N-Methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

Central <Emphasis Type="SmallCaps">l</Emphasis>-arginine reduced stress responses are mediated by <Emphasis Type="SmallCaps">l</Emphasis>-ornithine in neonatal chicks

Recently, we observed that central administration of L-arginine attenuated stress responses in neonatal chicks, but the contribution of nitric oxide (NO) to this response was minimal. The sedative and hypnotic effects of L-arginine may be due to L-arginine itself and/or its metabolites, excluding NO. To clarify the mechanism, the effect of intracerebroventricular (i.c.v.) injection of L-arginine metabolites on behavior under social separation stress was investigated. The i.c.v. injection of agmatine, a guanidino metabolite of L-arginine, had no effect during a 10 min behavioral test. In contrast, the i.c.v. injection of L-ornithine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior. The L-ornithine concentration in the telencephalon and diencephalon increased following the i.c.v. injection of L-arginine. In addition, several free amino acids including L-alanine, glycine, L-proline and L-glutamic acid concentrations increased in the telencephalon. In conclusion, it appears that L-ornithine, produced by arginase from L-arginine in the brain, plays an important role in the sedative and hypnotic effects of L-arginine observed during a stress response. In addition, several other amino acids having a sedative effect might partly participate in the sedative and hypnotic effects of L-arginine.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline is a sedative regulator of acute stress in the brain of neonatal chicks

The purpose of the present study was to clarify the central nervous system function of amino acids during acute stress. In Experiment 1, changes in free amino acid pattern were investigated in the brain of neonatal chicks exposed to either restraint with isolation-induced or fasting stress. l-Proline and l-arginine were decreased in the telencephalon and diencephalon under any stress. Since the central nervous system functions of l-arginine during the stress response has recently been reported, in Experiment 2, the effect of intracerebroventricular injection of l-proline (0.5, 1.0, 2.0 μmol) during isolation-induced stress was investigated. l-Proline induced sedative and hypnotic effects in a dose-dependent manner. It is suggested that l-proline may have an important role to attenuate the stress response in the central nervous system of chicks.     

N-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-d-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced sleep-like behavior or sedative effects, the potency of NMDA was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.     

A glycinergic intervention potentiates the antiseizure efficacies of MK-801, flurazepam,and carbamazepine

Twenty four hours after mice were forced to swim for up to 10 minutes in cold water, there was a reduction in the ability of MK-801 to antagonize the electrical precipitation of tonic hindlimb extension. Milacemide, a lipophilic prodrug of glycine, restored the antiseizure efficacy of MK-801 to the same level observed in unstressed animals treated with milacemide and MK-801. Stimulation of the glycine-gated chloride ionophore subsequent to the liberation of free glycine could explain milacemide's pharmacologic action as an adjuvant to MK-801. Consistent with this interpretation, milacemide was able to potentiate the antiseizure effects of flurazepam, a benzodiazepine agonist, in stressed and unstressed mice and carbamazepine in unstressed animals.d-cycloserine, a partial glycine agonist with greater specificity for the strychnine-insensitive modulatory site on the NMDA receptor complex, was examined for its effect on MK-801's antiseizure efficacy. At a high dose (320 mg/kg),d-cycloserine alone had an anticonvulsant effect. Moreover, this dose ofd-cycloserine administered with MK-801 showed a significantly greater anticonvulsant efficacy than MK-801 alone. The data support the development of glycinergic interventions as adjunctive agents in the pharmacotherapy of seizure disorders.     

Reduction in the mechanonociceptive response by intrathecal administration of glycine and related compounds

We have previously reported that enhanced glycine release is produced by epidural spinal cord stimulation, a clinical method for treating neuropathic pain. Our current hypothesis is that glycine administered intrathecally reduces neuropathic pain as measured by the Randall-Selitto method. Neuropathic rats created by unilateral partial ligation of the sciatic nerve were treated with intrathecal infusion of glycine, strychnine, MK-801, or 5,7-DKA at 0.1 μmol, or artificial CSF for 2 hours at a rate of 10 μl/min. Force required to produce the pain response was significantly increased after glycine administration and reduced using strychnine, a specific glycine receptor (Gly 1) antagonist. Strychnine blocked the response to glycine when infused together. Administration of the non-specific NMDA receptor MK-801 antagonist and 5,7-DKA, a specific glycine-NMDA receptor (Gly 2) antagonist, however, failed to block the response to glycine. Our results provide evidence for the use of glycine and related compounds to treat neuropathic pain.     

Reduction in Thermal Hyperalgesia by Intrathecal Administration of Glycine and Related Compounds

We have previously shown in animal models that enhanced segmental glycine release is produced by neuroaugmentation techniques commonly used to control pain in humans. Our current hypothesis is that glycine administered intrathecally reduces the pain response evoked by the hotplate analgesia meter method. Neuropathic rats created by unilateral partial ligation of the sciatic nerve were treated with intrathecal infusion of glycine, strychnine, MK-801, or 5–7 DKA at 0.1 mol for 2 hours at a rate of 10 l/min. Time required for limb withdrawal at 42°C was significantly increased after glycine administration but not altered by strychnine, a specific glycine receptor antagonist. Administration of the NMDA receptor antagonist, MK-801, blocked the influence of glycine, with a less obvious antagonistic response from 5,7 DKA. Our results provide evidence that glycine and related compounds significantly modify thermal hyperalgesia, and may operate primarily through the NMDA receptor complex.     

Solubilization of Spermidine-Sensitive (+)-[3H]5-Methyl-10,11 -Dihydro-5H-Dibenzo[a,d]cyclohepten-5,10-Imine ([3H]MK-801) Binding Activity from Rat Brain

The receptor-ionophore complex of the N-methyl-D-aspartate (NMDA)-sensitive receptor was solubilized by deoxycholic acid from rat brain using (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801) binding as a marker for the receptor. Gel filtration of the solubilized preparations on a Sephadex G-25 column revealed significant [3H]MK-801 binding sensitive to potentiation by glutamate and glutamate/glycine, which was prevented by competitive antagonists for the NMDA and strychnine-insensitive glycine (GlyB) sites. In contrast to NMDA and glycine, spermidine markedly potentiated the amount of [3H]MK-801 binding in solubilized preparations by increasing the apparent affinity of the ligand. In the presence of all three stimulants, the solubilized preparations exhibited pharmacological profiles similar to those in the membrane preparations. These results clearly indicate that the whole macromolecular NMDA receptor-ionophore complex is solubilized under the experimental conditions used.     

Effects of long-term antipsychotic treatment on NMDA receptor binding and gene expression of subunits

Postmortem studies in schizophrenic patients revealed alterations in NMDA receptor binding and gene expression of specific subunits. Because most of the patients had been treated with antipsychotics over long periods, medication effects might have influenced those findings. We treated animals with haloperidol and clozapine in clinical doses to investigate the effects of long-term antipsychotic treatment on NMDA receptor binding and gene expression of subunits. Rats were treated with either haloperidol (1,5 mg/kg/day) or clozapine (45 mg/kg/day) given in drinking water over a period of 6 months. Quantitative receptor autoradiography with [³H]-MK-801 was used to examine NMDA receptor binding. In situ hybridization was performed for additional gene expression studies of the NR1, NR2A, NR2B, NR2C, and NR2D subunits. [³H]-MK-801 binding was found to be increased after haloperidol treatment in the striatum and nucleus accumbens. Clozapine was shown to up-regulate NMDA receptor binding only in the nucleus accumbens. There were no alterations in gene expression of NMDA subunits in any of the three regions. However, the NR2A subunit was down-regulated in the hippocampus and prefrontal cortex by both drugs, whereas only clozapine induced a down-regulation of NR1 in the dorsolateral prefrontal cortex. NR2B, 2C, and 2D subunits did not differ between treatment groups and controls. Both altered NMDA receptor binding and subunit expression strengthen a hyperglutamatergic function after haloperidol treatment and may contribute to some of our postmortem findings in antipsychotically treated schizophrenic patients. Because the effects seen in different brain areas clearly vary between haloperidol and clozapine, they may also be responsible for some of the differences in efficacy and side effects.     

N-Methyl-D-Aspartate receptors in acquisition of the short-term memory in the honeybeeApis mellifera

Role of NMDA receptors in the process of associative learning has been studied in the honeybeeApis mellifera L. in behavior experiments, using method of conditional reflexes. To determine pharmacological profile of NMDA receptors, effects of Mg²⁺ ions, NMDA, glycine (Gly), antagonist of the glycine site 5,7-dichlorokynurenic acid (DCK), competitive antagonists of NMDA receptors: D,L-2-aminophosphovalerate (APV), L-2-aminophosphobutyrate (APB), and D-glutamyl-aminomethylphosphonic acid (GAMP) as well of antagonists of NMDA receptor ion channels MK-801 and ketamine (Ket) were studied on acquisition of alimentary conditional reflex and its retention in memory. NMDA increased capacity for learning by stimulating shortterm memory. The NMDA receptor co-agonist Gly activated this NMDA effect. DCK eliminated the NMDA and Gly stimulatoty effects. All tested antagonists at millimolar concentrations inhibited associative function. The data obtained confirm our hypothesis about participation of NMDA receptors in processes of formation of short-term memory in the honeybeeApis mellifera and suggest that functional characteristics of the NMDA receptors involved in the process of associative learning in the honeybee resemble those in mammals.     

Neurosteroids allosterically modulate the ion pore of the NMDA receptor consisting of NR1/NR2B but not NR1/NR2A

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer’s disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3α5βS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [³H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [³H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.     

NMDA Receptor Regulation by <Emphasis Type="SmallCaps">D</Emphasis>-serine: New Findings and Perspectives

The N-methyl-d-aspartate (NMDA) receptors play key roles in excitatory neurotransmission and are involved in several important processes, including learning, behavior, and synaptic plasticity. The regulation of NMDA receptor neurotransmission has been extensively studied, but many important questions still remain unsolved. One of the most debated aspects of the NMDA receptor regulation relates to the identity, role, and cellular origin of the NMDA coagonist(s). In addition to glutamate, the NMDA receptor activity was believed to be regulated by the coagonist glycine. More recently, d-serine has also been proposed to play a role as a key coagonist for NMDA receptor activity and neurotoxicity. A surprising unique biosynthetic pathway for d-serine has been demonstrated, indicating the conservation of d-amino acid metabolism in mammals. d-Serine was originally shown to be exclusively made in astrocytes, indicating a possible role as a gliotransmitter. Nevertheless, recent data indicate that d-serine has a neuronal origin as well, which raises several new questions on d-serine disposition. In this review, I discuss recent advances in the field and propose a novel model of d-serine signaling that includes a bidirectional flow of d-serine between astrocytes and neurons. This review is dedicated to the memory of Dr. Marcos Wolosker.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity

The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [³H]MK-801 labeled a uniform population of sites (K_d = 3.2 ± 0.3 nM) in a saturable manner (B_max = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [³H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [³H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.     

Muscarinic antagonists attenuate dizocilpine-induced hypermotility in mice.

Pretreatment of mice with the muscarinic receptor antagonists scopolamine and atropine attenuated the hypermotility (but not the depression of rearing) induced by a low dose of dizocilpine maleate [(+)-MK-801; 0.1 mg/kg, i.p.], a non-competitive NMDA antagonist. In contrast, the muscarinic blockers failed to affect hypermotility induced by equieffective doses of phencyclidine (1 mg/kg, i.p.) or d-amphetamine (2 mg/kg, i.p.). These results suggest differences between the mechanism of behavioral activation produced by dizocilpine and phencyclidine, and demonstrate the potential of muscarinic blockade for diminishing the behavioral toxicity of NMDA antagonists.     

<Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">D</Emphasis>-aspartate Receptors are Involved in the Quinolinic Acid,but not in the Malonate Pro-oxidative Activity <Emphasis Type="Italic">in vitro</Emphasis>

Oxidative stress plays a significant role in the neurotoxicity of a variety of agents that interact with the N-methyl-D-aspartate (NMDA) receptors. Here we investigated in a comparative way the pro-oxidative effects of quinolinic acid (QA) and malonate, two neurotoxic substances that act through distinct primary molecular mechanisms on the production of thiobarbituric acid reactive species (TBARS) by brain homogenates. In fact, QA is thought to activate directly the NMDA receptor, whereas malonate seems to act primarily by inhibiting oxidative metabolism. The malonate-induced TBARS formation was not modified by cyanide (CN^–) or 2,4-dinitrophenol. MK-801 did not reduce basal or malonate induced-TBARS production in fresh tissues preparations. However, in heat-treated preparations a significant effect of MK-801 against basal TBARS production was observed, but not on the malonate induced-TBARS production. QA induced-TBARS production was significantly prevented by MK-801 either in fresh or heat-treated preparations. The antioxidant effect of MK-801 on basal and QA-induced TBARS production increased as the temperatures used to treat S1 were increased. Succinate dehydrogenase (SDH) was inhibited by malonate but not by QA. Malonate was able to chelate iron(II) and the malonate-iron complex(es) is(are) active as measured by its(their) activity on deoxyribose degradation assay. These findings indicate that direct interactions of malonate with NMDA receptors are not involved in malonate pro-oxidative activity in vitro. QA pro-oxidative activity in vitro was related, at least in part, to its capability in stimulate NMDA receptors. Taken together, these findings indicated that malonate pro-oxidative activity in vitro could be attributed to its capability of changing the ratio Fe²⁺/ Fe³⁺, which is essential to TBARS production.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Effect of a noncompetitive antagonist (MK-801) of NMDA receptors on convulsions and brain amino acid level in E1 mice

Anticonvulsant action of MK-801, a novel noncompetitive antagonist of N-methyl-d-aspartate (NMDA) receptor, was examined in genetically epileptic E1 mice. Systemic injection of MK-801 (0.1–1.0 mg/kg) potently suppressed generalized tonic-clonic convulsions of in a dose-dependent manner (ED₅₀, 0.17 mg/kg). This anticonvulsant effect of MK-801 appeared at a dose which did not induced any obvious behavioral changes. Following the administration of a fully anticonvulsant dose of MK-801 (1 mg/kg), amino acid analysis revealed a significantly elevated level of glycine in the hippocampus. Levels of other amino acids including glutamate, aspartate, taurine, glutamine, alanine, and -aminobutyrate were not changed either in the hippocampus or in the cerebral cortex. This study suggests that NMDA system may play an essential role in seizure-triggering mechanisms in E1 mouse.     

Midazolam and theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7) attenuate stress-induced expression of c-fos mRNA in the dentate gyrus

Summary 1. The effects of restraint stress on c-fos mRNA expression in the dentate gyrus were investigated byin situ hybridization.2. Confirming previous findings, c-fos mRNA expression increased after 30 min of forced restraint.3. This effect was attenuated by a previous i.c.v. injection of the anxiolytic benzodiazepine midazolam (20 nmol/2 µl) or theN-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7; 5 nmol/2 µl).4. These results suggest that the dentate gyrus is activated during restraint stress and that this activation may be modulated by benzodiazepine -aminobutyric acid_A (GABA_A) or NMDA receptors.     

Regional Heterogeneity of Polyamine Effects on the N-Methyl-D-Aspartate Receptor in Rat Brain

Abstract: Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [³H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [³H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [³H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the K_D for [³H]MK-801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the E_max value in the ventromedial caudate putamen without changing the EC₅₀. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.