Glycine Site Of N-methyl-D-aspartate Receptor Research Articles

D-serine added to antipsychotics for the treatment of schizophrenia

Background: Hypofunction of N-methyl- D-aspartate (NMDA) subtype glutamate receptor has been implicated in the pathophysiology of schizophrenia. D-serine is a full agonist of the glycine site of NMDA receptor, an endogenous cotransmitter enriched in corticolimbic regions and distributed in parallel with NMDA receptor. Supplementation of D-serine may improve the symptoms of schizophrenia. Methods: Thirty-one Taiwanese schizophrenic patients enrolled in a 6-week double-blind, placebo-controlled trial of D-serine (30 mg/kg/day), which was added to their stable antipsychotic regimens. Of these, 28 completed the trial. Measures of clinical efficacy, side effects, and serum levels of amino acids and D-serine were determined every other week. Wisconsin Card Sorting Test (WCST) was performed at the beginning and end of the trial. Results: Patients who received D-serine treatment revealed significant improvements in their positive, negative, and cognitive symptoms as well as some performance in WCST. D-serine levels at week 4 and 6 significantly predicted the improvements. D-serine was well tolerated and no significant side effects were noted. Conclusions: The significant improvement with the D-serine further supports the hypothesis of NMDA receptor hypofunction in schizophrenia. Given the effects of D-serine on positive symptoms, a trial of D-serine alone in schizophrenia should be considered.

Evidence for functional native NMDA receptors activated by glycine or D-serine alone in the absence of glutamatergic coagonist.

In this study we have examined the effects of N-methyl-D-aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration-dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration-dependent manner the depolarization-evoked release of both CCK-LI and SRIF-LI. This effect of glycine was strychnine-insensitive and could be mimicked by D-serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/D-serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by D-serine was strongly inhibited by ifenprodil (0.3 microM) and by Zn2+ ions (50 nM), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK- and SRIF-releasing neurons possess non-conventional NMDA receptors whose channels can be operated by glycine or D-serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B.

Age- and dose-dependent facilitation of associative eyeblink conditioning by D-cycloserine in rabbits.

Normal aging selectively impairs some forms of learning. For example, aging rabbits require more than twice as many trials to acquire 500-ms trace eyeblink conditioning than do young rabbits. N-methyl-D-aspartate (NMDA) receptor antagonists also impair trace conditioning. The effects of daily D-cycloserine (DCS; a partial agonist of the NMDA receptor-glycine site) treatment were tested on trace conditioning of young or aging rabbits using a conservative quantitative approach. DCS dose dependently improved acquisition, maximally reducing trials to criterion by approximately 50%. Dose-response curves were right-shifted by aging (twice the dose was required to achieve the same enhancement compared with controls). DCS did not affect nonassociative performance but sharpened the conditioned stimulus tone intensity discrimination. DCS thus can functionally modulate NMDA receptors in normal aging, enhance associative learning at all ages, and reduce or reverse age-dependent learning deficits.

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons.

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.

Pharmacological characterization of MDL 105,519, an NMDA receptor glycine site antagonist

MDL 105,519, ( E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid, is a potent and selective inhibitor of [ 3H]glycine binding to the NMDA receptor. MDL 105,519 inhibits NMDA ( N-methyl- d-aspartate)-dependent responses including elevations of [ 3H] N-[1,(2-thienyl)cyclohexyl]-piperidine ([ 3H]TCP) binding in brain membranes, cyclic GMP accumulation in brain slices, and alterations in cytosolic Ca 2+ and Na +-Ca 2+ currents in cultured neurons. Inhibition was non-competitive with respect to NMDA and could be nullified with d-serine. Intravenously administered MDL 105,519 prevented harmaline-stimulated increases in cerebellar cyclic GMP content, providing biochemical evidence of NMDA receptor antagonism in vivo. This antagonism was associated with anticonvulsant activity in genetically based, chemically induced, and electrically mediated seizure models. Anxiolytic activity was observed in the rat separation-induced vocalization model, but muscle-relaxant activity was apparent at lower doses. Higher doses impair rotorod performance, but were without effect on mesolimbic dopamine turnover or prepulse inhibition of the startle reflex. This pattern of activities differentiates this compound from (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801) and indicates a lower psychotomimetic risk. © 1997 Elsevier Science B.V. All rights reserved.

Age- and dose-dependent facilitation of associative eyeblink conditioning by D-cycloserine in rabbits.

Normal aging selectively impairs some forms of learning. For example, aging rabbits require more than twice as many trials to acquire 500-ms trace eyeblink conditioning than do young rabbits. N-methyl-D-aspartate (NMDA) receptor antagonists also impair trace conditioning. The effects of daily D-cycloserine (DCS; a partial agonist of the NMDA receptor-glycine site) treatment were tested on trace conditioning of young or aging rabbits using a conservative quantitative approach. DCS dose dependently improved acquisition, maximally reducing trials to criterion by approximately 50%. Dose-response curves were right-shifted by aging (twice the dose was required to achieve the same enhancement compared with controls). DCS did not affect nonassociative performance but sharpened the conditioned stimulus tone intensity discrimination. DCS thus can functionally modulate NMDA receptors in normal aging, enhance associative learning at all ages, and reduce or reverse age-dependent learning deficits.

In vitro modulation of hippocampal pyramidal cell response by quinolones: effects of HA 966 and gamma-hydroxybutyric acid.

The influence of quinolones on electrically evoked pyramidal cell activity in the rat hippocampus in vitro was studied by using the slice technique. We hoped to learn more about the possible mechanisms for the development of side effects of different quinolones and to find a possible treatment. As reported earlier (W. Dimpfel, M. Spüler, A. Dalhoff, W. Hofmann, and G. Schlüter, Antimicrob. Agents Chemother. 35:1142-1146, 1991), the amplitude of the population spike increased in the presence of ciprofloxacin, lomefloxacin, or ofloxacin about twofold in comparison with reference values. This increase could be prevented in a concentration-dependent manner by the concomitant presence of 3-amino-1-hydroxy-2-pyrrolidone (HA 966), a compound acting at the so-called glycine site of the N-methyl-D-aspartate (NMDA) receptor, but not in the presence of aminophosphonovaleric acid (APV), which acts at a different recognition site of the NMDA receptor. Another tool, 6,7-dinitroquinoxaline-2,3-dione, an antagonist of the so-called AMPA receptor (named after the binding of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] to this site), could not antagonize the effect induced by the quinolones. Activation of the glycine site of the NMDA receptor induced by the presence of D-serine in the superfusion medium also resulted in a concentration-dependent increase in the population spike amplitude. This response remained unchanged in the presence of ciprofloxacin, whereas lomefloxacin and ofloxacin led to further increases in the amplitude, especially in the presence of higher concentrations of D-serine. These results also point to an involvement of the glycine site of the central NMDA receptor in the development of side effects by different quinolones. A complete attenuation of the quinolone-induced effects was obtained in the presence of 2.5 microM gamma-hydroxybutyric acid (GHB), a physiological neuromodulator which is marketed in some countries of Europe as a sedative. It is therefore concluded that the excitatory adverse effects of quinolones might be treated by the administration of GHB.

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

Presence of the N‐Methyl‐D‐Aspartate‐Associated Glycine Receptor Agonist, D‐Seine, in Human Temporal Cortex: Comparison of Normal, Parkinson, and Alzheimer Tissues

The amino acid D-serine (D-Ser), previously recognized as a pharmacological tool for potentiating neuronal activity mediated by the N-methyl-D-aspartate (NMDA) receptor complex, in vitro and in vivo, has been observed in several brain regions of the rat and mouse, most prominently in cortex. In addition to reconfirming the presence and distribution of D-Ser in rat brain, we have observed, for the first time, endogenous, free D-Ser in temporal cortex of normal human brains at a level of 2.18 +/- 0.12 nmol/mg of protein, representing 15 +/- 2% of the free L-Ser pool. The D- and L-Ser specific content and the D/L-Ser ratio obtained from temporal cortex of Parkinson and Alzheimer brains did not differ significantly from those of controls. However, at the levels observed here, and considering its specificity and affinity for the NMDA-associated glycine receptor, endogenous D-Ser is a plausible NMDA receptor glycine site agonist.

Polyamine and ifenprodil interactions with the NMDA receptor's glycine site

Ifenprodil partially inhibited [ 3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor in rat telencephalic membranes. The polyamine spermine significantly enhanced [ 3H]glycine binding and decreased ifenprodils inhibitory potency. However, ifenprodil was unable to completely reverse the stimulation of binding produced by spermine. Also, ifenprodil was found to reduce the maximum degree to which spermine enhanced binding but had no effect on the polyamine's potency. These results show that ifenprodil and spermine do not competitively interact with respect to their abilities to modify [ 3H]glycine binding to the NMDA receptor complex.

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

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl- d-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC 50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC 50s of 175 and 18 μM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Allosteric modulation of nmda receptors: Significance for therapeutics

Neboglamine is a functional modulator of the glycine site on the N-methyl-d-aspartate (NMDA) receptor. Dysfunction of this receptor has been associated with negative and cognitive symptoms in schizophrenia. Thus, we tested the hypothesis that neboglamine behaves as a potential antipsychotic. We compared the effects of neboglamine, d-serine, clozapine, and haloperidol on the expression of Fos-like immunoreactivity (FLI), a marker of neuronal activation, in rat forebrain. We also studied the effects of these agents on phencyclidine (PCP)-induced behaviour in rats, a model predictive of potential antipsychotic activity. Neboglamine, like haloperidol and clozapine, significantly increased the number of FLI-positive cells in the prefrontal cortex, nucleus accumbens, and lateral septal nucleus (3.2-, 4.8-, and 4.5-fold over control, respectively). Haloperidol dramatically increased FLI (390-fold over control) in the dorsolateral striatum, a brain region in which neboglamine and clozapine had no effect. The pattern of FLI induced by neboglamine closely matched that of d-serine, an endogenous agonist at the glycine site of NMDA receptors. Consistent with this finding, neboglamine restored NMDA-mediated neurotransmitter release in frontal cortex punches exposed to the NMDA antagonist PCP. In the behavioural model, all test compounds significantly inhibited PCP-induced hyperlocomotion. Unlike haloperidol and clozapine, neither neboglamine nor d-serine affected the basal levels of locomotor activity. Moreover, oral neboglamine dose-dependently inhibited both the hyperlocomotion and the frequency of rearing behaviour induced by PCP. These results, while confirming that the NMDA glycine site is a feasible target for activating the frontostriatal system, support the clinical evaluation of neboglamine as a treatment for schizophrenia.