the polyamine site of the NMDAR. However, spermine did not enhance NMDA-induced [(3)H]noradrenaline release from synaptosomes, and the potentiation of the same response by tele-methylhistamine was not antagonized by the polyamine antagonist arcaine. In hippocampal membranes, like spermine, tele-methylhistamine enhanced [(3)H]dl-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding to the glutamate site. In contrast, spermine increased nonequilibrium [(3)H]5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) binding, and suppressed [(3)H]ifenprodil binding, whereas histamine and tele-methylhistamine had no effect. In conclusion, the histamine-induced potentiation of NMDARs occurs in the brain under normal conditions. Histamine does not bind to the polyamine site, but to a distinct entity, the so-called histamine site of the NMDAR.

Topics: 2-Amino-5-phosphonovalerate; Allosteric Site; Animals; Binding Sites; Calcium; Dizocilpine Maleate; Drug Synergism; Hippocampus; Histamine; In Vitro Techniques; Intracellular Space; Male; Methylhistamines; N-Methylaspartate; Neurons; Norepinephrine; Piperidines; Polyamines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermine; Synaptosomes

Presynaptic stimulation stochastically recruits transmission according to the release probability (P(r)) of synapses. The majority of central synapses have relatively low P(r), which includes synapses that are completely quiescent presynaptically. The presence of presynaptically dormant versus active terminals presumably increases synaptic malleability when conditions demand synaptic strengthening or weakening, perhaps by triggering second messenger signals. However, whether modulator-mediated potentiation involves recruitment of transmission from dormant terminals remains unclear. Here, by combining electrophysiological and fluorescence imaging approaches, we uncovered rapid presynaptic awakening by select synaptic modulators. A phorbol ester phorbol 12,13-dibutyrate (PDBu) (a diacylglycerol analog), but not forskolin (an adenylyl cyclase activator) or elevated extracellular calcium, recruited neurotransmission from presynaptically dormant synapses. This effect was not dependent on protein kinase C activation. After PDBu-induced awakening, these previously dormant terminals had a synaptic P(r) spectrum similar to basally active synapses naive to PDBu treatment. Dormant terminals did not seem to have properties of nascent or immature synapses, judged by NR2B NMDAR (NMDA receptor) receptor subunit contribution after PDBu-stimulated awakening. Strikingly, synapses rendered inactive by prolonged depolarization, unlike basally dormant synapses, were not awakened by PDBu. These results suggest that the initial release competence of synapses can dictate the acute response to second messenger modulation, and the results suggest multiple pathways to presynaptic dormancy and awakening.

Topics: Analysis of Variance; Animals; Animals, Newborn; Calcium; Carbazoles; Carcinogens; Cells, Cultured; Colforsin; Dizocilpine Maleate; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Indoles; Maleimides; Neurons; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Piperidines; Presynaptic Terminals; Rats; Sucrose; Sweetening Agents; Vesicular Glutamate Transport Protein 1

In the retina, it is not well understood how visual processing depends on AMPA- and NMDA-type glutamate receptors. Here we investigated how these receptors contribute to contrast coding in identified guinea pig ganglion cell types in vitro. NMDA-mediated responses were negligible in ON alpha cells but substantial in OFF alpha and delta cells. OFF delta cell NMDA receptors were composed of GluN2B subunits. Using a novel deconvolution method, we determined the individual contributions of AMPA, NMDA, and inhibitory currents to light responses of each cell type. OFF alpha and delta cells used NMDA receptors for encoding either the full contrast range (alpha), including near-threshold responses, or only a high range (delta). However, contrast sensitivity depended substantially on NMDA receptors only in OFF alpha cells. NMDA receptors contribute to visual contrast coding in a cell-type-specific manner. Certain cell types generate excitatory responses using primarily AMPA receptors or disinhibition.

Topics: Animals; Contrast Sensitivity; Dizocilpine Maleate; Electric Conductivity; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Guinea Pigs; In Vitro Techniques; Light; Mice; Mice, Inbred C57BL; N-Methylaspartate; Patch-Clamp Techniques; Photic Stimulation; Piperidines; Receptors, N-Methyl-D-Aspartate; Retina; Retinal Ganglion Cells; Visual Perception

There is accumulating evidence that excitotoxicity and oxidative stress resulting from excessive activation of glutamate (N-methyl-D-aspartate) NMDA receptors are major participants in striatal degeneration associated with 3-nitropropionic acid (3NP) administration. Although excitotoxic and oxidative mechanisms are implicated in 3NP toxicity, there are conflicting reports as to whether NMDA receptor antagonists attenuate or exacerbate the 3NP-induced neurodegeneration. In the present study, we investigated the involvement of NMDA receptors in striatal degeneration, protein oxidation and motor impairment following systemic 3NP administration. We examined whether NMDA receptor antagonists, memantine and ifenprodil, influence the neurotoxicity of 3NP. The development of striatal lesion and protein oxidation following 3NP administration is delayed by memantine but not affected by ifenprodil. However, in behavioral experiments, memantine failed to improve and ifenprodil exacerbated the motor deficits associated with 3NP toxicity. Together, these findings suggest caution in the application of NMDA receptor antagonists as a neuroprotective agent in neurodegenerative disorders associated with metabolic impairment.

Topics: Adenosine Diphosphate; Animals; Corpus Striatum; Dizocilpine Maleate; Drug Interactions; Male; Memantine; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Nitro Compounds; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Propionates; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tyrosine

In this study, we evaluated the involvement of N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) system on the antidepressant-like effects of paroxetine in the mouse forced swimming test.. Swim sessions were conducted by placing mice in individual glass cylinders filled with water for 6 min. The duration of behavioral immobility during the last 4 min of the test was evaluated.. Paroxetine (8 and 16 mg/kg, intraperitoneal [i.p.]) significantly reduced the immobility times of mice, whereas lower doses (2 and 4 mg/kg) had no effect. NMDA antagonists MK-801 (0.1 and 0.25 mg/kg, i.p.) and ifenprodil (1 and 3 mg/kg, i.p.) and the NO synthase inhibitor NG-L-arginine methyl ester (L-NAME; 30 and 100 mg/kg, i.p.) significantly decreased the immobility time. Lower doses of MK-801 (0.01 and 0.05 mg/kg), ifenprodil (0.1 and 0.5 mg/kg), and L-NAME (10 mg/kg) had no effect. Combined treatment of subeffective doses of paroxetine (4 mg/kg) and MK-801 (0.05 mg/kg), ifenprodil (0.5 mg/kg), and L-NAME (10 mg/kg) robustly exerted an antidepressant-like effect. The noneffective dose of a NO precursor L: -arginine (750 mg/kg, i.p.) prevented the antidepressant-like effect of paroxetine (30 mg/kg).. We suggested, for the first time, a possible role for NMDAR/NO signaling in the antidepressant-like effects of paroxetine, providing a new approach for the treatment of depression.

Topics: Analysis of Variance; Animals; Antidepressive Agents, Second-Generation; Behavior, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Exploratory Behavior; Immobility Response, Tonic; Male; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Paroxetine; Piperidines; Receptors, N-Methyl-D-Aspartate; Swimming

The nucleoside adenosine (ADO) is a neuromodulator in brain. ADO and its metabolite inosine (INO) have been shown to increase cell viability in stroke models. During ischemia, extracellular levels of both ADO and INO are increased. In this study, we treated rat cortical neurons with N-methyl-D-aspartate (NMDA) to initiate excitotoxicity and then investigated the mechanisms of ADO and INO release. NMDA induced a significant increase in ADO and INO production. The effect of NMDA receptor antagonists on NMDA-evoked ADO and INO release was examined. MK-801 (1 micromol/L), a potent antagonist that lacks receptor subunit selectivity, completely blocked evoked release of both ADO and INO. Memantine (10 micromol/L), a lower affinity antagonist that also lacks subunit selectivity, blocked INO, but not ADO, release. Ifenprodil (10 micromol/L), an inhibitor selective for NMDA receptors containing the NR2B subunit, completely blocked evoked ADO and INO release. NVP-AAM077 (NVP, 0.4 micromol/L), an inhibitor selective for NMDA receptors containing the NR2A subunit, did not significantly block evoked release of either ADO or INO. Removal of extracellular Ca2+ abolished NMDA-evoked release of both ADO and INO. BAPTA (25 micromol/L), which chelates intracellular Ca2+, had no significant effect on either ADO or INO release unless extracellular Ca2+ was also removed. Inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) prevented NMDA-evoked ADO and INO release and decreased nucleoside transporter function. These data indicate that NMDA-evoked ADO and INO release is dependent on subunit composition of NMDA receptors. As well, NMDA-evoked ADO and INO release requires nucleoside transporters and extracellular Ca2+ and is enhanced by activation of CaMKII.

Topics: Adenosine; Animals; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cerebral Cortex; Chelating Agents; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; Inosine; N-Methylaspartate; Neurons; Piperidines; Purines; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (GABA(A)Rs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca(2+) dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca(2+) signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 muM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABA(A)R antagonism by a high dose of 200 microM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABA(A)R profiles of Ca(2+) signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca(2+) surges and NCAM-180 signaling.

Topics: Animals; Calcium; Cell Differentiation; Cell Line; Dizocilpine Maleate; Gene Expression Regulation; Growth Cones; Mice; Neurites; Neurotransmitter Agents; Piperidines; Polyamines; Protein Subunits; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate

Intrathecal (i.t.) administration of D-cycloserine (100 and 300 fmol), a partial agonist of the glycine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, produced a behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank in mice, which peaked at 5 - 10 min and almost disappeared at 15 min after the injection. The behavior induced by D-cycloserine (300 fmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.5-2 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also dose-dependently inhibited by i.t. co-administration of 7-chlorokynurenic acid (0.25-4 nmol), a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex; D-(-)-2-amino-5-phosphonovaleric acid (62.5-500 pmol), a competitive NMDA receptor antagonist; MK-801 (62.5-500 pmol), an NMDA ion-channel blocker; ifenprodil (0.5-8 nmol); arcaine (31-125 pmol); and agmatine (0.1-10 pmol), all being antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex. However, [D-Phe7,D-His9]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10,376, a tachykinin NK2-receptor antagonist, had no effect on D-cycloserine-induced nociceptive behavior. These results in the mouse spinal cord suggest that D-cycloserine-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the glycine recognition site and that it does not involve the tachykinin receptor mechanism.

Topics: 2-Amino-5-phosphonovalerate; Agmatine; Animals; Cycloserine; Dizocilpine Maleate; Dose-Response Relationship, Drug; Injections, Spinal; Ion Channels; Kynurenic Acid; Mice; Morphine; Neurokinin A; Nociceptors; Pain; Peptide Fragments; Piperidines; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Tachykinin; Substance P

To assess the neuroprotective effects of different glutamate modulation strategies, with a nonselective (MK801) and a selective (ifenprodil) NMDA receptor antagonist and a metabotropic glutamate receptor agonist (mGluR Group II, LY354740), in glaucoma-related in vivo rat models of retinal ganglion cell (RGC) apoptosis.. RGC apoptosis was induced in Dark Agouti (DA) rats by staurosporine (SSP) treatment. Single agents MK801, ifenprodil, or LY354740, or MK801 and LY354740 combined, were administrated intravitreally at different doses. Eyes were imaged in vivo using a recently established technique and the results confirmed histologically. The most effective combined therapy regimen of MK801 and LY354740 was then assessed in a chronic ocular hypertension (OHT) rat model with application at 0, 1, and 2 weeks after OHT surgery and the effects assessed as described before.. All strategies of glutamate modulation reduced SSP-induced-RGC apoptosis compared with the control, in a dose-dependent manner: MK801 (R2= 0.8863), ifenprodil (R2= 0.4587), and LY354740 (R2= 0.9094), with EC50s of 0.074, 0.0138, and 19 nanomoles, respectively. The most effective combination dose of MK801 and LY354740 was 0.06 and 20 nanomoles (P < 0.05), respectively, and the optimal timing of the therapy was 0 weeks after OHT surgery (P < 0.05).. This novel SSP model was validated as a useful tool for screening neuroprotective strategies in vivo. Group II mGluR modulation may be a useful treatment for RGC death. Combination therapy optimized to limit neurotoxic effects of MK801 may be an effective neuroprotective approach in retinal degenerative disease. Furthermore, treatments that minimize secondary RGC degeneration may be most useful in glaucoma.

Topics: Animals; Apoptosis; Bridged Bicyclo Compounds; Disease Models, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Therapy, Combination; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glaucoma; Glutamic Acid; Intraocular Pressure; Male; Neuroprotective Agents; Ocular Hypertension; Piperidines; Rats; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Retinal Ganglion Cells; Staurosporine

Excitotoxicity is generally studied in dissociated neurons, cultured hippocampal slices, or intact animals. However, the requirements of dissociated neurons or cultured slices to use prenatal or juvenile rats seriously limit the advantages of these systems, whereas the complexity of intact animals prevents detailed molecular investigations. In the present experiments, we studied developmental changes in NMDA neurotoxicity in acute hippocampal slices with lactate dehydrogenase (LDH) release in medium, propidium iodide (PI) uptake, and Nissl staining as markers of cell damage. Calpain-mediated spectrin degradation was used to test calpain involvement in NMDA neurotoxicity. NMDA treatment produced increased LDH release, PI uptake, and spectrin degradation in slices from juvenile rats but not adult rats. NMDA-induced changes in slices from young rats were blocked completely by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801) and by the antagonists of NR2B receptor ifenprodil and R-(R, S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol and were partly blocked by calpain inhibitor III but were not affected by the NR2A-specific antagonist [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid. NMDA-induced changes in Nissl staining were also different in slices from young and adult rats and blocked by NR2B but not NR2A antagonists. In contrast to NMDA treatment, oxygen/glucose deprivation (OGD) induced neurotoxicity in slices from both young and adult rats, although OGD-induced toxicity was attenuated by MK-801 only in slices from young rats. Our results are consistent with the idea that NMDA-mediated toxicity is caused by activation of NR2B- but not NR2A-containing NMDA receptors leading to calpain activation and that developmental changes in NMDA toxicity reflect developmental changes in NMDA receptor subunit composition.

Topics: Age Factors; Animals; Biomarkers; Calpain; Dipeptides; Disks Large Homolog 4 Protein; Dizocilpine Maleate; Enzyme Activation; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Gene Expression Regulation, Developmental; Glucose; Hippocampus; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; L-Lactate Dehydrogenase; Membrane Proteins; N-Methylaspartate; Neurons; Oxygen; Phenols; Piperidines; Propidium; Protein Subunits; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spectrin; Valine

Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.

Topics: Animals; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Gene Targeting; Hippocampus; Humans; Male; Mice; Mice, Transgenic; Motor Activity; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Piperidines; Protein Subunits; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Xenopus laevis

While some species of birds retain the ability to learn new songs as adults, many species can only learn during a restricted period when young. Previous studies have suggested that one potential mechanism of such a limited learning period, an alteration in the composition of postsynaptic NMDA receptors, does not competely block further song learning. Here, we examined whether presynaptic function could play a role in the regulation of learning capacity. We first showed that the participation of NMDA receptor NR2B subunits in synaptic currents in the robust nucleus of the arcopallium (RA), a critical location for integration of signals during song learning by young birds, decreases from young birds to adults. Using release-dependent block of postsynaptic NMDA receptors by an open-channel antagonist to assay presynaptic function, we showed that transmitter release at RA synapses from both HVC and the lateral magnocellular nucleus of the anterior nidopallium systematically decreases during the period of song learning, and in adults is about half that of juveniles. Further, activation of postsynaptic NMDA receptors could induce an acute depression of transmitter release, while lack of exposure to a normal learning environment could delay the developmental reduction in transmitter release. These results suggest that regulation of learning capacity may occur in part by coordination of presynaptic and postsynaptic function.

Topics: Age Factors; Animals; Behavior, Animal; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Finches; In Vitro Techniques; Learning; Male; Membrane Potentials; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Piperidines; Prosencephalon; Receptors, N-Methyl-D-Aspartate; Synapses; Vocalization, Animal

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Topics: Agmatine; Analysis of Variance; Animals; Behavior, Animal; Biguanides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Dynorphins; Enkephalins; Enzyme Inhibitors; Ethylmaleimide; Excitatory Amino Acid Antagonists; Immune Sera; Injections, Spinal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Narcotics; Nociceptin Receptor; Piperidines; Protein Precursors; Receptors, Opioid; Spinal Cord; Time Factors

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor.

Topics: Alanine; Binding, Competitive; Cell Membrane; Cerebral Cortex; Conotoxins; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Humans; Neurons; Neurotoxins; Piperidines; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Spermine

Neurosteroids are produced de novo in neuronal and glial cells, which begin to express steroidogenic enzymes early in development. Studies suggest that neurosteroids may play important roles in neuronal circuit maturation via autocrine and/or paracrine actions. However, the mechanism of action of these agents is not fully understood. We report here that the excitatory neurosteroid pregnenolone sulfate induces a long-lasting strengthening of AMPA receptor-mediated synaptic transmission in rat hippocampal neurons during a restricted developmental period. Using the acute hippocampal slice preparation and patch-clamp electrophysiological techniques, we found that pregnenolone sulfate increases the frequency of AMPA-mediated miniature excitatory postsynaptic currents in CA1 pyramidal neurons. This effect could not be observed in slices from rats older than postnatal day 5. The mechanism of action of pregnenolone sulfate involved a short-term increase in the probability of glutamate release, and this effect is likely mediated by presynaptic NMDA receptors containing the NR2D subunit, which is transiently expressed in the hippocampus. The increase in glutamate release triggered a long-term enhancement of AMPA receptor function that requires activation of postsynaptic NMDA receptors containing NR2B subunits. Importantly, synaptic strengthening could also be triggered by postsynaptic neuron depolarization, and an anti-pregnenolone sulfate antibody scavenger blocked this effect. This finding indicates that a pregnenolone sulfate-like neurosteroid is a previously unrecognized retrograde messenger that is released in an activity-dependent manner during development.

Topics: Age Factors; Animals; Animals, Newborn; Antibodies; Calcium; Calcium Channel Blockers; Chelating Agents; Dizocilpine Maleate; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Egtazic Acid; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Membrane Potentials; Neuronal Plasticity; Patch-Clamp Techniques; Piperidines; Pregnenolone; Presynaptic Terminals; Quinolinic Acids; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Synapses; Synaptic Transmission; Tetrodotoxin; Time Factors

Neuronal activity regulates the synaptic strength of neuronal networks. However, it is still unclear how diminished activity changes connection patterns in neuronal circuits. To address this issue, we analyzed neuronal connectivity and relevant mechanisms using hippocampal cultures in which developmental synaptogenesis had occurred. We show that diminution of network activity in mature neuronal circuit promotes reorganization of neuronal circuits via NR2B subunit-containing NMDA-type glutamate receptors (NR2B-NMDARs), which mediate silent synapse formation. Simultaneous double whole-cell recordings revealed that diminishing neuronal circuit activity for 48 h increased the number of synaptically connected neuron pairs with both silent and functional synapses. This increase was accompanied by the specific expression of NR2B-NMDARs at synaptic sites. Analysis of miniature EPSCs (mEPSCs) showed that the frequency of NMDAR-mediated, but not AMPAR-mediated, mEPSCs increased, indicating that diminished neuronal activity promotes silent synapse formation via the surface delivering NR2B-NMDARs in mature neurons. After activation of neuronal circuit by releasing from TTX blockade (referred as circuit reactivation), the frequency of AMPAR-mediated mEPSCs increased instead, and this increase was prevented by ifenprodil. The circuit reactivation also caused an increased colocalization of glutamate receptor 1-specfic and synaptic NR2B-specific puncta. These results indicate that the circuit reactivation converts rapidly silent synapses formed during activity suppression to functional synapses. These data may provide a new example of homeostatic circuit plasticity that entails the modulation of neuron-neuron connectivity by synaptic activity.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cell Count; Cells, Cultured; Dizocilpine Maleate; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamate Decarboxylase; Hippocampus; Immunohistochemistry; Isoenzymes; Ketamine; Membrane Potentials; N-Methylaspartate; Nerve Net; Neural Inhibition; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Statistics, Nonparametric; Synapses; Synaptic Transmission; Synaptophysin; Tetrodotoxin; Time Factors

A significant impairment of learning and memory-related behavior was induced in mice on the 7th and 14th days after olfactory bulbectomy (OBX), as measured by a passive avoidance task. The involvement of the N-methyl-D-aspartate (NMDA) receptor ion-channel complex for learning and memory-related behavior impairment was examined by the intracerebroventricular administration of several NMDA receptor-related agonists and in combination with antagonists. The NMDA receptor agonist NMDA (1 ng/mouse) and the polyamine site agonist spermidine (1 micro g/mouse) improved learning and memory-related behavior impairment. In contrast, the glycine agonist D-cycloserine (0.2, 1 and 5 micro g/mouse) had no effect on learning and memory-related behavior impairment. The improved effects by NMDA and spermidine were reversed by the coadministration of D-APV, a competitive NMDA receptor antagonist, MK-801, an NMDA ion-channel blocker and ifenprodil, a polyamine site antagonist, respectively. These results suggest that the degeneration of NMDA receptors and polyamine sites in the NMDA receptor ion-channel complex may be involved in the OBX-induced impairment of learning and memory-related behavior.

Topics: 2-Amino-5-phosphonovalerate; Animals; Avoidance Learning; Cycloserine; Disease Models, Animal; Dizocilpine Maleate; Drug Therapy, Combination; Injections, Intraventricular; Male; Memory Disorders; Mice; N-Methylaspartate; Olfactory Bulb; Piperidines; Receptors, N-Methyl-D-Aspartate; Spermidine; Time Factors

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.

Topics: Animals; Dizocilpine Maleate; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Hippocampus; Male; Piperazines; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Status Epilepticus

The increase of Fos expression in the striatum induced by haloperidol, an antagonist of the dopamine D2 receptor, might be related to the activation of glutamatergic neurotransmission, especially that of N-methyl-D-aspartate (NMDA) receptors. In this study, using behavioral and immunohistochemical techniques, we examined the effects of a noncompetitive NMDA antagonist, (+)-MK-801, and an NMDA receptor NR2B subunit antagonist, ifenprodil, on catalepsy, an extrapyramidal symptom; in this context, we also considered the expression of Fos protein in the forebrain after the administration of haloperidol. Catalepsy in mice, induced by the administration of haloperidol (1 mg/kg), was inhibited by pretreatment with (+)-MK-801 (0.2 mg/kg) or ifenprodil (10 mg/kg). Furthermore, pretreatment with (+)-MK-801 (0.2 mg/kg) significantly attenuated the induction of Fos-immunoreactive (IR) cells in the dorsomedial, dorsolateral, and ventrolateral striatum, but not in the shell region of the nucleus accumbens after the administration of haloperidol, whereas pretreatment with ifenprodil (10 mg/kg) significantly attenuated the induction of Fos-IR cells in all of these areas. It is known that ifenprodil binds sigma receptors and alpha-1 adrenergic receptors with high affinity. Pretreatment with the sigma receptor antagonist BD-1407 (3 mg/kg) or the alpha-1 adrenergic receptor antagonist prazosin (3 mg/kg) affected neither catalepsy nor the expression of Fos-IR cells after the administration of haloperidol. However, pretreatment with CP-101,606 (1 mg/kg), a selective antagonist for the NR2B subunit of the NMDA receptor, significantly attenuated catalepsy and the expression of Fos-IR cells in the forebrain after the administration of haloperidol. These results suggest that the NMDA receptor antagonists attenuated the induction of catalepsy and Fos-IR cells in forebrain after the administration of haloperidol. It was also suggested that haloperidol-induced expression of Fos-IR cells in the shell region of the nucleus accumbens might be differentially regulated by NMDA receptor subunits. Therefore, it appears that selective antagonists for the NR2B subunit of the NMDA receptor (e.g., CP-101,606) might be useful drugs for the treatment of extrapyramidal side effects (EPS) associated with the chronic use of typical antipsychotics such as haloperidol.

Topics: Adrenergic alpha-Antagonists; Animals; Behavior, Animal; Catalepsy; Cell Count; Corpus Striatum; Dizocilpine Maleate; Dopamine Antagonists; Drug Interactions; Ethylenediamines; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Haloperidol; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Piperidines; Prazosin; Prosencephalon; Proto-Oncogene Proteins c-fos; Reaction Time; Receptors, N-Methyl-D-Aspartate; Time Factors

Cells that survive traumatic brain injury are exposed to changes in their neurochemical environment. One of these changes is a prolonged (48 h) uptake of calcium which, by itself, is not lethal. The N-methyl-D-aspartate receptor (NMDAR) is responsible for the acute membrane flux of calcium following trauma; however, it is unclear if it is involved in a flux lasting 2 days. We proposed that traumatic brain injury induced a molecular change in the NMDAR by modifying the concentrations of its corresponding subunits (NR1 and NR2). Changing these subunits could result in a receptor being more sensitive to glutamate and prolong its opening, thereby exposing cells to a sustained flux of calcium. To test this hypothesis, adult rats were subjected to a lateral fluid percussion brain injury and the NR1, NR2A and NR2B subunits measured within different regions. Although little change was seen in NR1, both NR2 subunits decreased nearly 50% compared with controls, particularly within the ipsilateral cerebral cortex. This decrease was sustained for 4 days with levels returning to control values by 2 weeks. However, this decrease was not the same for both subunits, resulting in a decrease (over 30%) in the NR2A:NR2B ratio indicating that the NMDAR had temporarily become more sensitive to glutamate and would remain open longer once activated. Combining these regional and temporal findings with 45calcium autoradiographic studies revealed that the degree of change in the subunit ratio corresponded to the extent of calcium accumulation. Finally, utilizing a combination of NMDAR and NR2B-specific antagonists it was determined that as much at 85% of the long term NMDAR-mediated calcium flux occurs through receptors whose subunits favor the NR2B subunit. These data indicate that TBI induces molecular changes within the NMDAR, contributing to the cells' post-injury vulnerability to glutamatergic stimulation.

Topics: Animals; Autoradiography; Behavior, Animal; Blotting, Western; Brain Injuries; Calcium; Calcium Radioisotopes; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fluoresceins; Fluorescent Dyes; Male; Occipital Lobe; Organic Chemicals; Parietal Lobe; Piperidines; Protein Isoforms; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors; Trauma Severity Indices; Wounds, Nonpenetrating

It is widely believed that long-term depression (LTD) and its counterpart, long-term potentiation (LTP), involve mechanisms that are crucial for learning and memory. However, LTD is difficult to induce in adult cortex for reasons that are not known. Here we show that LTD can be readily induced in adult cortex by the activation of NMDA receptors (NMDARs), after inhibition of glutamate uptake. Interestingly there is no need to activate synaptic NMDARs to induce this LTD, suggesting that LTD is triggered primarily by extrasynaptic NMDA receptors. We also find that de novo LTD requires the activation of NR2B-containing NMDAR, whereas LTP requires activation of NR2A-containing NMDARs. Surprisingly another form of LTD, depotentiation, requires activation of NR2A-containing NMDARs. Therefore, NMDARs with different synaptic locations and subunit compositions are involved in various forms of synaptic plasticity in adult cortex.

Topics: 2-Amino-5-phosphonovalerate; Animals; Aspartic Acid; Cerebral Cortex; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Long-Term Potentiation; Long-Term Synaptic Depression; N-Methylaspartate; Neurons; Neurotransmitter Uptake Inhibitors; Phenols; Picrotoxin; Piperidines; Protein Subunits; Pyrrolidines; Quinoxalines; Rats; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate

In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50-200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2' cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Culture Media, Serum-Free; Dizocilpine Maleate; Ethanol; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; Kynurenic Acid; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Piperidines; Protein Subunits; Rats; Receptors, N-Methyl-D-Aspartate; RNA Splicing

Excitatory neuronal activity produces beneficial influences on neuronal survival under several circumstances. We show that cultivation of rat midbrain slices in the presence of elevated extracellular Mg(2+) resulted in a marked decrease in the number of dopaminergic neurons. The effect was prominent when Mg(2+) was added to the medium during the first week of cultivation. Chronic treatment with antagonists of N-methyl-D-aspartate receptors such as 2-amino-5-phosphonovaleric acid, MK-801 and ifenprodil also resulted in a marked loss of dopaminergic neurons, whereas nicotinic receptor antagonists showed no effect. The effect of MK-801 was abolished by chronic depolarization by elevated extracellular K(+), or by application of forskolin or dibutyryl cyclic AMP. Thus, tonic activation of N-methyl-D-aspartate receptors driven by neuronal activity may play an important role in the maintenance of dopaminergic neurons.

Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Colforsin; Culture Techniques; Dizocilpine Maleate; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Immunohistochemistry; Magnesium; Mesencephalon; Neurons; Piperidines; Potassium; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sodium; Time Factors; Tyrosine 3-Monooxygenase

The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine, is a dissociative anesthetic with antihyperalgesic properties. However, its clinical use is compromised by psychotomimetic side-effects. As ketamine and other noncompetitive NMDA antagonists, such as phencyclidine and dizocilpine, are not selective for the NR2A-2D subunits of the NMDA receptor, it is unclear which of these subunits is responsible for the psychotomimetic side-effects. This study investigated the role of the NR2B subunit in the ketamine drug discrimination model, a possible correlate for such side-effects. In a first experiment aimed at assessing general potency and time dependency, ketamine, dizocilpine, phencyclidine and the NR2B-selective antagonists ifenprodil and Ro 25-6981, dose-dependently suppressed fixed ratio 10 food-reinforced responding in rats, with peak efficacy obtained around 15-40 min. In rats trained to discriminate ketamine from vehicle in a two-lever fixed ratio 10 food-reinforced procedure, ketamine, dizocilpine, phencyclidine and Ro 25-6981 induced complete generalization (>80%); whereas ifenprodil induced partial generalization (33%). These findings suggest that the NR2B subunit is involved in the discriminative stimulus effects of noncompetitive NMDA antagonists, and that selective NR2B antagonists may also induce psychotomimetic side-effects.

Topics: Animals; Conditioning, Operant; Discrimination Learning; Discrimination, Psychological; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Ketamine; Male; Phencyclidine; Phenols; Piperidines; Protein Subunits; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reinforcement, Psychology

Responding of rats was maintained under a 120-response fixed ratio (FR) schedule of food delivery, and animals received individual and combined injections of N-methyl-D-aspartic acid (NMDA), phencyclidine hydrochloride, (+)-MK-801 hydrogen maleate (MK-801), (+/-)-2-amino-5-phosphonopentanoic acid (AP5), 7-chlorokynurenic acid (7CK), ifenprodil tartrate, N(G)-nitro-L-arginine methyl ester hydorchloride (L-NAME), 7-nitroindazole, aminoguanidine hemisulfate, L-arginine, molsidomine, sodium nitroprusside, and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Behavioral suppression after NMDA was completely and dose-dependently reversed by MK-801, phencyclidine, AP5, and aminoguanidine; partially and dose-dependently attenuated by molsidomine, ifenprodil, and 7CK; and not attenuated at all by L-NAME, 7-nitroindazole, or TMB-8. These findings suggested that behavioral suppression after NMDA was associated with nitric oxide from the inducible synthase. In a second series of experiments, comparable behavioral suppression by 0.1 mg/kg MK-801, but not 3 mg/kg phencyclidine, was attenuated by nitroprusside, molsidomine, and L-arginine, suggesting that suppressions from MK-801 and phencyclidine were mediated by different final common pathways, and that behavioral suppression from MK-801, but not phencyclidine, may be associated with Ca(2+)-dependent nitric oxide.

Topics: 2-Amino-5-phosphonovalerate; Animals; Arginine; Conditioning, Operant; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Gallic Acid; Guanidines; Indazoles; Injections, Intraperitoneal; Kynurenic Acid; Molsidomine; N-Methylaspartate; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroprusside; Phencyclidine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

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.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Conotoxins; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Mexiletine; Neurons; Neuroprotective Agents; Piperidines; Pregnancy; Prosencephalon; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Staurosporine

It is well established that the NMDA receptor antagonists block hippocampal long-term potentiation and impair acquisition in the Morris watermaze task, although the role of individual NMDA receptor subtypes is largely unknown. In the present study, we compared the effects of (+/-)-CP-101,606, an antagonist selective for NMDA receptor NR1/NR2B subunit-containing receptors and the nonselective NMDA receptor antagonist MK-801, on acquisition in the Morris watermaze. Male hooded Lister rats were given 4 trials/day to find a fixed hidden platform submerged beneath the opaque water of the Morris watermaze. Twenty-four hours after the last acquisition trial, a 'probe trial' was conducted to assess the rat's spatial memory for the location of the hidden platform. Those rats treated with MK-801 (0.1 mg/kg, i.p.) 60 min prior to the acquisition and probe trials took significantly longer to find the hidden platform during training and spent significantly less time searching the platform's location during the probe trial than vehicle-treated rats. In contrast, 60-min pretreatment with (+/-)-CP-101,606 (60 mg/kg, p.o.), a dose that fully occupied hippocampal NR1/NR2B subunit-containing receptors, as determined by ex vivo NMDA receptor-specific [3H]ifenprodil binding immediately following watermaze experiments, had no effect on acquisition or the probe trial. These results suggest that antagonists selective for NR1/NR2B subunit-containing receptors may not impair spatial memory in rats in the Morris watermaze.

Topics: Administration, Oral; Animals; Behavior, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Injections, Intraperitoneal; Male; Maze Learning; Memory; Piperidines; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Swimming

It has been suggested that abnormalities seen in fetal alcohol syndrome are linked with NMDA receptor malfunction. Our laboratory has previously shown that prenatal ethanol treatment decreases [3H]MK-801 binding density at postnatal day 21, when NMDA receptor subunit protein levels were unaltered. Thus, the focus of the present study was to examine whether prenatal ethanol modifies native NMDA receptor levels.. Cerebral cortices were taken from offspring born to three treatment groups of pregnant Sprague Dawley(R) rats: an ethanol group given an ethanol liquid diet during the gestational period, a pair-fed control group that received a liquid diet without ethanol, and an ad libitum group fed rat chow and tap water. Western blot studies were carried out at postnatal days 1, 7, 14, and 21 to examine total protein expression of NR1 and NR1b splice variants. NR2 subunit levels were examined by [3H]MK-801 binding studies using spermidine, an endogenous polyamine, and ifenprodil, a selective NR2B antagonist.. [3H]MK-801 binding density was significantly reduced in prenatal ethanol-treated groups compared with ad libitum and pair-fed control groups. Spermidine increased [3H]MK-801 binding, although potentiation by spermidine was not significantly different among all three experimental groups. Furthermore, no significant differences in total protein expression of NR1 or NR1b splice variants were observed in cortical membrane homogenates at postnatal days 1 through 21. [3H]MK-801 binding in the presence of ifenprodil showed that prenatal ethanol treatment significantly decreased low-affinity ifenprodil binding. High-affinity ifenprodil binding was reduced in both pair-fed and ethanol-treated groups.. These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.

Topics: Alternative Splicing; Animals; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Ethanol; Female; Piperidines; Pregnancy; Prenatal Exposure Delayed Effects; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine; Tritium

The glucose deprivation-induced release of [3H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [3H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca2+ because L- and N-type Ca2+-channel blockers, such as diltiazem (1 microM), nitrendipine (1 microM), and omega-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [3H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [3H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca2+ and is similar to previous reports pertaining to brain tissues.

Topics: Alanine; Animals; Biguanides; Calcium Channel Blockers; Cattle; D-Aspartic Acid; Diltiazem; Dizocilpine Maleate; Drug Synergism; Glucose; Glutamic Acid; Glycine; Humans; Kainic Acid; N-Methylaspartate; Nitrendipine; omega-Conotoxins; Perfusion; Piperidines; Polyamines; Receptors, Glutamate; Retina; Tritium; Verapamil

Staurosporine-induced apoptosis was associated with a 20% cellular survival rate in primary rat forebrain cultures. Treatment with the NR2B subunit-selective NMDA receptor antagonist conantokin-G (0.1-1 microM) increased the survival rate up to 78%. No protection was provided by the nonselective NMDA receptor antagonist dizocilpine (0.01-10 microM) but 34-64% cellular survival was provided by ifenprodil (0.01-10 microM), another NR2B subunit-selective antagonist. These results suggest a novel anti-apoptotic mechanism linked to the NR2B receptor subunit.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Conotoxins; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Nerve Degeneration; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Staurosporine

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analgesics, Opioid; Animals; Behavior, Animal; Dizocilpine Maleate; Dynorphins; Endorphins; Enkephalins; Excitatory Amino Acid Antagonists; Injections, Spinal; Kynurenic Acid; Male; Mice; Morphine; Neurokinin A; Nociceptors; Peptide Fragments; Piperidines; Protein Precursors; Receptors, N-Methyl-D-Aspartate; Receptors, Tachykinin; Substance P

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.

Topics: Agmatine; Animals; Biguanides; Binding Sites; Binding, Competitive; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermidine; Tritium

The antirelapse drug acamprosate has previously been reported to inhibit activating effects of polyamines on -methyl-D-aspartic acid receptor (NMDAR) function. Because increased synthesis of polyamines has been suggested as a mechanism for potentiation of NMDAR function during ethanol withdrawal, we evaluated the effects of acamprosate, MK-801, and ifenprodil in a cell culture model of ethanol withdrawal-induced neurotoxicity.. Organotypic hippocampal cultures from 8-day-old neonatal rats were maintained in vitro for 23 days before experimental use. The ethanol withdrawal model consisted of exposing cultures to ethanol (70-100 mM) for 4 days before being "withdrawn" into Calcium-Locke's buffer for 1 hr and then into minimal medium for 23 hr. Uptake of (45)CaCl(2) and propidium iodide by damaged cells was assessed 1 hr and 24 hr after the start of ethanol withdrawal, respectively. Additional studies examined effects of exposure to NMDA (50 microM) or spermidine (100 microM) on withdrawal-induced hippocampal damage. Last, these studies examined the ability of the sodium salt of acamprosate (Na-acamprosate, 200 microM), ifenprodil (50 microM), or MK-801 (30 microM) to inhibit neurotoxicity and (45)Ca(2+) entry produced by these insults.. Ethanol withdrawal was associated with significantly greater toxicity and (45)Ca(2+) entry, relative to controls. Exposure to spermidine and NMDA during ethanol withdrawal further increased neurotoxicity and (45)Ca(2+) entry. Acamprosate, ifenprodil, and MK-801 almost completely prevented ethanol withdrawal-induced toxicity and (45)Ca(2+) entry. Acamprosate also reduced spermidine-induced neurotoxicity during ethanol withdrawal but was ineffective against NMDA-induced toxicity or (45)Ca(2+) entry at this time.. The results support the contention that acamprosate, like ifenprodil, interacts with polyamines and that these compounds may be effective in reducing consequences of ethanol withdrawal. NMDAR activation is also strongly implicated in ethanol withdrawal neurotoxicity, but whether acamprosate causes any of these effects in this preparation directly via the NMDAR remains uncertain.

Topics: Acamprosate; Animals; Animals, Newborn; Calcium Signaling; Dizocilpine Maleate; Ethanol; Female; Hippocampus; Male; Organ Culture Techniques; Piperidines; Rats; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Taurine

In the present study we show that chronic exposure to low levels of lead (Pb(2+)) during development alters the type of N-methyl-D-aspartate receptor (NMDAR) expressed in the developing and young adult rat brain. Ifenprodil inhibition of [3H]MK-801 binding to the NMDAR channel in cortical and hippocampal neuronal membranes expressed high and low affinity components. Previous studies have shown that the high affinity component is associated with NR1/NR2B receptor complexes while the low affinity component is associated with the appearance and insertion of the NR2A subunit to NMDAR complexes. Pb(2+)-exposed rats express a greater number of [3H]MK-801 binding sites associated with the high affinity and low affinity components of ifenprodil inhibition. Further, [3H]ifenprodil saturation isotherms and Scatchard analysis in cortical and hippocampal membranes showed a higher number of binding sites (B(max)) with no change in binding affinity (K(d)) in Pb(2+)-exposed animals relative to controls. Quantitative [3H]MK-801 autoradiography in response to glutamate and glycine provided evidence that NMDAR complexes in Pb(2+)-exposed rat brain were maximally activated in situ. Higher levels of ifenprodil-sensitive binding sites and increased sensitivity to agonists are properties characteristic of NR1/NR2B recombinant receptors. Thus, our results strongly suggest that a greater proportion of the total number of NMDAR are NR1/NR2B receptors in the Pb(2+)-exposed rat brain. This Pb(2+)-induced change in NMDAR subtypes in the rat brain was associated with reduced CREB phosphorylation in cortical and hippocampal nuclear extracts. These findings demonstrate that chronic exposure to environmentally relevant levels of Pb(2+) altered the subunit composition of NMDAR complexes with subsequent effects on calcium-sensitive signaling pathways involved in CREB phosphorylation.

Topics: Animals; Animals, Newborn; Binding Sites; Brain; Calcium Signaling; Cell Differentiation; Cyclic AMP Response Element-Binding Protein; Dizocilpine Maleate; Environmental Exposure; Female; Lead; Lead Poisoning, Nervous System; Male; Neurons; Phosphorylation; Piperidines; Pregnancy; Prenatal Exposure Delayed Effects; Protein Subunits; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Binding, Competitive; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hindlimb; Hyperalgesia; Injections, Spinal; Male; Mice; Mice, Mutant Strains; Morphine; Naloxone; Narcotic Antagonists; Nerve Tissue Proteins; Pain Measurement; Piperazines; Piperidines; Pyrrolidinones; Reaction Time; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid; Spinal Cord; Subarachnoid Space

The N-methyl-D-aspartate (NMDA) antagonist ifenprodil and several structurally related compounds are highly selective for the NR2B-containing receptor subtype. This selectivity could provide an explanation for the reported difference of the analgesic and side-effect profile of ifenprodil-like compounds from other NMDA antagonists. In this work, we have queried if the ifenprodil-induced antinociception can be attributed to the block of NMDA receptors in the spinal cord. Ifenprodil and some other NMDA antagonists (MK-801, memantine) were tested in a model of inflammatory pain (Randall-Selitto) in rats. The in vivo NMDA antagonism was assessed in anaesthetised rats on responses of spinal dorsal horn (DH) neurones to iontophoretic NMDA and in the model of single motor unit (SMU) wind-up. Ifenprodil, MK-801 and memantine dose-dependently increased nociceptive thresholds in the Randall-Selitto model. Antinociceptive doses of the channel blockers selectively antagonised NMDA responses of DH neurones and inhibited wind-up. In contrast, antinociceptive doses of ifenprodil did not show any NMDA antagonism in electrophysiological tests. Although ifenprodil did not inhibit the SMU responses to noxious stimuli in spinalised rats, it markedly and dose-dependently inhibited nociceptive SMU responses in sham-spinalised rats. These results argue against the spinal cord being the principal site of antinociceptive action of ifenprodil; supraspinal structures seem to be involved in this effect.

Topics: Analgesics; Animals; Decerebrate State; Dizocilpine Maleate; Electric Stimulation; Excitatory Amino Acid Antagonists; Male; Memantine; Motor Activity; N-Methylaspartate; Naloxone; Narcotic Antagonists; Pain; Piperidines; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spinal Cord

We used ligand binding to ascertain whether the pharmacological actions of RO 25-6981 [(R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol] match those of other NR2B (epsilon2) subunit specific agents. RO 25-6981 inhibited binding of 125I-MK801 [iodo-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to receptors made from NR1a/epsilon2 but not NR1a/epsilon1. Increasing the concentration of spermidine did not change the efficacy of RO 25-6981 and minimally changed the IC(50) value. Chimeric epsilon1/epsilon2 receptors demonstrated that the structural determinants for high affinity actions of RO 25-6981 were contained completely within the first 464 amino acids, but no receptor retained wildtype features when the size of the epsilon2 component was decreased further. Epsilon1Q336R receptors were more inhibited by ifenprodil and RO 25-9681 than wildtype epsilon1 receptors in ligand binding assays but not in functional assays. Selected mutations of epsilon2E200 and epsilon2E201 also decreased the sensitivity of receptors to ifenprodil and RO 25-6981. These results suggest that RO 25-6981 shares structural determinants with ifenprodil and other modulators in the NR2B subunit.

Topics: Animals; Brain; Cell Line; Dizocilpine Maleate; Dopamine Antagonists; Excitatory Amino Acid Antagonists; Haloperidol; Humans; Kinetics; Mice; Mutation; Phenols; Piperidines; Protein Structure, Tertiary; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Spermidine

Ketamine, which is a non-competitive NMDA receptor antagonist, has been used as a dissociative anesthetic agent. However, chronic use of ketamine produces psychotomimetic effects, such as nightmares, hallucination and delusion. Therefore, the present study was designed to ascertain the role of the NMDA receptor and sigma receptor in the discriminative stimulus effect induced by ketamine. Fischer 344 rats were trained to discriminate between ketamine (5 mg/kg, i.p.) and saline under a fixed-ratio 10 food-reinforced procedure. Non-competitive antagonists for both NR2A- and NR2B-containing NMDA receptors, such as phencyclidine (0.1--1 mg/kg, i.p.) and dizocilpine (3--30 microg/kg, i.p.), and the NR2A-containing NMDA receptor-preferred antagonist dextromethorphan (3--56 mg/kg, i.p.) fully substituted for the ketamine cue in a dose-dependent manner. By contrast, the NR2B-containing NMDA receptor antagonist ifenprodil (5--20 mg/kg, i.p.) exhibited no generalization. Additionally, the competitive NMDA antagonist 3-[(+/-)-2-carboxypiperazine-4-yl] propyl-1-phosphonic acid ((+/-)-CPP; 0.3--5.6 mg/kg, i.p.) and a sigma receptor ligand DTG (0.3--3 mg/kg, s.c.) displayed no generalization to the ketamine cue. These results suggest that NR1/NR2A subunit containing NMDA antagonism may be critical for the production of the ketamine cue.

Topics: Animals; Dextromethorphan; Discrimination Learning; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Guanidines; Ketamine; Ligands; Male; Phencyclidine; Piperazines; Piperidines; Protein Subunits; Rats; Rats, Inbred F344; Receptors, N-Methyl-D-Aspartate; Receptors, sigma

The pharmacology of N-methyl-D-aspartate (NMDA) receptors shows regional differences in affinity for various agonists and antagonists. We have investigated the modulatory mechanisms acting via the polyamine, redox and proton sites in the cerebral cortex and the spinal cord of adult, male rats using [3H]MK-801 binding. The affinity for glycine-independent spermine stimulation was one magnitude higher in cerebrocortical than in spinal cord membranes while the affinity for the spermine antagonist arcaine was similar. Spermine abolished the inhibiting effect of low pH in both regions. Thus, the difference in the polyamine site between the two regions seems to be restricted to agonist binding. The proportion of high affinity/total ifenprodil binding was approximately 35% both in the spinal cord and the cerebral cortex, suggesting similar relative amounts of the NMDA receptor subunit 2B. The affinity of ifenprodil to the high affinity site was however significantly higher in the cerebral cortex. Redox modulatory agents had similar effects in the two regions but spermine fully counteracted the inhibiting effect of 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in the cerebral cortex while there was only a partial effect in the spinal cord. These data show that the regional pharmacological heterogeneity involves several of the mechanisms regulating the function of the NMDA receptor. The data also indicate that the NMDA receptor subunit 2B is much more common in spinal cord than previously suggested.

Topics: Animals; Biguanides; Cerebral Cortex; Dizocilpine Maleate; Hydrogen-Ion Concentration; Male; Neuroprotective Agents; Oxidation-Reduction; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine; Spinal Cord; Tritium

The purification, characterization, and synthesis of conantokin-R (Con-R), an N-methyl-D-aspartate (NMDA) receptor peptide antagonist from the venom of Conus radiatus, are described. With the use of well defined animal seizure models, Con-R was found to possess an anticonvulsant profile superior to that of ifenprodil and dizocilpine (MK-801). With voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs, Con-R exhibited the following order of preference for NR2 subunits: NR2B approximately NR2A > NR2C >> NR2D. Con-R was without effect on oocytes expressing the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 or the kainate receptor subunit GluR6. In mouse cortical neurons voltage-clamped at -60 mV, Con-R application produced a slowly developing block of inward currents evoked by 10 microM NMDA and 1 microM glycine (IC(50) = 350 nM). At 3 microM, Con-R did not affect gamma-aminobutyric acid- or kainate-evoked currents. Con-R prevented sound-induced tonic extension seizures in the Frings audiogenic seizure-susceptible mice at i.c.v. doses below toxic levels. It was also effective at nontoxic doses in CF#1 mice against tonic extension seizures induced by threshold (15 mA) and maximal (50 mA) stimulation, and it partially blocked clonic seizures induced by s.c. pentylenetetrazol. In contrast, MK-801 and ifenprodil were effective only at doses approaching (audiogenic seizures) or exceeding (electrical and pentylenetetrazol seizures) those required to produce significant behavioral impairment. These results indicate that the subtype selectivity and other properties of Con-R afford a distinct advantage over the noncompetitive NMDA antagonists MK-801 and ifenprodil. Con-R is a useful new pharmacological agent for differentiation between the anticonvulsant and toxic effects of NMDA antagonists.

Topics: Animals; Anticonvulsants; Behavior, Animal; Binding, Competitive; Cerebral Cortex; Conotoxins; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Electroshock; Evoked Potentials; Female; gamma-Aminobutyric Acid; Glutamic Acid; In Vitro Techniques; Kainic Acid; Male; Mice; Mollusk Venoms; Oocytes; Pentylenetetrazole; Piperidines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Seizures; Sound; Xenopus

Glutaryl-CoA dehydrogenase deficiency is a neurometabolic disorder with a specific age- and region-dependent neuropathology. Between 6 and 18 mo of age, unspecific illnesses trigger acute encephalopathic crises resulting in acute striatal and cortical necrosis. We hypothesized that acute brain damage in glutaryl-CoA dehydrogenase deficiency is caused by the main pathologic metabolites 3-hydroxyglutaric and glutaric acids through an excitotoxic sequence. Therefore, we investigated the effect of 3-hydroxyglutaric acid and glutaric acid on primary neuronal cultures from chick embryo telencephalons and mixed neuronal and glial cell cultures from neonatal rat hippocampi. Exposure to glutaric acid and 3-hydroxyglutaric acid decreased cell viability in a concentration- and time-dependent fashion. This neurotoxic effect could be totally prevented by preincubation with an N-methyl-D-aspartate receptor subunit 2B (NR2B)-specific antagonist, NR2B antibodies, and an unspecific N-methyl-D-aspartate receptor blocker and was partially blocked with an NR2A-specific antagonist but not with NR2A antibodies or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor and metabotropic glutamate receptor antagonists. Furthermore, metabolite toxicity increased in parallel with the increasing expression of the NR2B subunit on cultured neurons from second to sixth day in vitro. We conclude from these results that 3-hydroxyglutaric acid and glutaric acid act as false neurotransmitters, in particular through NR1/2B, and that the extent of induced neurotoxicity is dependent on the temporal and spatial expression of NR1/2B in the CNS during maturation. Beyond favorable implications for treatment and long-term prognosis, glutaryl-CoA dehydrogenase deficiency is the first neurologic disease in which specific neuropathology could be experimentally linked to ontogenetic expression of a particular neurotransmitter receptor subtype.

Topics: Animals; Antibodies; Brain Diseases, Metabolic, Inborn; Cells, Cultured; Chick Embryo; Dizocilpine Maleate; Glutarates; Glutaryl-CoA Dehydrogenase; Neurons; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Piperidines; Rats

(+/-)-2-(4-Benzylpiperidino)-1-(4-hydroxyphenyl)propan-1-ol ([3H]ifenprodil) binding to a subcellular fraction of porcine hippocampus, which was obtained by centrifugation on a discontinuous sucrose gradient, was investigated with the objective to label selectively the ifenprodil recognition site of native NMDA receptors. Saturation experiments revealed high-affinity sites for [3H]ifenprodil in this membrane fraction which could be characterised by a K(d) value of 23.0+/-1.8 nM using a one-site model. Calculation of saturation isotherms on the basis of a two-site model yielded a K(d1) value of 10.4+/-2.4 nM and a K(d2) value of 2200+/-1300 nM, respectively. Inhibition of [3H]ifenprodil binding by NR2B subunit-selective NMDA receptor antagonists, by polyamines, by sigma receptor ligands, by a variety of ligands acting at different NMDA receptor recognition sites and by several cations was studied and compared with the effects of these compounds on (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) binding under non-equilibrium conditions. It turned out that sigma receptor ligands such as 1, 3-di(2-tolyl)-guanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (R)-3-PPP, (S)-3-PPP and (1-¿2-[bis(4-fluorophenyl)methoxy]ethyl¿)(-4-[3-phenylpropyl]piperazi ne) (GBR-12909) did not affect [3H]ifenprodil binding in the nanomolar range or only slightly. In contrast, ifenprodil, eliprodil, nylidrin and haloperidol inhibited [3H]ifenprodil binding in the nanomolar range and in the same rank order and with the same potency as observed for the inhibition of the high-affinity fraction of [3H]MK-801 binding. The polyamines, which activate NMDA receptors, inhibited [3H]ifenprodil binding in a biphasic manner. Their potency to inhibit the high-affinity fraction of [3H]ifenprodil binding was found to be in the same range as their potency to enhance [3H]MK-801 binding. In the presence of 10 microM spermine a significantly enhanced (P=0.0097) rate of dissociation of [3H]ifenprodil binding was found, suggesting that inhibition of [3H]ifenprodil binding by spermine is not, or at least not exclusively mediated by a competitive interaction.

Topics: Animals; Azides; Binding, Competitive; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Guanidines; Hippocampus; In Vitro Techniques; Kinetics; Membranes; Piperidines; Protein Binding; Receptors, N-Methyl-D-Aspartate; Receptors, sigma; Subcellular Fractions; Swine

NMDA receptors undergo drastic changes in their subunit composition during development of the mammalian neocortex. An increase in the expression of the NR2A subunit correlates with developmental changes in the properties of synaptic NMDA receptors. In this study, we investigated whether these developmental alterations are restricted to synaptic NMDA receptors or whether similar developmental changes also occur at extrasynaptic NMDA receptors. To analyse the properties of extrasynaptic receptors, glutamate-evoked ion currents mediated by extrasynaptic NMDA receptors were isolated by irreversibly blocking synaptic NMDA receptors with MK-801. Whole-cell ion currents mediated by extrasynaptic receptors showed developmental changes in their sensitivity against the NR2B subunit-specific antagonist ifenprodil similar to that of synaptic receptors. In summary, our results strongly suggest that NR2A subunit-containing NMDA receptors increasingly contribute also to extrasynaptic NMDA receptors during in vitro differentiation.

Topics: Animals; Cells, Cultured; Coculture Techniques; Dizocilpine Maleate; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Gene Expression Regulation, Developmental; Glutamic Acid; Glycine; Ion Channels; Neocortex; Neurons; Patch-Clamp Techniques; Picrotoxin; Piperidines; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Stimulation, Chemical; Synapses

Ifenprodil has been widely used as an antagonist selective for NMDA receptors containing the NR2B subunit. Evidence suggests, however, that ifenprodil also increases NMDA receptor affinity. Using rat brain slices, we found that ifenprodil enhanced NMDA-induced current in both cortical and subcortical areas examined. To test whether the effect is due to an increase in NMDA receptor affinity, we compared the effect of ifenprodil on currents induced by different concentrations of NMDA. Consistent with the hypothesis, the enhancing effect (percent increase) was relatively constant at low NMDA concentrations. As NMDA concentration increased, however, the effect decreased. To test whether the effect is blocked when NMDA binding sites are saturated with NMDA, high concentrations of NMDA were applied. To partially block Ca(2+) influx and prevent cells from deteriorating, the experiments were performed in the presence of either MK801 or kynurenate, two noncompetitive antagonists. Under such conditions, ifenprodil not only failed to potentiate NMDA currents, but consistently suppressed the current. When the same concentration of NMDA was applied in the presence of the competitive antagonist CGP37849, ifenprodil regained its ability to potentiate NMDA currents. Furthermore, the higher the concentration of CGP37849 the more the NMDA current was potentiated by ifenprodil. These results, combined with previous studies, suggest that the enhancing effect is due to an increase in NMDA receptor affinity and is specific for responses induced by low NMDA concentrations. As NMDA concentration increases, the affinity-enhancing effect decreases. Consequently, the channel-suppressing effect becomes more prominent.

Topics: 2-Amino-5-phosphonovalerate; Animals; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Synergism; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Kynurenic Acid; Male; Membrane Potentials; N-Methylaspartate; Organ Culture Techniques; Piperidines; Prefrontal Cortex; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; Animals; Cell Survival; Cells, Cultured; Chick Embryo; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Methylmalonic Acid; Neurons; Neuroprotective Agents; Neurotoxins; NG-Nitroarginine Methyl Ester; Piperidines

Thalamic afferents and efferents utilize glutamate as their primary neurotransmitter. There are four families of glutamate receptors that can transduce this activity, as well as regulate glutamate release from thalamic relay neurons. The three ionotropic subtypes are of particular importance, because subunit composition confers variability in functional properties of each subtype. We have quantified the expression of NMDA, AMPA and kainate receptors in the thalamus of the macaque using receptor autoradiography and in situ hybridization. NMDA receptors are multimeric associations of NR1 and NR2A-NR2D subunits that form ligand-gated ion channels. Particular subunits are associated with modulatory binding sites that affect receptor activity. NR1 was the most abundant subunit mRNA; NR2A, NR2B, and NR2D subunit mRNAs were also present, but were expressed in nucleus-specific patterns. Very high levels of [3H]ifenprodil binding to the polyamine site of the NMDA complex were detected in a fairly homogeneous distribution. Binding of the ion channel ligand [3H]MK-801 was also abundant, and limbic nuclei expressed higher levels than motor nuclei or the reticular nucleus. [3H]CGP39653 binding to the glutamate site of the NMDA receptor was the least abundant of the NMDA receptor binding sites. There was variability in the stoichiometric relationships of binding sites across nuclei, suggesting that there is heterogeneity in the pharmacological properties of NMDA receptors expressed in the thalamus. AMPA and kainate are also multimeric associations of specific subunits that form ligand-gated ion channels. These subunits are encoded by specific genes: gluR1-gluR4 for AMPA receptors, and gluR5-gluR7 and KA1-KA2 for kainate receptors. GluR4 and gluR6 mRNAs were, respectively the most abundant of the AMPA and kainate receptor subunit transcripts. Both AMPA and kainate receptor subunit transcripts were expressed in a nucleus-specific pattern. The binding of [3H]kainate was higher than that of [3H]AMPA throughout the thalamus, but AMPA subunit mRNA levels were three to five orders of magnitude higher than those encoding the kainate receptor subunits. The mismatch between the levels of expression of kainate receptor subunit transcripts and binding sites is suggestive of a presynaptic localization of kainate receptors on thalamic afferents. These results suggest that ionotropic glutamate receptors are heterogeneously expressed in the thalamus of the primate, and that their diff

Topics: 2-Amino-5-phosphonovalerate; Animals; Autoradiography; Binding Sites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Macaca nemestrina; Neurons; Organ Specificity; Piperidines; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Thalamic Nuclei; Transcription, Genetic; Tritium

The objective of this study was to investigate the effects of the HIV-1 envelope protein gp120 and its peptide fragments on the function of N-methyl-D-aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or D-serine 'only'. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU-C-34-A, but not by RP-135 (a central portion of BRU-C-34-A). The EC50 values of gp120 were 0.02 nM (CCK-LI release) and 0.01 nM (SRIF-LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK- and SRIF-releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near-maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cerebral Cortex; Cholecystokinin; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; HIV Envelope Protein gp120; HIV-1; Indoles; Kynurenic Acid; Male; Maleimides; Neurons; Nitroarginine; Peptide Fragments; Pipecolic Acids; Piperazines; Piperidines; Potassium; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Somatostatin; Synaptic Transmission; Synaptosomes; Zinc

Current symptomatic treatment for Parkinson's disease is based largely on dopamine-replacing agents. The fact that long-term treatment with these drugs is characterized by many side effects has lead to widespread interest in nondopaminergic therapies. To date, however, it has proved difficult to devise a nondopaminergic therapy with significant antiparkinsonian efficacy when administered as monotherapy. Overactivity of the striatolateral pallidal pathway, the "indirect" striatal output pathway, is thought be responsible for the generation of parkinsonian symptoms. Indeed, it has been suggested that selective reduction in the activity of the "indirect" pathway may be achieved by blockade of NR2B-containing NMDA receptors. In the present study, we demonstrate that selective blockade of NR2B-containing NMDA receptors with the polyamine antagonists ifenprodil and eliprodil causes a significant increase in locomotor activity in the reserpine-treated rat model of Parkinson's disease (30 mg/kg ifenprodil, 221.2 +/- 54 mobile counts compared to vehicle, 19.6 +/- 6.87, P < 0.001). Additionally, we show that, subsequent to dopamine depletion, the ability of ifenprodil to bind to the polyamine site and inhibit binding of the NMDA channel blocker [3H] MK-801 is increased fourfold (IC50 3.7 +/- 0.4 microM compared to vehicle, IC50 14.3 +/- 2.34 microM, P < 0.01). We suggest that ifenprodil selectively targets the polyamine site on overactive NR2B-containing NMDA receptors. Thus, we propose that NR2B-selective NMDA receptor antagonists may prove useful in the treatment of Parkinson's disease.

Topics: Animals; Antiparkinson Agents; Binding, Competitive; Corpus Striatum; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Male; Parkinson Disease, Secondary; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reserpine

In this study, we investigated the hypothesis that inhibition of the N-methyl-D-aspartate (NMDA) receptor complex by zinc involves a polyamine-sensitive regulatory site. We found that the specific binding of the open channel ligand [3H]MK-801 to rat hippocampal membranes 1) was inhibited by low concentrations of Zn2+ (IC50 = 5.5 microM) by 65%. 2) This high-affinity component of inhibition was reversed by the polyamine spermine to an extent that could be reconciled with competitive interaction between Zn2+ and spermine. 3) Partial inhibition by Zn2+ was additive with partial inhibition by ifenprodil, an inhibitor of the NMDA receptor complex supposed to act at a polyamine-sensitive regulatory site, and 4) in membranes prepared from several other brain regions, inhibition of [3H]MK-801 binding by Zn2+ and by ifenprodil was either less than additive, or superadditive. Our observation that ifenprodil, at concentrations saturating its high-affinity component of inhibition, prevented spermine from reversing the inhibition by Zn2+ indicates that spermine did not increase [3H]MK-801 binding by competition with Zn2+ but rather via another polyamine regulatory site not sensitive to zinc but sensitive to ifenprodil. We conclude that Zn2+ reduces channel opening of the NMDA receptor complex by allosteric inhibition of a polyamine-sensitive regulatory site different from that inhibited by ifenprodil and that these two allosteric sites influence each other in a manner dependent on the brain region investigated. The different proportions of zinc/ifenprodil inhibition in different regions could reflect different percentages of various NMDA receptor subtypes.

Topics: Allosteric Regulation; Animals; Binding, Competitive; Brain; Cell Membrane; Dentate Gyrus; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Kinetics; Male; Neurons; Organ Specificity; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermine; Tritium; Zinc

The present study investigated the regional distribution of the N-methyl-D-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibers and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02-1 mg/kg i.p., L-687,414 10-300 mg/kg i.p., and L-701,324 1-10 mg/kg i.p.) and the anticonvulsant, gabapentin (10-500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (+/-)-CP-101,606 (1-100 mg/kg p.o.) and (+/-)-Ro 25-6981 (3-100 mg/kg i.p.) showed a significant dose window. (+/-)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (+/-)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3-500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.

Topics: Acetates; Amines; Animals; Anticonvulsants; Cyclohexanecarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Gabapentin; gamma-Aminobutyric Acid; Hyperalgesia; Lamotrigine; Male; Motor Activity; Pain Measurement; Phenols; Piperidines; Pyrrolidinones; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Triazines

The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (epsilon2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high-affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (epislon1/epsilon2) receptors. Mutation of epsilon2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit 125I-MK-801 binding. Epsilon2E201 does not govern the interactions of ifenprodil, because all of the mutants at epsilon2E201 exhibited wild-type affinity for ifenprodil. Mutation of epsilon2R337 causes a 400-fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.

Topics: Amino Acid Sequence; Binding, Competitive; Chimera; Dizocilpine Maleate; Dopamine Antagonists; Excitatory Amino Acid Antagonists; Haloperidol; Humans; Mutagenesis, Site-Directed; Piperidines; Receptors, N-Methyl-D-Aspartate; Spermidine

The present study investigated the effects of the NMDA receptor antagonists dizocilpine (MK-801) and ifenprodil on the appearance of diazepam withdrawal signs caused by discontinuation of long-term diazepam treatment using a drug-admixed food (DAF) method in Fischer 344 rats. The total withdrawal score was significantly decreased by after-withdrawal treatment with dizocilpine or ifenprodil. Dizocilpine, in particular, markedly suppressed the motor withdrawal signs and body weight loss, while ifenprodil suppressed the motor and emotional withdrawal signs. Furthermore, the decrease in the food intake during withdrawal (anorexia) was significantly reduced by dizocilpine, but not by ifenprodil. These behavioral results indicated that the activation of NMDA receptors during withdrawal may play an important role in the appearance of withdrawal signs (in particular motor withdrawal signs) caused by discontinuation of chronic diazepam treatment, and that inhibitory agents for NMDA receptors may be effective in alleviation of the appearance of benzodiazepine withdrawal signs.

Topics: Administration, Oral; Animals; Anorexia; Anti-Anxiety Agents; Behavior, Animal; Diazepam; Dizocilpine Maleate; Eating; Emotions; Excitatory Amino Acid Antagonists; Male; Motor Activity; Piperidines; Rats; Rats, Inbred F344; Receptors, N-Methyl-D-Aspartate; Substance Withdrawal Syndrome; Time Factors; Weight Loss

Recent pharmacological studies indicate that aminoglycoside-induced hearing loss may be an excitotoxic process modulated by a polyamine-like activation of cochlear NMDA receptors. Aminoglycoside antibiotics are constituted by a series of glycosidically linked aminocyclitols and aminosugars. We report here on the actions of these individual aminocyclitols and aminosugars on wild type NMDA receptors from rat brain. Compared to the parent molecules, neither aminocyclitols (e.g., 2-deoxystreptamine, streptamine, and streptidine) nor aminosugars (e.g., D-glucosamine and kanosamine) were effective at enhancing [3H]dizocilpine ([3H]MK-801) binding or inhibiting [3H]ifenprodil at NMDA receptors. Moreover, the appropriate combinations of aminosugars and aminocyclitols did not reconstitute the activity of the parent aminoglycoside at NMDA receptors. These data indicate that the polyamine-like actions of aminoglycosides are attributable to the conformation of the parent molecule rather than a particular amine containing constituent. Thus, it may be possible to synthesize or isolate aminoglycoside antibiotics devoid of ototoxicity.

Topics: Adrenergic alpha-Antagonists; Animals; Anti-Bacterial Agents; Carbohydrate Sequence; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; In Vitro Techniques; Kanamycin; Male; Molecular Sequence Data; Neomycin; Piperidines; Polyamines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine

Dopamine D1 receptor regulation of striatal gene expression is dependent on N-methyl-D-aspartate (NMDA) receptors. To determine whether pharmacologically distinct NMDA receptors are differentially involved, we examined the effects of different NMDA receptor antagonists on D1-induced immediate early gene expression. Systemic administration of the channel blocking antagonists MK-801 and dextrorphan and the competitive antagonist CGS 19755 blocked gene expression induced by the D1 agonist SKF 82958. The NMDA polyamine site antagonist ifenprodil, however, potentiated the effect of SKF 82958. Since ifenprodil is selective for receptors containing the NR2B subunit, the data suggest that subtypes of NMDA receptors may be differentially involved in regulating striatal function.

Topics: Animals; Corpus Striatum; Dextrorphan; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Genes, Immediate-Early; Male; Pipecolic Acids; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, N-Methyl-D-Aspartate

Haloperidol and ifenprodil are N-methyl-D-aspartate (NMDA) receptor (NR) antagonists with preference for the NR1/NR2B subunit combination. Previous investigations utilizing 125I-MK801 binding assays with recombinant receptors distinguished certain structural determinants on the NR2B subunit for these two drugs, with glutamate 201 being critical for haloperidol sensitivity and arginine 337 being important for ifenprodil block. Other studies, however, suggested that these two sites pharmacologically overlap. In an attempt to resolve these discrepancies, we have characterized the actions of haloperidol and CP101,606, an ifenprodil analog, on the single-channel properties of NR1/NR2B(E201R) receptors transiently expressed in Chinese hamster ovary cells, because receptors formed by NR1/NR2B(R337K) appear to be nonfunctional. Haloperidol (10 microM) inhibited wild-type NR1/NR2B channels by decreasing the frequency of channel opening, whereas CP101,606 (0.5 microM) antagonized NR1/NR2B channel activity by decreasing both the open dwell time and the frequency of channel opening. The inhibitory actions of both drugs were virtually absent in the mutant NR1/NR2B(E201R) receptors. These results suggest that glutamate 201 is critical for both haloperidol and CP101,606 inhibition, thus demonstrating common features in the action of these two antagonists.

Topics: Animals; Arginine; CHO Cells; Cricetinae; Dizocilpine Maleate; Dopamine Antagonists; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamic Acid; Haloperidol; Iodine Radioisotopes; Piperidines; Point Mutation; Receptors, N-Methyl-D-Aspartate

The function of striatopallidal neurons is regulated by N-methyl-D-aspartate (NMDA) and dopamine D2 receptors. Previous studies show that immediate early gene induction by D2 receptor blockade is suppressed by NMDA receptor antagonists. Because the pharmacology of NMDA receptors depends on the incorporation of different NR2 subunits and NR2 subunits show regional and cellular differences in their expression in striatum, our study examined whether different NMDA receptor antagonists would have differential effects on eticlopride-induced immediate early gene expression in striatum. Male Sprague-Dawley rats were pretreated with vehicle, CGS 19755, MK-801 or ifenprodil. Rats then received injections of eticlopride and were killed 40 min later. In situ hybridization histochemistry was used to determine the expression of c-fos and zif268 in the striatum. Eticlopride increased immediate early gene expression in striatum, with the increase generally being greater in lateral than in medial striatum. Pretreatment with each of the NMDA receptor antagonists dose-dependently decreased the expression of the immediate early genes. This suppression of eticlopride-induced gene expression was significant only in the medial-central aspect of striatum. Although there was a trend toward suppression of the gene induction in lateral striatum, it did not reach statistical significance and was not typically dose dependent. The data suggest that different types of NMDA receptor antagonists do not exert differential effects on D2 dopamine receptor-mediated function in the striatum. In addition, the data indicate that eticlopride-induced gene expression in the striatum is not uniformly dependent on NMDA receptor activation.

Topics: Animals; Corpus Striatum; Dizocilpine Maleate; Dopamine Antagonists; Excitatory Amino Acid Antagonists; Gene Expression; Genes, Immediate-Early; Male; Pipecolic Acids; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Salicylamides

This study examined the effects on rat behaviour of antagonists acting at various sites on the N-methyl-D-aspartate (NMDA) receptor complex, i.e. the glutamate recognition site (CPP), ion channel (dizocilpine), glycine recognition site [(+)-HA-966] and the NR2B subunit-selective compound ifenprodil. Specifically, the effects of these agents were examined on working memory, assessed using the operant delayed match-to-position task (DMTP), and overt behaviour, assessed (a) in animals responding for food under a variable interval 20-s (VI20) schedule and (b) by spontaneous behaviour. Dizocilpine, CPP and (+)-HA-966 each reduced accuracy in the DMTP task independent of delay. At equivalent doses, changes in locomotor behaviour and VI20 responding were evident. In contrast, ifenprodil failed to impair accuracy in the DMTP task, even at doses that affected other performance measures and reduced VI20 responding. The relevance of these observations to neuroprotective and anticonvulsant doses of these compounds is considered.

Topics: Animals; Behavior, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Memory, Short-Term; Motor Activity; Piperazines; Piperidines; Pyrrolidinones; Rats; Receptors, N-Methyl-D-Aspartate; Reinforcement Schedule

We investigated the role of the NMDA receptor complex in DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate)-induced seizures in mice. The seizure threshold of DMCM was evaluated using an i.v. infusion technique. Pretreatment with the non-competitive NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cycloheptan-5,10-imine maleate) or phencyclidine (PCP) significantly increased the seizure threshold for DMCM. Furthermore, the seizure threshold of DMCM was increased by intracerebroventricular (i.c.v.), but not intrathecal (i.t.), pretreatment with MK-801. Moreover, 7-chlorokynurenic acid, a glycine site antagonist, also increased the seizure threshold of DMCM, whereas ifenprodil, a non-competitive polyamine site antagonist, did not. These findings indicate that the ion-channel binding site and the glycine binding site on the NMDA receptor complex in the brain may be involved in the expression of seizures induced by DMCM.

Topics: Animals; Carbolines; Cerebral Ventricles; Convulsants; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Infusions, Intravenous; Injections, Intraventricular; Injections, Spinal; Kynurenic Acid; Male; Mice; Mice, Inbred Strains; Phencyclidine; Piperidines; Receptors, N-Methyl-D-Aspartate; Seizures; Spinal Cord

The effects of several NMDA receptor antagonists on pentylenetetrazole-induced diazepam-withdrawal seizure were examined in mice. The decrease in the seizure threshold for pentylenetetrazole during diazepam withdrawal was inhibited by pretreatment with MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate), 7-chlorokynurenic acid and ifenprodil. Furthermore, MK-801 and ifenprodil, at doses which did not affect the threshold of pentylenetetrazole-induced seizure in control mice, also significantly suppressed the decrease in the seizure threshold during diazepam withdrawal, whereas 7-chlorokynurenic acid did not. These findings suggest that overactivity of an ion channel site and an ifenprodil binding site on the NMDA receptor may play an important role in the hypersensitivity of pentylenetetrazole-induced seizure in diazepam-withdrawn mice.

Topics: Animals; Anticonvulsants; Convulsants; Diazepam; Disease Models, Animal; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Infusions, Intravenous; Injections, Intraperitoneal; Injections, Subcutaneous; Kynurenic Acid; Male; Mice; Neuroprotective Agents; Pentylenetetrazole; Piperidines; Receptors, N-Methyl-D-Aspartate; Seizures; Substance Withdrawal Syndrome

Some of the effects of glucocorticoids on the function and neuronal plasticity of the hippocampus are mediated by N-methyl-D-aspartate receptor activation. We tested the hypothesis that chronic corticosterone administration increases N-methyl-D-aspartate receptor expression in the hippocampus of the rat. We used in situ hybridization histochemistry to measure the messenger RNA levels for the NR1, NR2A and NR2B subunits of the N-methyl-D-aspartate receptor and [3H]dizocilpine maleate (a non-competitive antagonist) binding to measure N-methyl-D-aspartate receptor density. Since corticosterone depresses circulating testosterone levels, we also examined whether the effects of corticosterone are mediated by or interact with the effects of testosterone. In the intact animal, corticosterone increased messenger RNA levels for NR2A and NR2B but not NR1 subunits of the N-methyl-D-aspartate receptor in all regions of the hippocampus. Testosterone had no significant effect on messenger RNA levels of any of the subunits. The subunit composition determines the functional and pharmacological properties of the N-methyl-D-aspartate receptor. We used ifenprodil inhibition of [3H]dizocilpine maleate binding, which has been used to distinguish NR2A- from NR2B-containing receptors, to determine whether corticosterone altered the proportion of high- and low-affinity sites for ifenprodil in parallel with the changes in subunit messenger RNA levels. Corticosterone increased the density of [3H]dizocilpine maleate binding sites without changing the dissociation constant for [3H]dizocilpine maleate or the proportion of high- and low-affinity sites for ifenprodil. These data suggest that the effects of corticosterone on hippocampal function are mediated, in part, by parallel increases in NR2A and NR2B subunit levels and the number of receptor channel binding sites.

Topics: Animals; Anti-Inflammatory Agents; Binding Sites; Binding, Competitive; Cell Membrane; Corticosterone; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glucocorticoids; Hippocampus; Image Processing, Computer-Assisted; In Situ Hybridization; Male; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Testosterone

Topics: Animals; Blood-Brain Barrier; Capillary Permeability; Cerebellum; Dizocilpine Maleate; Encephalomyelitis, Autoimmune, Experimental; Male; Piperidines; Putrescine; Rats; Rats, Inbred Lew; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Vasodilator Agents

Topics: 2-Amino-5-phosphonovalerate; Animals; Binding, Competitive; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Kainic Acid; Kynurenic Acid; Male; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

We have studied in a well-characterized in vitro neuronal system, cultures of cerebellar granule cells, the toxicity of polyamines endogenously present in the brain: spermine, spermidine, and putrescine. Twenty-four-hour exposure of mature (8 days in vitro) cultures to 1-500 microM spermine resulted in a dose-dependent death of granule cells, with the half-maximal effect being reached below 50 microM concentration. Putrescine was moderately toxic but only at 500 microM concentration. Spermidine was tested at 50 and 100 microM concentration and its toxicity was evaluated to be about 50% that of spermine. Neuronal death caused by spermine occurred, at least in part, by apoptosis. Spermine toxicity was completely prevented by competitive (CGP 39551) and noncompetitive (MK-801) antagonists of the NMDA receptor, but was unaffected by a non-NMDA antagonist (NBQX) or by antagonists of the polyamine site present on the NMDA receptor complex, such as ifenprodil. A partial protection from spermine toxicity was obtained through the simultaneous presence of free radical scavengers or through inhibition of the free radical-generating enzyme nitric oxide synthase, known to be partially effective against direct glutamate toxicity. The link between spermine toxicity and glutamate was further strengthened by the fact that, under culture conditions in which glutamate toxicity was ineffective or much reduced, spermine toxicity was absent or very much decreased. Exposure to spermine was accompanied by a progressive accumulation of glutamate in the medium of granule cell cultures. This was attributed to glutamate leaking out from dying or dead cells and was substantially prevented by the simultaneous presence of MK-801 or CGP 39551. The present results demonstrate that polyamines are toxic to granule cells in culture and that this toxicity is mediated through the NMDA receptor by interaction of exogenously added polyamines with endogenous glutamate released by neurons in the medium. The involvement of brain polyamines, in particular spermine and spermidine, in excitotoxic neuronal death is strongly supported by our present results.

Topics: 2-Amino-5-phosphonovalerate; Animals; Apoptosis; Aspartic Acid; Butylated Hydroxytoluene; Cells, Cultured; Cerebellar Cortex; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Free Radical Scavengers; Glutamic Acid; L-Lactate Dehydrogenase; Male; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Nitric Oxide Synthase; Nitroarginine; Piperidines; Putrescine; Quinoxalines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine; Vitamin E

The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.

Topics: Animals; Calcium Channels; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Phenols; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Sodium Channels

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Dizocilpine Maleate; Guinea Pigs; Hair Cells, Auditory, Outer; Hearing Loss, Sensorineural; Humans; Neuroprotective Agents; Piperidines; Risk Factors

The developmental change in the antiseizure effect of ifenprodil against pentylenetetrazole (PTZ) was examined in mice. Ifenprodil (i.p.) significantly increased the latency to seizure induced by PTZ in 7- and 10-day-old mice, but not in 14- or 21-day-old mice. Intracerebroventricular administration of ifenprodil also failed to modify the latency to PTZ-induced seizure in 21-day-old mice. Dizocilpine produced an increase in the latency to PTZ-induced seizure in 7- and 21-day-old mice. In an NMDA receptor binding assay using [3H]dizocilpine, ifenprodil was clearly more potent in inhibiting [3H]dizocilpine binding in a forebrain membrane preparation from 7- rather than 21-day-old mice. These results suggest that the remarkable antiseizure effect of ifenprodil against PTZ in 7-day-old mice may be related to the high proportion of ifenprodil-sensitive NMDA receptors in the brain.

Topics: Aging; Animals; Anticonvulsants; Convulsants; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Injections, Intraventricular; Male; Mice; Mice, Inbred Strains; Neuroprotective Agents; Pentylenetetrazole; Piperidines; Reaction Time

Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which demonstrates a 140-fold preference for NR2B over NR2A subunits, although the molecular basis for this subunit specificity is unknown. We have made chimeric receptors by fusing the murine forms of NR2A (epsilon 1) and NR2B (epsilon 2) to localize the high affinity determinants of ifenprodil inhibition on the 2B subunit. Binding experiments with 125I-MK-801 implicated the region between amino acids 198 and 356 of NR2B for high affinity ifenprodil interaction. Site-directed mutants at Arg-337 showed that this residue is absolutely required for high affinity ifenprodil inhibition. Polyamines also modulate the NMDA receptor with a preference for NR2B subunits, and the pharmacology of these agents overlaps with ifenprodil. Although the determinants of the polyamine enhancement of iodo-MK-801 binding also localize to the NH2 terminus of NR2B, the point mutants at Arg-337 form receptors that are polyamine-stimulated at wild type levels. In addition, polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Arginine; Base Sequence; Binding Sites; Cell Line; Cloning, Molecular; Dizocilpine Maleate; DNA Primers; Excitatory Amino Acid Antagonists; Genetic Variation; Humans; Kidney; Kinetics; Macromolecular Substances; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Piperidines; Polymerase Chain Reaction; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Spermidine; Transfection

Polyamines can modulate activation of N-methyl-D-aspartate (NMDA) receptors by binding to a specific polyamine site associated with a NMDA receptor macrocomplex. Polyamines induce histamine release from mast cells, although the mechanism had not been defined. We have examined whether spermine, a natural polyamine, and compound 48/80, regarded as a synthetic polyamine, activate mast cells by a polyamine site associated with a NMDA receptor macrocomplex. Spermine induced secretion of histamine from rat peritoneal mast cells and rat brain mast cells in a concentration-dependent manner. Rat peritoneal mast cells were used as a model system to explore the effects of NMDA antagonists on polyamine-induced histamine release. Ifenprodil, MK801 and arcaine inhibited histamine secretion from mast cells exposed to polyamines; the percentage inhibition was greater against spermine than compound 48/80. These data support the proposal that spermine (and possibly compound 48/80) induce histamine release from mast cells by interacting with a specific polyamine site on a NMDA receptor complex.

Topics: Animals; Binding Sites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Histamine Release; Mast Cells; p-Methoxy-N-methylphenethylamine; Peritoneum; Piperidines; Polyamines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spermine; Thalamus

1. The involvement of the N-methyl-D-aspartate (NMDA) receptor macrocomplex in the development of spermine-induced CNS excitation in vivo was investigated. 2. Injection of 100 micrograms of spermine into the left lateral cerebral ventricle of female Laca mice (20-25 g) resulted in the development of two distinct phases of CNS excitatory effects which were quantified by a scoring system. 3. The first phase effects occurred within minutes of injection and generally lasted for about 1 h. Most mice showed scratching of the upper body, frequent face washing and some mice developed clonic convulsions. By about 2 h after injection, the second phase of effects began to develop in the form of body tremor which worsened with time and culminated in fatal tonic convulsions, generally within 8 h of injection. 4. Pretreatment of the mice with dizocilpine (0.3 mg kg-1, i.p.) resulted in antagonism of the first phase of spermine-induced effects, but a higher dose (0.3 mg kg-1, (x2), i.p.) was necessary to inhibit the second phase effects. 5. Whereas the glutamate antagonist, 3-((R)-2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (D-CPP) (10, 20 mg kg-1, i.p.), the glycine antagonist 7-chlorokynurenate (10, 30, 50 nmol, i.c.v.), or the polyamine antagonist ifenprodil (30, 60 mg kg-1, i.p.) antagonized the first phase of effects produced by spermine, these agents given as monotherapy, were ineffective against the development of the second phase of effects. 6. Co-administration of ifenprodil with either D-CPP or 7-chlorokynurenate resulted in a dose-dependent antagonism of the development of the second phase of spermine-induced effects. 7. It is concluded that the development of the two temporally distinct phases of spermine-induced effects may be mediated by pharmacologically distinct mechanisms, although the results suggest that the NMDA receptor macrocomplex may be involved in both phases of effects. Furthermore, a moderate dose of D-CPP or 7-chlorokynurenate appears to enhance the inhibitory potential of ifenprodil in vivo.

Topics: Animals; Brain; Cerebral Ventricles; Dizocilpine Maleate; Female; Kynurenic Acid; Mice; Neuroprotective Agents; Piperazines; Piperidines; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Seizures; Spermine; Tremor

We have recently demonstrated that a single administration of m-chlorophenylpiperazine (m-CPP, a preferential 5-HT2C receptor agonist) produces tolerance to its stimulatory effect on adrenocorticotropic hormone (ACTH) concentrations when challenged 24 h later with the same dose of m-CPP. In the present study, we studied the effects of pretreatment with various N-methyl-D-aspartate (NMDA) receptor antagonists on development of tolerance to m-CPP's stimulatory effect on ACTH concentrations. Pretreatment with various NMDA receptor antagonists such as 5.7-dichlorokynurenic acid (1.0 mg/kg), 3-amino-1-hydroxy 2-pyrrolidone (1.0 mg/kg), dizocilpine (0.1 mg/kg) and ifenprodil (1.0 mg/kg) injected 30 min before the first injection of m-CPP (2.5 mg/kg) blocked development of tolerance to m-CPP's stimulatory effect on ACTH concentrations in rats injected 24 h later with the same dose (2.5 mg/kg) of m-CPP. These findings suggest that tolerance to postsynaptic 5-HT2C receptor-mediated response is initiated though stimulation of NMDA receptor complex and, furthermore, demonstrate a functional interaction between the 5-HT and glutamate systems.

Topics: Adrenocorticotropic Hormone; Animals; Dimethyl Sulfoxide; Dizocilpine Maleate; Drug Tolerance; Kynurenic Acid; Male; Phencyclidine; Piperazines; Piperidines; Pyrrolidinones; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Serotonin Receptor Agonists

Antagonists at four distinct regulatory sites on the N-methyl-D-aspartate (NMDA) receptor were tested for their ability to attenuate NMDA-mediated chronic excitotoxicity and the consequences on AT8 tau immunoreactivity in neuronal cultures. Excitotoxicity was monitored in cultures by diacetate fluorescein staining. Immunoreactivity of tau phosphorylated at serine 202 was quantified by laser confocal microscopy. The NMDA-receptor antagonists MK801, AP7 and 7-chlorokynurenate significantly blocked NMDA-induced cell death and significantly reduced AT8 tau immunoreactivity. NMDA antagonism by the polyamine site antagonist, ifenprodil, did not completely reverse the increase in AT8 tau immunolabeling induced by NMDA and did not completely protect NMDA-sensitive neurons, suggesting an heterogeneity in the NMDA receptor population.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Fetus; Fluorescent Antibody Technique; Kynurenic Acid; Microscopy, Confocal; Neurons; Piperidines; Pregnancy; Rats; Receptors, N-Methyl-D-Aspartate; tau Proteins

In midbrain dopamine neurons in vitro, N-methyl-D-aspartate (NMDA) evokes oscillation of membrane potential and burst firing which are dependent on a ouabain-sensitive sodium pump. In the present study, we investigated the ionic dependence and pharmacological modulation of NMDA-mediated currents which might be important in burst firing. By use of patch pipettes to record membrane currents in whole-cell voltage clamps, we found that NMDA (10 microM) evoked inward currents that were significantly reduced in a low extracellular concentration of Na+ (25 mM), but not when extracellular Ca+2 was decreased from 2.5 to 0.5 mM. The current-voltage relationship for subtracted NMDA currents showed a prominent region of negative slope conductance which was absent when the slice was perfused with solution containing zero Mg++. 7-Chlorokynurenic acid, an antagonist at the nonstrychnine-sensitive glycine binding site, produced a concentration-dependent reduction in amplitude of excitatory postsynaptic currents mediated by NMDA receptors (IC50 = 15 +/- 3 microM). NMDA-activated currents were blocked by phencyclidine (IC50 = 130 +/- 65 nM), dizocilpine maleate (MK-801) (1 microM) and ketamine (100 microM), but not by amantadine (1 mM). Spermine (100 microM), a polyamine which reportedly modulates NMDA currents in other neurons, presynaptically inhibited excitatory postsynaptic currents mediated by NMDA receptors but had no effect on the currents mediated by NMDA. We conclude that the most important factors for NMDA-induced burst firing are the relatively large Na+ influx through NMDA-gated channels and the strong voltage-dependent block of conductance by Mg++.

Topics: Animals; Dizocilpine Maleate; Excitatory Amino Acid Agonists; In Vitro Techniques; Kynurenic Acid; Magnesium; Male; Membrane Potentials; N-Methylaspartate; Phencyclidine; Piperidines; Rats; Rats, Sprague-Dawley; Sodium; Spermine; Substantia Nigra; Ventral Tegmental Area

The use of aminoglycoside antibiotics is limited by ototoxicity that can produce permanent hearing loss. We report that concurrent administration of N-methyl-D-aspartate (NMDA) antagonists markedly attenuates both the hearing loss and destruction of cochlear hair cells in guinea pigs treated with aminoglycoside antibiotics. These findings indicate that aminoglycoside-induced hearing loss is mediated, in part, through an excitotoxic process. The high correlation (Spearman correlation coefficient: 0.928; P < 0.01) obtained between the relative cochleotoxicities of a series of aminoglycosides in humans and the potencies of these compounds to produce a polyamine-like enhancement of [3H]dizocilpine binding to NMDA receptors is consistent with this hypothesis, and provides a simple in vitro assay that can predict this aspect of aminoglycoside-induced ototoxicity.

Topics: Animals; Anti-Bacterial Agents; Cochlea; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Guinea Pigs; Hair Cells, Auditory; Hearing Loss; Hearing Tests; Kanamycin; Male; N-Methylaspartate; Neomycin; Piperidines; Prosencephalon; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

The N-methyl-D-aspartate (NMDA) receptor polyamine site antagonist, ifenprodil, had no effect on spontaneous alteration or locomotor activity in the Y-maze when given alone. The NMDA receptor/ion channel blocker, dizocilpine, induced a deficit in spontaneous alteration, but when ifenprodil was co-administered with dizocilpine, it showed a strong tendency to attenuate the dizocilpine-induced deficit. In the plus-maze, ifenprodil had an anxiolytic profile which was accompanied by an increase in locomotion. Dizocilpine had an anxiolytic profile in this model and increased locomotor activity. When co-administered with dizocilpine, ifenprodil reduced both the anxiolytic and locomotor effects of dizocilpine. When co-administered with ifenprodil, cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentylxanthine (CPX) reduced the anxiolytic effect of ifenprodil. CPA and CPX in combination did not reverse the anxiolytic effect of ifenpropil. It is concluded that NMDA antagonists with different sites of action can show distinct behavioural profiles, with dizocilpine but not ifenprodil inducing a deficit in working memory, while both are anxiolytic. Blockade of NMDA receptors by ifenprodil, however, can preclude any response to dizocilpine. The anxiolytic activity of ifenprodil may involve the release of purines acting at adenosine receptors.

Topics: Animals; Anxiety; Disease Models, Animal; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Male; Memory; Mice; Mice, Inbred Strains; Piperidines

Four hours following cryo-injury rat cerebral pericapillary astrocytes from the perilesional area were markedly swollen occupying 17% of the pericapillary space as compared to 11% in sham operated controls. Ornithine decarboxylase activity and polyamine levels were increased over sham controls. The astrocytic swelling, the percentage of the pericapillary space occupied by astrocytic processes, and polyamine levels were reduced to near control levels by the following: (1) alpha-difluoromethylornithine; (2) Ifenprodil; and (3) MK-801. alpha-Difluoromethylornithine is a specific inhibitor of ornithine decarboxylase, Ifenprodil is an inhibitor of the polyamine binding site on the n-methyl-d-aspartate receptor, and MK-801 is an antagonist to n-methyl-d-aspartate binding to the n-methyl-d-aspartate receptor. Addition of putrescine, the product of ornithine decarboxylase activity, reversed the effect of alpha-difluoromethylornithine and restored the pericapillary swelling. Putrescine did not affect the MK-801-induced reduction in pericapillary astrocytic swelling. Therefore, polyamines and the n-methyl-d-aspartate receptor modulate excitotoxic responses to cryo-injury in pericapillary cerebral astrocytes.

Topics: Animals; Astrocytes; Biogenic Polyamines; Brain; Brain Injuries; Capillaries; Cold Temperature; Dizocilpine Maleate; Eflornithine; Ornithine Decarboxylase; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Statistics as Topic

The behavioural and convulsant effects of imipenem (Imi), a carbapenem derivative, were studied after intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. The anticonvulsant effects of some excitatory amino acid antagonists and muscimol (Msc), a GABAA agonist, against seizures induced by i.p. or i.c.v. administration of Imi were also evaluated. The present study demonstrated that the order of anticonvulsant activity in our epileptic model, after i.p. administration, was (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclohepten-5,10-imine maleate (MK-801) > (+/-)(E)-2-amino-4-methyl-5-phosphono-3-pentenoate ethyl ester (CGP 39551) > 3-((+/-)-2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid (CPPene) > 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CCP) > 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxaline (NBQX). Ifenprodil, a compound acting on the polyamine site of NMDA receptor complex was unable to protect against seizures induced by Imi, suggesting that the poliamine site did not exert a principal role in the genesis of seizures induced by Imi. In addition, the order of anticonvulsant potency in our epileptic model, after i.c.v. administration, was CPPene > MK-801 > Msc > (-)-2-amino-7-phosphonic acid (AP7) > gamma-D-glutamylaminomethylsulphonate (gamma-D-GAMS) > NBQX > kynurenic acid (KYNA) > 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). The relationship between the different site of action and the anticonvulsant activity of these derivatives was discussed. Although the main mechanism of Imi induced seizures cannot be easily determined, potential interactions with the receptors of the excitatory amino acid neurotransmitters exists. In fact, antagonists of excitatory amino acids are able to increase the threshold for the seizures or to prevent the seizures induced by Imi. In addition, Imi acts on the central nervous system by inhibition of GABA neurotransmission and Msc, a selective GABAA agonist, was able to protect against seizures induced by Imi.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acids; Animals; Anticonvulsants; Dizocilpine Maleate; Excitatory Amino Acids; Glutamine; Imipenem; Kynurenic Acid; Mice; Mice, Inbred DBA; Muscimol; Piperazines; Piperidines; Quinoxalines; Seizures

Developmental changes in the levels of N-methyl-D-aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Topics: Aging; Animals; Animals, Newborn; Brain; Dizocilpine Maleate; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; RNA Splicing; RNA, Messenger

Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125I-MK-801 binding assays and electrophysiological measures. Binding of 125I-MK-801 to cells transfected with NR 1a/2B is saturable with a KD of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.

Topics: Animals; Brain; Cell Survival; Dizocilpine Maleate; Electrophysiology; Membranes; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Transfection

The strychnine-insensitive glycine site on the N-methyl-D-aspartate (NMDA) receptor complex is a target for development of a host of therapeutic agents including anxiolytics, antidepressants, antiepileptics, anti-ischemics and cognitive enhancers. In the present experiments, the discriminative stimulus effects of (+)-HA-966 [R-(+)-3-amino-1-hydroxypyrrolid-2-one], a low-efficacy partial agonist of the glycine site, was explored. Male, Swiss-Webster mice were trained to discriminate (+)-HA-966 (170 mg/kg i.p.) from saline in a T-maze under which behavior was controlled by food. Other glycine partial agonists, 1-amino-1-cyclopropanecarboxilic acid and D-cycloserine, fully substituted for the discriminative stimulus effects of (+)-HA-966 despite known differences in other pharmacological effects of these compounds. The glycine site antagonist, 7-chlorkynurenic acid, did not substitute for (+)-HA-966. Likewise other functional NMDA antagonists acting at nonglycine sites of the NMDA receptor also did not substitute: neither the high (dizocilpine) or low affinity (ibogaine) ion-channel blocker, the competitive antagonist, NPC 17742 [2R,4R,5S-2-amino-4,5-(1, 2-cyclohexyl)-7-phosphonoheptanoic acid], nor the polyamine antagonist, ifenprodil, substituted for (+)-HA-966. Although the full agonist, glycine, did not substitute, this compound fully blocked the discriminative stimulus effects of (+)-HA-966. In a separate group of mice trained to discriminate 0.17 mg/kg of dizocilpine from saline, (+)-HA-966 produced a maximum of only 50% dizoclipine-appropriate responses. These data suggest that the discriminative stimulus effects of (+)-HA-966 are based upon its partial agonist actions at the strychnine-insensitive glycine site.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Animals; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Ibogaine; Male; Mice; Piperidines; Pyrrolidinones; Receptors, N-Methyl-D-Aspartate; Strychnine

Topics: Animals; Biguanides; Dizocilpine Maleate; Histamine Release; In Vitro Techniques; Kinetics; Mast Cells; p-Methoxy-N-methylphenethylamine; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermine

Topics: Animals; Cerebral Ventricles; Dizocilpine Maleate; Guanidines; Injections, Intraventricular; Male; Mice; Mice, Inbred Strains; Motor Activity; Neurotoxins; Oxidoreductases Acting on CH-NH Group Donors; Piperidines; Putrescine; Receptors, N-Methyl-D-Aspartate; Spermidine; Stereotyped Behavior

The present study was undertaken to characterize [3H]ifenprodil binding in rat brain. [3H]Ifenprodil showed saturable, high-affinity binding at 4 degrees C. Specific binding, defined with 10 microM ifenprodil as a competitor, was inhibited biphasically by the s receptor ligands, GBR 12909, 1,3-di-o-tolylguanidine (DTG), and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP). At 4 degrees C, 3 microM GBR 12909, which inhibited about 50% of specific binding of [3H]ifenprodil was used to mask sigma receptors. Under these conditions, specific binding of [3H]ifenprodil was inhibited potently by ifenprodil, SL 82.0715, poly(L-arginine), poly(L-lysine), neomycin, ruthenium red, spermine, arcaine and spermidine. In the presence of 3 microM GBR 12909, Zn2+ and Mg2+ partially inhibited specific binding of [3H]ifenprodil at 4 degrees C. In contrast, in the absence of GBR 12909, at 37 degrees C specific binding of [3H]ifenprodil was partially inhibited by Zn2+, but not by Mg2+. The anatomical distribution of [3H]ifenprodil binding at 4 degrees C (GBR 12909 included) in rat brain closely paralleled that of [3H]MK-801 (dizocilpine) binding (r = 0.971, P < 0.005). Without GBR 12909, specific [3H]ifenprodil binding at 37 degrees C was inhibited potently by sigma ligands. In the presence of 3 microM GBR 12909, [3H]ifenprodil binding at 4 degrees C was highest in synaptosomal and myelin fractions; however, without GBR 12909, [3H]ifenprodil binding at 37 degrees C was highest in microsomal and myelin fractions, consistent with the subcellular distribution of sigma receptors. The results suggest that, in the presence of 3 microM GBR 12909, at 4 degrees C, [3H]ifenprodil binds to sites that are sensitive to polyamines and related compounds; and that without GBR 12909, at 37 degrees C, [3H]ifenprodil interacts with sigma receptors in rat brain.

Topics: Adrenergic alpha-Antagonists; Animals; Binding Sites; Binding, Competitive; Brain; Dizocilpine Maleate; Dopamine Agents; Dose-Response Relationship, Drug; Guanidines; In Vitro Techniques; Male; Neurotransmitter Uptake Inhibitors; Piperazines; Piperidines; Rats; Rats, Inbred F344; Receptors, sigma

KCl (20-100 mM) and N-methyl-D-aspartate (NMDA, 100-1,000 microM) produce concomitant concentration-dependent increases in the release of previously captured [14C]acetylcholine and [3H]spermidine from rat striatal slices in vitro. The effects of NMDA (300 microM) on striatal [14C]acetylcholine and [3H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [14C]acetylcholine release was antagonized by ifenprodil (IC50 = 5.3 microM) and MgCl2 (IC50 = 200 microM), neither compound antagonized the NMDA-evoked release of [3H]spermidine at concentrations up to 100 microM (ifenprodil) or 1 mM (MgCl2). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striatum.

Topics: 2-Amino-5-phosphonovalerate; Acetylcholine; Aminoquinolines; Animals; Carbon Radioisotopes; Corpus Striatum; Dizocilpine Maleate; Dose-Response Relationship, Drug; In Vitro Techniques; Kinetics; Magnesium; Magnesium Chloride; N-Methylaspartate; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Tritium

Spermine and other polyamines both stimulate and inhibit N-methyl-D-aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [3H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 microM) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [3H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 mM), spermine markedly decreased equilibrium [3H]dizocilpine binding by decreasing both affinity and Bmax, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [3H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 microM) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC50 = 123 microM) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [3H]dizocilpine binding.

Topics: Animals; Biguanides; Binding Sites; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; Kinetics; Male; Piperidines; Polyamines; Prosencephalon; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine; Tritium

The selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) and the selective N-methyl-D-aspartate (NMDA) receptor antagonists MK 801 and ifenprodil were administered to Mongolian gerbils following a 5 min period of bilateral carotid artery occlusion. NBQX when given 4, 6 or 24 h after ischaemia gave a reduced loss of hippocampal CA1 neurones compared to control animals receiving vehicle only. Dizocilipine (MK 801) (1-10 mg/kg i.p.) and ifenprodil (a total of 45 mg/kg i.p.) gave no protection. The peak levels of NBQX obtained in the cerebrospinal fluid of gerbils receiving the neuroprotective dose (3 x 30 mg/kg i.p.) was 1 microM. In gerbil cortex slices, this concentration had no effect on NMDA-evoked depolarization, but had a moderate effect on kainate and gave a total blockade of AMPA depolarizations. It is concluded that antagonists of non-NMDA glutamate receptor subtypes, possibly AMPA, may be a useful therapeutic approach for cerebral ischaemia-related brain damage following global ischaemia.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Gerbillinae; Hippocampus; Ibotenic Acid; Ischemic Attack, Transient; Kinetics; Male; Neurons; Piperidines; Quinoxalines; Receptors, AMPA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

Some non-competitive antagonists of N-methyl-D-aspartate (NMDA) were evaluated for potency to antagonize audiogenic seizures in genetically epilepsy-prone rats, following intraperitoneal administration. Phencyclidine (PCP), dizocilpine (MK-801), ketamine, ifenprodil and dextromethorphan, displayed anticonvulsant activity at doses similar to those which impaired performance in the rotarod equilibrium procedure. The noncompetitive NMDA receptor antagonists, at doses which slightly overlapped with the doses required for a full anticonvulsant protection against audiogenic seizures in genetically epilepsy-prone rats, induced profound untoward behavioural effects. This behavioural syndrome was characterized by marked ataxia, hyperactivity, stereotypes and wet dog shakes. In contrast, the effective anticonvulsant dose of 3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid (CPPene) was less than that required to impair rotarod performance and did not produce the PCP-like syndrome. A potential use in antiepileptic therapy, of CPPene or other new selective NMDA antagonists, with fewer neurotoxic effects but not for non-competitive antagonists such as MK-801, is suggested.

Topics: Acoustic Stimulation; Adrenergic alpha-Antagonists; Animals; Anticonvulsants; Dextromethorphan; Dizocilpine Maleate; Epilepsy; Injections, Intraperitoneal; Ketamine; Male; Movement; Phencyclidine; Piperazines; Piperidines; Postural Balance; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

The properties of many ligand-gated ion channels are altered during development. We have characterized a developmental switch in the sensitivity of NMDA receptors to the novel antagonist ifenprodil using ligand binding assays with rat brain membranes and voltage-clamp recording of Xenopus oocytes expressing NMDA receptors after injection of RNA from rat brain and from cloned subunits of the receptor. In neonatal rat brain, NMDA receptors have a uniformly high affinity for ifenprodil. During postnatal development, a second population of receptors having a 100-fold lower affinity for ifenprodil is expressed and represents 50% of NMDA receptors in adult rat brain. This developmental change also occurred in cortical neurons maintained in primary culture. Ifenprodil potently inhibited responses of homomeric NR1 and heteromeric NR1/NR2B receptors but not NR1/NR2A receptors expressed in oocytes, suggesting that inclusion of different NR2 subunits in native NMDA receptors can control the sensitivity to ifenprodil.

Topics: Animals; Animals, Newborn; Brain; Cell Membrane; Cells, Cultured; Cerebral Cortex; Cloning, Molecular; Dizocilpine Maleate; Female; Gene Expression; Neurons; Oocytes; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Transfection; Xenopus laevis

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)

Topics: Amino Acids; Animals; Biogenic Polyamines; Chick Embryo; Dizocilpine Maleate; Kynurenic Acid; N-Methylaspartate; Pipecolic Acids; Piperidines; Receptors, Glutamate; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine; Retina

The N-methyl-D-aspartate (NMDA) antagonists ifenprodil and SL-82,0715 were examined for neuroprotective efficacy against glutamate toxicity of hippocampal neurons in culture. Hippocampal cells were grown on 96-well culture plates for 2 to 3 weeks and then exposed for a 15-min period to glutamate or NMDA. Neurodegeneration was quantified 24 hr after the excitotoxin exposure, by measuring the activity of lactate dehydrogenase leaked into the culture medium by the damaged cells. Glutamate induced a concentration-dependent increase in lactate dehydrogenase that reached 3-fold the activity of control cultures. The NMDA antagonists MK-801 and AP-7 blocked this neurotoxicity when added either during or after the glutamate exposure. Ifenprodil and SL-82,0715 blocked the neurotoxicity only when added during the excitotoxin exposure. Ifenprodil was 3 times more potent than SL-82,0715 in blocking glutamate or NMDA-induced neurotoxicity. Glycine did not reverse the neuroprotective effects of these antagonists. The neuroprotective effect of ifenprodil or SL-82,0715 did not appear to result from actions at alpha-1 adrenergic or sigma receptor sites because the alpha-1 adrenergic antagonist prazosin and the sigma ligands haloperidol, 3-(3-hydroxyphenyl)-N-propylpiperidine) and 1,3-di-o-tolylguanidine) showed no neuroprotective activity. We conclude that ifenprodil and SL-82,0715 protect cultured hippocampal neurons from excitotoxic damage by antagonizing NMDA receptors.

Topics: Animals; Cells, Cultured; Culture Media; Dizocilpine Maleate; Drug Antagonism; Glutamates; Glutamic Acid; Hippocampus; L-Lactate Dehydrogenase; N-Methylaspartate; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

We investigated the effect of ifenprodil on excitotoxic cell death induced by acute exposure to glutamate receptor agonists in primary cultures of foetal mouse cerebral cortex. L-Glutamate and N-methyl-D-aspartate (NMDA) but not kainate and quisqualate-induced toxicity was attenuated in the presence of ifenprodil. In addition, ifenprodil and MK-801 blocked NaCN-induced toxicity. It is concluded that the cerebro-protective properties of ifenprodil in these models are mediated by NMDA receptor antagonism.

Topics: Animals; Cell Death; Cells, Cultured; Cerebral Cortex; Cyanates; Dizocilpine Maleate; Glutamates; Glutamic Acid; Kainic Acid; Mice; N-Methylaspartate; Neurons; Piperidines; Quisqualic Acid; Receptors, N-Methyl-D-Aspartate

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevai

Topics: Adrenergic alpha-Antagonists; Animals; Binding, Competitive; Brain; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; Kinetics; Male; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes; Tritium

The mechanism of neurodegeneration and the possible therapeutic amelioration were investigated in a model induced by successive carbon monoxide (CO) exposures. Successive CO exposures resulted in a consistent pattern of degeneration of hippocampal CA1 pyramidal cells, which was quantified using an image analyzer. Competitive and noncompetitive antagonists of N-methyl-D-aspartate (NMDA) receptors, cyclopentenophenanthrene, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten,5,10-imine maleate and an antagonist of glycine binding sites, 7-chlorokynurenic acid, significantly reduced the CO-induced neurodegeneration. Ifenprodil (a antagonist of polyamine binding sites) and glycine had no effect. From these results, it is clear that NMDA receptor/ion channel complex is involved in the mechanism of CO-induced neurodegeneration, and that glycine binding site antagonist as well as NMDA competitive and noncompetitive antagonists may have neuroprotective properties in neurological disorders associated with overactivation of NMDA receptors.

Topics: Animals; Binding Sites; Carbon Monoxide Poisoning; Dizocilpine Maleate; Glycine; Hippocampus; Male; Mice; Piperidines; Receptors, N-Methyl-D-Aspartate; Vasodilator Agents

In the present investigation, the effects of polyamines, spermidine and spermine on events mediated by the N-methyl-D-aspartate (NMDA) receptor complex were examined. Spermine and spermidine did not alter basal levels of cyclic GMP (cGMP) in the cerebellum of the mouse, over a wide range of concentrations. However, exogenously added spermine, spermidine, D- and L-ornithine and putrescine attenuated the increases in cGMP seen after the administration of D-serine, an agonist of the NMDA receptor-associated glycine recognition sites. Spermine and/or spermidine also antagonized harmaline-, methamphetamine- and pentylenetetrazol-induced increases in the levels of cGMP. Spermidine also potentiated (+)-MK-801 (dizocilipine)-induced attenuation of basal levels of cGMP. Intracerebroventricular administration of ifenprodil, a suggested polyamine antagonist, did not antagonize spermine- and spermidine-induced attenuation of the response to D-serine. These data suggest that exogenously added polyamines attenuate events mediated by the NMDA receptor complex, in an ifenprodil-insensitive manner.

Topics: Animals; Biogenic Polyamines; Cerebellum; Cyclic GMP; Dizocilpine Maleate; Injections, Intraventricular; Male; Methamphetamine; Mice; Neural Pathways; Ornithine; Pentylenetetrazole; Piperidines; Putrescine; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine

The administration of methamphetamine (METH) to experimental animals results in damage to nigrostriatal dopaminergic neurons. We have demonstrated previously that the excitatory amino acids may be involved in this neurotoxicity. For example, several compounds which bind to the phenyclidine site within the ion channel linked to the N-methyl-D-aspartate (NMDA) receptor protected mice from the METH-induced loss of neostriatal tyrosine hydroxylase activity and dopamine content. The present study was conducted to characterize further the role of the excitatory amino acids in mediating the neurotoxic effects of METH. The administration of three or four injections of METH (10 mg/kg) every 2 hr to mice produced large decrements in neostriatal dopamine content (80-84%) and in tyrosine hydroxylase activity (65-74%). A dose-dependent protection against these METH-induced decreases was seen with two noncompetitive NMDA antagonists, ifenprodil and SL 82.0715 (25-50 mg/kg/injection), both of which are thought to bind to a polyamine or sigma site associated with the NMDA receptor complex, and with two competitive NMDA antagonists, CGS 19755 (25-50 mg/kg/injection) and NPC 12626 (150-300 mg/kg/injection). Moreover, an intrastriatal infusion of NMDA (0.1 mumol) produced a slight but significant loss of neostriatal dopamine which was potentiated in mice that also received a systemic injection of METH. The results of these studies strengthen the hypothesis that the excitatory amino acids play a critical role in the nigrostriatal dopaminergic damage induced by METH.

Topics: Amino Acids; Animals; Corpus Striatum; Dizocilpine Maleate; Male; Methamphetamine; Mice; N-Methylaspartate; Pipecolic Acids; Piperidines; Receptors, Dopamine; Receptors, N-Methyl-D-Aspartate; Tyrosine 3-Monooxygenase

1. Agonists may act at any one of three sites on the N-methyl-D-aspartate (NMDA) receptor-effector complex to promote opening of the associated ion channel. The three sites are activated by i) NMDA, L-glutamate, aspartate, and other dicarboxylic amino acids; ii) glycine, D-serine, D-cycloserine, and others; iii) the polyamines spermine or spermidine, but not cadaverine or putrescine. 2. This opening by exogenous agonists is reflected by an enhanced binding of the phencyclidine-like dissociative anesthetic [3H]MK-801 to rat cortical membranes (well washed to remove endogenous agonists, e.g., L-glutamate, glycine). 3. The effects of adding combinations of agonists yielded stimulation approximately equal to the sum of each agonist's effect, suggesting that in the first approximation the three classes act at independent sites. 4. When the glutamate (E) site was antagonized with D-2-amino-5-phosphonopentanoate (D-AP5), no stimulation in binding could be elicited by agonists at the two other sites. Activation of the E site is therefore necessary but not sufficient for channel opening. 5. When the glycine (G) site was antagonized with 7-chlorokynurenate, no stimulation in binding could be elicited by agonists at the other two sites. Activation of the G site is therefore necessary but not sufficient for channel opening. 6. Of the two putative antagonists for the polyamine (PA) site, ifenprodil fails to completely inhibit the binding of [3H]MK-801, whereas arcaine inhibited [3H]MK-801 binding completely. We present data which question the selectivity of arcaine for the polyamine site, and propose that the polyamine site is merely modulatory, but neither necessary nor sufficient, for channel opening.

Topics: 2-Amino-5-phosphonovalerate; Adrenergic alpha-Antagonists; Animals; Biguanides; Binding, Competitive; Biogenic Polyamines; Biotransformation; Brain; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; In Vitro Techniques; Kynurenic Acid; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine

Injection of the N-methyl-D-aspartate receptor agonist quinolinate, or N-methyl-D-aspartate itself, into the rat brain produces neurodegeneration which can be prevented by N-methyl-D-aspartate receptor antagonists administered up to 5 h after excitotoxin injection. The present study was designed to investigate aspects of the mechanisms involved in this delayed form of neurodegeneration. Following its injection into the rat striatum, extracellular levels of [3H]quinolinate were monitored using a microdialysis probe located 1 mm from the site of injection. Peak concentrations were observed 10-20 min after injection and [3H]quinolinate levels decayed in a biexponential fashion, the initial component having an apparent t1/2 of 13.7 +/- 5.2 min (n = 3). Estimations of the extracellular concentrations of quinolinate after an injection of 200 nmol indicated a peak level of 13.7 +/- 6.0 mM (n = 3) at 10-20 min which declined to 1.2 +/- 0.13 mM (n = 3) by 2 h and substantial levels were present up to 5 h, the period over which N-methyl-D-aspartate receptor antagonists are effective in this model. Administration of dizocilpine at 1, 2, 3 or 5 h after injection of 100, 200 or 400 nmol quinolinate resulted in a similar temporal profile of neuroprotection, as assessed by measuring the activities of choline acetyltransferase and glutamate decarboxylase in striatal homogenates, which was independent of the degree of neurodegeneration produced by the different excitotoxin doses. Overall, these results suggest that the neuronal degeneration caused by quinolinate in vivo is critically dependent upon events occurring after the initial peak of excitoxin levels in the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic alpha-Antagonists; Animals; Anticonvulsants; Convulsants; Corpus Striatum; Dialysis; Diazepam; Dizocilpine Maleate; Glutamate Decarboxylase; Haloperidol; Ibotenic Acid; Injections; Male; Nerve Degeneration; Pipecolic Acids; Piperazines; Piperidines; Quinolinic Acid; Quinolinic Acids; Rats; Rats, Inbred Strains; Stereotaxic Techniques

The non-competitive N-methyl-D-aspartate (NMDA) antagonists (channel blockers), MK 801, phencyclidine (PCP) and ketamine induced spontaneous tail-flicks in rats. Their order of relative potency (MK 801 greater than PCP greater than ketamine) corresponds to their relative affinities for the ion channel coupled to NMDA receptors. Drugs interacting with their other potential targets (sigma receptors as well as dopamine, serotonin and noradrenaline uptake sites) failed to induce spontaneous tail-flicks. In addition, the catecholamine stimulants, methylphenidate and cocaine were inactive. CPP and CGS 19755, antagonists at the NMDA recognition site, also dose dependently elicited spontaneous tail-flicks: their maximal effect was equal to that of the channel blockers. In contrast, HA-966 and ifenprodil, putative antagonists at the glycine and polyamine recognition sites, respectively, failed to elicit spontaneous tail-flicks. These data demonstrate that both antagonists of the NMDA recognition site and non-competitive blockers of the associated channel induce spontaneous tail-flicks in rats.

Topics: Animals; Behavior, Animal; Dizocilpine Maleate; Dose-Response Relationship, Drug; Ion Channels; Ketamine; Male; N-Methylaspartate; Organophosphorus Compounds; Phencyclidine; Pipecolic Acids; Piperazines; Piperidines; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.

Topics: Animals; Binding Sites; Brain; Chromatography, Gel; Desipramine; Dizocilpine Maleate; Glycine; Ligands; Male; Piperidines; Polyamines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Solubility; Tritium

The effects of spermidine on the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex was studied in human cerebral cortical membranes. [3H]MK-801 binding was increased from 56 +/- 5 fmol/mg protein (mean +/- S.E.M., n = 7) to 319 +/- 71 fmol/mg protein in the presence of 200 microM spermidine. The ED50 for spermidine stimulation of [3H]MK-801 binding was 89 +/- 22 microM (mean +/- S.E.M., n = 6). In the presence of glutamate (1 microM) plus glycine (1 microM) the ED50 was reduced to 5.5 +/- 0.7 microM. The increase in binding in the presence of spermidine was characterised by an increase in the rate of association of [3H]MK-801. In the presence of spermidine. [3H]MK-801 was inhibited by AP5. 7-chlorokynurenic acid and ifenprodil with IC50 values of 0.5 +/- 0.3 24 +/- 19 and 91 +/- 28 microM, respectively. None of these antagonists was a competitive inhibitor of the spermidine stimulation of [3H]MK-801 binding. Thus spermidine modulates the NMDA receptor complex in human brain, providing further evidence that the complex is similar in rat and human cortex.

Topics: Adrenergic alpha-Antagonists; Aged; Aged, 80 and over; Cerebral Cortex; Dizocilpine Maleate; Female; Humans; In Vitro Techniques; Kynurenic Acid; Male; Membranes; Piperidines; Radioligand Assay; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Spermidine

We have investigated the interactions of polyamines and the N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil with the binding of [3H]MK801 to the NMDA receptor. Spermine and spermidine but not putrescine substantially increase [3H]MK801 binding to well washed rat brain membranes in the absence or presence of saturating concentrations of glutamate and glycine. Spermine also increased the association and dissociation of [3H]MK801 from its binding site, suggesting that polyamines activate the NMDA receptor in a similar manner to glycine. Ifenprodil inhibited the binding of [3H]MK801 in a biphasic fashion. The high affinity phase of binding (Ki of approximately 15 nM) accounted for 50-60% of total [3H]MK801 binding in the nominal absence of glutamate, glycine, and polyamines or in the presence of 100 microM glutamate. This fraction was reduced to 20% by the addition of 30 microM glycine and could be abolished by the addition of 50 microM spermine. However, ifenprodil apparently did not act by binding to the polyamine recognition site. The low affinity phase (Ki of 20-40 microM) was insensitive to the presence of positive modulators and may represent binding to the Zn2+ regulatory site. Ifenprodil decreased NMDA and glycine-induced Ca2+ influx into cultured rat brain neurons. The potency of ifenprodil suggests that spermine may activate NMDA receptors in vivo. These data indicate that ifenprodil may bind to the NMDA receptor in a state-dependent fashion and preferentially stabilize an inactivated form of the channel.

Topics: Acetamides; Animals; Aspartic Acid; Brain; Calcium; Cell Membrane; Dibenzocycloheptenes; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glutamates; Glycine; In Vitro Techniques; Ketamine; Kinetics; N-Methylaspartate; Phencyclidine; Piperidines; Polyamines; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

The behavioral effects of phencyclidine (PCP) were compared with those of several compounds known to antagonize the actions of N-methyl-D-aspartate using two patterns of schedule-controlled responding in rats. Rates of variable interval responding suppressed by punishment were increased greatly by the benzodiazepine chlorodiazepoxide and showed small increases after MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine] . However, no consistent increases in response rates were produced by PCP, by the stereoisomers of N-allylnormetazocine (NANM: SKF 10,047) or by the anti-ischemic drug, ifenprodil. Small doses of PCP did increase rates of unpublished variable interval responding, as did a low dose of MK-801. Timing behavior maintained by a differential reinforcement of low rate schedule was disrupted by all the compounds studied. Response rates were increased by at least one dose of PCP, MK-801, (+)-NANM and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. The effect of MK-801, however, was considerably greater than that of the other compounds. Ifenprodil and (-)-NANM did not increase rates of responding but, at high doses, produced decreases in reinforcement frequency indicating that efficient timing behavior had been disrupted. These results show that although PCP, MK-801 and (+)-NANM produce generally similar behavioral effects, there may also be some differences between the compounds, notably a more consistent effect of MK-801 on punished responding. These behavioral effects may be related to antagonism of N-methyl-D-aspartate but ifenprodil, which is also an N-methyl-D-aspartate antagonist, does not show a similar behavioral profile.

Topics: Animals; Aspartic Acid; Conditioning, Psychological; Dibenzocycloheptenes; Dizocilpine Maleate; Male; N-Methylaspartate; Phenazocine; Phencyclidine; Piperazines; Piperidines; Punishment; Rats; Rats, Inbred Strains; Reinforcement Schedule; Stereoisomerism

Rats were trained to discriminate phencyclidine (PCP) from saline at doses of 2 and 4 mg/kg, using a two-lever food reinforced operant technique. +/- N-allylnormetazocine (+/- SKF 10047), +5-methyl-10,11-dihydro-5H-dibenzo[A,D]cyclohepten-5,10-imine MK 801), 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) and ifenprodil, which have been shown to antagonise the effects of N-methyl-D-aspartate (NMDA), were tested for their ability to give rise to PCP-appropriate responding. In rats trained at both doses of PCP, +/- SKF 10047 (2-12 mg/kg) and MK 801 (0.0125-0.2 mg/kg) produced dose-related responding on the lever associated with PCP injection. The relative potency of these two compounds was the same in the two groups of animals, but their absolute potencies to produce a PCP-like discriminative stimulus were dependent on the training dose of PCP. In contrast, neither the competitive NMDA antagonist CPP (4-20 mg/kg) nor the non-competitive antagonist ifenprodil (2-12 mg/kg) produced PCP-appropriate responding and ifenprodil (4 mg/kg) neither potentiated nor antagonised PCP. These findings are discussed in the light of the hypothesis that the behavioural effects of PCP are mediated via a reduction of neurotransmission at the NMDA-subtype of glutamate receptors.

Topics: Adrenergic beta-Antagonists; Animals; Anticonvulsants; Chlordiazepoxide; Dibenzocycloheptenes; Discrimination, Psychological; Dizocilpine Maleate; Drug Interactions; Generalization, Stimulus; Male; Phenazocine; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter